Anopheline Species Complexes in South and South-East Asiaapps.searo.who.int/pds_docs/B2406.pdf · Anopheline Species Complexes in South and South-East Asia v Foreword Differences

Anopheline SpeciesComplexes

in South and South-East Asia

SEARO Technical Publication No. 57

Anopheline Species Complexes in South and South-East Asiaii

© World Health Organization 2007

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The designations employed and the presentation of material in this publication do not imply the expressionof any opinion whatsoever on the part of the Secretariat of the World Health Organization concerningthe legal status of any country, territory, city or area or of its authorities, or concerning the delimitationof its frontiers or boundaries.

Printed in India

WHO Library Cataloguing-in-Publication data

World Health Organization, Regional Office for South-East Asia.

Anopheline Species Complexes in South and South-East Asia.

1. Anopheles 2. Species Specificity 3. Sibling Relations 4. Insect Vectors 5. South-East Asia6. Asia, Western

ISBN 978-92-9022-294-1 NLM Classification No. QX 515

Anopheline Species Complexes in South and South-East Asia iii

Contents

Foreword ............................................................................................................... v

Acknowledgements ............................................................................................... vi

1. Introduction ..................................................................................................... 1

2. Techniques used in the recognition of Species Complexes ................................ 7

3. Species Complexes ......................................................................................... 17

3.1 The Annularis Complex ........................................................................ 17

3.2 The Barbirostris Complex ...................................................................... 20

3.3 The Culicifacies Complex ..................................................................... 22

3.4 The Dirus Complex .............................................................................. 33

3.5 The Fluviatilis Complex......................................................................... 41

3.6 The Leucosphyrus Complex .................................................................. 46

3.7 The Maculatus Complex ....................................................................... 48

3.8 The Minimus Complex ......................................................................... 55

3.9 The Philippinensis-Nivipes Complex ..................................................... 62

3.10 The Punctulatus Complex ..................................................................... 65

3.11 The Sinensis Complex........................................................................... 69

3.12 The Subpictus Complex ........................................................................ 73

3.13 The Sundaicus Complex ....................................................................... 76

3.14. The Anopheles stephensi variants .......................................................... 79

4. Prospects for the future .................................................................................. 84

5. References and select bibliography ................................................................. 87

Anopheline Species Complexes in South and South-East Asia v

ForewordDifferences in the biological characteristics ofmembers of the complexes have an importantbearing on malaria transmission dynamics. Itis, therefore, imperative to determine siblingspecies composition and their bionomics aswell as their roles in the transmission ofmalaria.

In 1998, WHO published as a technicalpublication* Anopheline species complexes inSouth-East Asia authored by Dr Sarala K.Subbarao. This book has received muchappreciation both from researchers andprogramme managers. Since its publication,several papers on species complexesidentification tools, especially molecular-basedtools, formal designation of members ofcomplexes, and the phylogenetic relationshipbetween members of a complex and alsobetween the complexes have been published.In view of the importance of species complexesin malaria control operations, an updatededition has been prepared to provide the latestinformation on this important subject. This ispart of our commitment to highlight anddisseminate the knowledge on speciescomplexes which is so vital to malaria controlstrategy, especially when target-specificselective and sustainable vector control isurgently needed. In addition to the South-EastAsia Region, the present edition covers thework done on the species complexes prevalentin the South Asian countries as well. It presentsa clear summary of the work done onanopheline cryptic species, and I am sure itwill be very useful for field malariaentomologists, malaria control programmemanagers and basic researchers working onspecies complexes.

Vector-borne diseases continue to be amajor health problem in the world. Theworsening malaria situation during the

1980s led the World Health Organization(WHO) to declare the control of malaria as aglobal priority. The World Declaration onMalaria, adopted in Amsterdam in October1992, committed WHO Member States to theworldwide intensification of control effortsagainst this disease. Accordingly, a globalMalaria Control Strategy was developed whichlaid emphasis on the following key elements:case management; capacity building forcontrol; containment of epidemics; and basicand applied research. Halting the incidenceof malaria is also highlighted as one of thetargets to be achieved under the UnitedNations Millennium Development Goals(MDGs).

It is very important that vector control, as apart of the global as well as the regional malariacontrol strategy, should succeed. Its successwould depend on a systematic review of theavailable information on vector species andtheir biology, and of the vector control optionsand their selective use. Most of theanophelines that are involved in thetransmission of malaria in the South and South-East Asian countries have been identified asspecies complexes. Species complexes are ofcommon occurrence among anopheline taxa.More than 30 Anopheles taxa have beenidentified so far as species complexes and theyare important vectors of malaria in differentparts of the world. Members of a speciescomplex, commonly known as sibling species,are reproductively-isolated evolutionary unitswith distinct gene pools and, hence, differ intheir biological characteristics.

Samlee Plianbangchang, M.D., Dr.P.H.Regional Director

*WHO Technical Publication, SEARO No. 18 (1998).

Anopheline Species Complexes in South and South-East Asiavi

Acknowledgements

This edition of Anopheles Species Complexes in South and South-East Asia has been produced bythe World Health Organization's South-East Asia Regional Office, Department of CommunicableDiseases, Communicable Diseases Control group. The author of the earlier Anopheles SpeciesComplexes in South-East Asia (1998), Dr Sarala K. Subbarao, was commissioned to prepare therevised edition. The issuing of a new edition reflects the fact that new identification tools havebeen developed and identification of species is critical in control programmes for severalcomplexes.

Professor Chris Curtis, Dr Catherine Walton, Professor Nora J. Besanky and Dr Yeya Torrehave made very valuable suggestions that have enriched the quality of the monograph. ProfessorCurtis also provided detailed editorial corrections of the manuscript. Dr K. Raghavendra,Dr Suprabha G. Pulipparacharuvil and Mr O.P. Singh have provided necessary information andhelp, and Mr U. Sreehari is acknowledged for his help in the preparation of the document.Dr Subbarao also wishes to recognize support given by family members.

Anopheline Species Complexes in South and South-East Asia 1

Mosquitoes are ubiquitous and havea tremendous reproductivepotential and great adaptability to

different ecological conditions. Adding totheir innate ability to adapt, humans areproviding them with conditions, which arehighly congenial for their multiplication.There are about 4500 mosquito species indifferent parts of the world, belonging to 34genera in the family Culicidae, order Diptera,class Insecta and phylum Arthropoda.

Some of the mosquito species transmitdiseases and consequently form an importanttarget for control in public healthprogrammes. Species belonging to the genusAnopheles transmit malaria. Approximately424 formally designated anophelines havebeen identified morphologically, out of whichonly about 70 species are considered to bethe main vectors of malaria in the world. Thetotal number of species has now reachedmore than 500 because of the identificationof biological species within morphologicallyindistinguishable taxa.

Among many Diptera genera, such asDrosophila, Simulium, Anopheles, Aedes,Sciara and Chironomus, the populations withina morphologically defined species do notinterbreed. These morphologically-similar,reproductively-isolated species within a taxonare known as cryptic, sibling or isomorphicspecies, and the taxon as a whole as a speciescomplex. Sibling species are found in otheranimal groups also. Mayr (1970) gives adetailed account of the groups where siblingspecies have been found. Most of theanophelines that are implicated in thetransmission of malaria in the South and South-

East Asian countries have been identified asspecies complexes, which include Annularis,Barbirostris, Culicifacies, Dirus, Fluviatilis,Leucosphyrus, Macaulatus, Minimus,Philippinenisis-niyipes, Punctualatus, Sinensis,Subpictus and Sundaicus.

Maculipennis was the first complexdescribed in the genus Anopheles. Thediscovery of this complex resolved theepidemiological paradox that prevailed in the1930s when there was malaria transmissionin Europe and North America. In some areasin southern Europe, there was no malaria inspite of the presence of An. maculipennis,which led to the expression “anophelismwithout malaria”. Detailed studies on thebiological and cytogenetic characters of thesepopulations have now identified eight siblingspecies in this taxon in Europe. Recognizingthe significance of species complexes inmalaria epidemiology, Mayr (1970) goes onto state that, “Perhaps the most celebratedcase of sibling species is that of the malariamosquito complex in Europe,” referring tothe Maculipennis Complex. So far, about 30complexes have been described in differentregions of the world. The number of siblingspecies varies in each complex and a total ofabout 145 species have been identifiedamong these complexes (Table 1).

Among the members of the MaculipennisComplex in Europe, An. atroparvus, An.labranchiae, An. messeae, An. sacharovi andAn. subalpinus were vectors because theywould at least sometimes bite humans, whileAn. maculipennis sensu stricto, An. melanoonand An. beklemishevi were non-vectorsbecause they were generally entirely

1. Introduction

Anopheline Species Complexes in South and South-East Asia2

zoophilic. Some of these species breed infresh water and others in brackish water. Inthe Gambiae Complex in Africa, while fivesibling species were recognized as vectorswith varying levels of efficiency in transmittingmalaria, An. quadriannulatus was found to bea non-vector (Coluzzi, 1988). Later, withinAn. quadriannulatus, two zoophagic siblingspecies, A and B, were more recentlyrecognized (Hunt, Coetzee and Fetenne,1998). In India, of the five An. culicifaciessibling species, species A, C, D and E arevectors while species B is a non- vector(Subbarao, Adak and Sharma, 1980;Subbarao et al., 1988, 1992; and Subbarao,Nanda and Raghavendra, 1999). In areaswhere An. culicifacies A and B are sympatric,DDT spraying in many areas has caused anepidemiological impact on the transmission(Sharma et al., 1986) due to reduction inspecies A, which is a vector (Subbarao et al.,1988) and is more susceptible to DDT thanspecies B (Subbarao, Vasantha and Sharma,1988). These are a few examples thatdemonstrate the differences between siblingspecies within a species complex andhighlight the importance of identifying siblingspecies in malaria control programmes.

The genetic distinctness of each siblingspecies comes from the definition of thebiological species concept whereby eachspecies is an actually interbreeding naturalpopulation that is reproductively isolated fromother such populations. Reproductiveisolation between sibling species ismaintained either by pre- or post-matingbarriers or both. The post-mating barrier isexpressed in the form of non-viability ofhybrid progeny at immature stages or hybridsterility or both. The pre-mating barrier(s) isdue to failure in copulation because ofphysical incompatibilities or behaviouraldifferences in mating procedures. Thus, eachsibling species has a specific mate recognitionsystem that is distinctly different from that ofthe other sibling species in the complex.

Population genetic studies involvingchromosomal inversions and DNA markers

(Besansky et al., 1994, 1997; Garcia et al.,1996) have suggested the possibility of geneflow occurring between members of theGambiae Complex. Clear evidence forunidirectional introgression leading to geneflow from An. arabiensis to An. gambiaecame from a multilocus molecular markerstudy (Besansky et al., 2003) and a novelpopulation genetic analysis (Donelly et al.2004). It is intriguing that gene flow is notuniform throughout the genome, i.e.genomes are mosaic with respect to gene flow(Garcia et al., 1996; Besansky et al., 2003).Similarly, introgression was observed betweenmembers of the Dirus Complex found inSouth-East Asia (Walton et al., 2001).

The fact that introgressive hybridizationoccurs between sibling species, this may againraise the issue of whether the sibling speciesare full biological species or not and whethersuch introgression would change thebiological characterstics of these species andaffect vector control strategies. There is,however, so far no evidence that An. gambiaeand An. arabiensis have mixed and thecharacterstic differences of these two specieshave disappeared, and also very few hybridswere found in nature in spite of theirsympatric association over large areas of theirdistribution (Besansky et al., 2003). Similarly,only a single hybrid of An. culicifacies speciesA and B was found among several thousandsof specimens screened over largegeographical areas where these two speciesare sympatric (Subbarao, unpublished). Theseobservations indicate that introgressivehybridization, even if it occurs between siblingspecies, is a rare event and pre-matingisolation barriers are strong.

Thus, the sibling species should continueto be considered as full biological species andthere does not seem to be any likelihood ofany of the sibling species changing theirbiological characters in the near future thatshould lead to changes in vector controlstrategies that are being contemplated. Coluzzi(1988) highlights the importance ofidentification of sibling species by saying that


failure to recognize sibling species ofanopheline taxa may result in failure todistinguish between a vector and a non-vector;hence, the assessment of the impact of controlmeasures may be seriously misleading if theyare carried out on a morphologically definedtaxon which could be a mixture of two or moresibling species. The discovery of sibling speciesadds a new dimension to vector control.

With this background, an effort wasmade to compile the information availableon anopheline species complexes prevalentin South and South-East Asia in a singledocument. The main objective of this effortwas to bring to the notice of researchers, fieldworkers and programme organizers, up-to-date information available on speciescomplexes prevalent in South and South-EastAsian countries.

The first edition of the document waspublished in 1998. The present documentcontains the distribution of malaria vectors inSouth Asia, South-East Asia and neighbouringcountries (Figure 1). Most of these anophelinesimplicated in malaria transmission are speciescomplexes. The species complexes and siblingspecies discovered in each of the complexes,the formal designations given to sibling speciesand their prevalence in the South and South-East Asian countries are shown in Tables 2aand 2b. The techniques currently being usedfor the identification of species complexes aredescribed in Chapter 2 and the details of thecomplexes are given in Chapter 3. Thecomplexes covered in this chapter are:Annularis, Barbirostris, Culicifacies, Dirus,Fluviatilis, Leucosphyrus, Maculatus, Minimus,Philippinensis-nivipes, Sinensis, Subpictus andSundaicus. An. stephensi, though not yetidentified as a species complex, is includedbecause it is an important vector and is acomplex of different variants/ecological races.For each of the complexes, information on thetypes of evidence used for the identificationof sibling species, the number of sibling speciesidentified, the techniques that have beendeveloped for the identification of siblingspecies and the distribution and biologicalcharacteristics of the members are presented.

Malaria control strategies are not uniform andat different times and in different areas,programme organizers demand specificstrategies to cope with local situations. To meetthese challenges, there is a need to generatefield data to establish the prevalence of speciesat the lowest administrative units possible forthe implementation of effective controlstrategies. These aspects are covered inChapter 4. The references cited are listed bychapter and by complex in Chapter 5.

Table 1 : Anopheline species complexesidentified so far

Complexes No. of Distribution inspecies zoogeographical

identified regions

Coustani 2 AfrotropicalGambiae 7 AfrotropicalFunestus 9* AfrotropicalMarshallii 4 AfrotropicalNili 4 EthiopianLungae 3 AustralasianPunctulatus 11 AustralasianAnnulipes 7 PalearcticClaviger 2 PalearcticMaculipennis 8 Palearctic

5 NearcticQuadrimaculatus 5 NearcticAlbitarsis 4 Neotropical and NearcticCrucians 6 NeotropicalFreeborni 2 NeotropicalNuneztovari 2 NeotropicalPseudopunctipennis 2 NeotropicalPunctimacularus 2 NeotropicalOswaldi 2 NeotropicalAnnularis 2 OrientalBarbirostris 3 OrientalCulicifacies 5 OrientalDirus 7 OrientalFluviatilis 4 OrientalGigas 3 OrientalLeucosphyrus 4 OrientalLindesayi 4 OrientalMaculatus 9 OrientalMinimus 5 OrientalPhilippinensis-nivipes 3 OrientalSinensis 4 OrientalSubpictus 4 OrientalSundaicus 4+1** Oriental

Source: Information in this table has been compiled fromHarbach (2004) and other published and unpublisheddocuments.* The Funestus Group consists of nine species that are

morphologically similar at adult stage and, of these, fourbelonging to the Funestus Subgroup are morphologicallyindistinguishable at all stages.

** New cytotype


Table 2a : Species complexes recognized and number of sibling species identified in South Asian countries

Anopheles Sri Lanka Iran Afghanistan Pakistan India Nepal Bhutan BangladeshComplexes

Annularis (2) + + + 2 + + +A, B

Barbirostris (4) + + + + + +

Culicifacies (5) 2 1 1 2 5 1B, E A A A, B A, B, C, D, E B + +

Dirus (7)2 2 + + 1D, E D

Fluviatilis (4) 1 + + 4 + + +T S, T, U, V

Leucosphyrus (4)2

Maculatus(9)2 1 3 2 4 3 + 1B B, H, I B, H B, C, H, I B, H, I B

Minimus (5) + 1 + +A

Philippinensis/ 2 + +nivipes (3) n(A), p

Punctulatus (11)

Sinensis (4)

Subpictus (4) 2 + + 4 + + +A, B A, B, C, D

Sundaicus(4+1)2 1 +cytotype D


Table 2b : Species complexes recognized and number of sibling species identified in South-East Asian countries

Anopheles Myanmar Thailand Laos Viet Nam Cambodia Malaysia Indonesia Timor- PhilippinesComplexes LesteAnnularis (2) + + + + + + + + +

Barbirostris (4) + + + + + 3 +A, B, C

Culicifacies (5) + 1 1 1B B B

Dirus (7)2 1 5 1 1 2 1D A, B, C. A A B, F B

D, F

Fluviatilis (4) +

Leucosphyrus (4)2 1 2 3A A, b A, B, b

Maculatus (9)2 3 7 + 3 1 1 1 + 2A, B, C A, B, C, A, B, I B B B D, J

G, H, I, K

Minimus (5) C 4 2 2 1 + +A, B, A, C A, C AC, D

Philippinensis/ + 3 n, p n, p n, p + + +nivipes (3) n (A, B), p

Punctulatus (11) 2f, c

Sinensis (4) + 2 + + + 1s, sin 1

Subpictus (4) + + + + + + + +

Sundaicus(4+1)2 + 1 1 1 1 3 +A A A s. s A, B, C

( ) No. of sibling species identified in the complex; + Species present but sibling species composition not known; b—balabacensis, n—nivipes (This taxon has two sibling species A and B), p— philippinensis, f—faurauti, c—clowi, s—sineroides, sin—sinensis, nim—nimophilous, s. s.—senso stricto1 Newly identified sibling species are initially designated either with letters of the English alphabet or occasionally with

numbers which are subsequently dropped and are formally designated using binomial nomenclature2 Sibling species of the following complexes have been given the formal designations:

Dirus Leucosphyrus Maculatus SundaicusComplex Complex Complex Complexdirus (A) leutens (A) sawadwangporni (A) epiroticus (A)cracens (B) leucosphyrus s.s. (B) maculatus s.s.(B) sundaicus sensu stricto (s. s.)scanloni (C) dravidicus (C)baimaii (D) greeni (D)elegans (E) notonandai (G)nemophilous (F) willmori (H)

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Group(s) of individuals within a speciessometimes exhibit distinct differences withreference to resting habitats, preference tofeed on a host, the rate of development ofresistance to insecticides, susceptibility toacquiring infection, and so on. All thesedifferences may indicate the presence ofisomorphic species within a taxonomicspecies (defined morphologically), but thesedifferences cannot confer species status onpopulations. Hence, genetic techniques thatcan demonstrate reproductive isolation withina morphologically similar taxonomic speciesare needed. Table 3 gives the methods whichare available to researchers. Crossingexperiments, chromosomal variations andelectrophoretic variations at enzyme loci havebeen extensively used in studies to recognizespecies complexes. The chromosomalvariation and electrophoretic variation atenzyme loci provide evidence for therecognition of species complexes; later thevariations are also used in the developmentof diagnostic techniques/assays to identifysibling species. Cuticular hydrocarbonanalysis and molecular approaches aregenerally used to develop diagnostic assaysfor the identification of sibling species whichhave already been recognized by othertechniques. Sibling species by definition arespecies without easily observablemorphological differences. A carefulexamination may sometimes revealmorphological differences that are minuteand may be restricted to a particular stage inthe life-cycle.

The techniques (Table 3) and theprinciples behind these techniques have been

described in several papers. Reviews byWhite, Coluzzi and Zahar (1975), Miles (1981),Green (1985), Coluzzi (1988), Green et al.(1985), Service (1988), Subbarao (1996) andBlack and Munstermann (1996, 2004) are afew that describe and discuss these techniques.Readers are also referred to a WHO documentprepared by Zahar (1996). This is a review ofliterature published between 1974 and 1994on vector bionomics and the epidemiologyand control of malaria. The document alsoincludes a compilation of species complexesof the South-East Asia and West Asia regions.Another article recommended is by Harbach(2004). This article is an update of the internalclassification of the genus Anopheles, whichwas earlier reported by the same author in1994 (Harbach, 1994). The article lists species,species complexes, subgroups, groups and

Table 3: Techniques used in the identificationof species complexes

Morphological variationsCrossing experimentsMitotic and meiotic karyotypes– Structural variations– Heterochromatin variationsPolytene chromosomesElectrophoretic variationsCuticular hydrocarbon profilesMolecular approaches– DNA or RNA probesAllele specific polymerase chain reaction (ASPCR)– Restriction fragment length polymorphism

(RFLP)– Random amplified polymorphic DNA (RAPD)– Sigle strand conformational polymorphism

(SSCP)– Heteroduplex analysis (HDA)

2. Techniques used inthe recognition

of Species Complexes


series recognized (formally and informally) sofar in the genus. Reviews by Green (1985) andColuzzi (1988) are strongly recommended forall those who work in this area. Reviews byBesansky, Finnerty and Collins (1992), Hill andCrampton (1994), Collins et al. (2000),Kryzywinski and Besansky (2003) and Blackand Munstermann (2004) are alsorecommended for those who intend to use ordevelop molecular techniques for theidentification of sibling species, and by Philippset al. (1988) for cuticular hydrocarbon analysis.White (1977) and Service (1988) discussed anddescribed the role of morphological charactersin the investigation of species complexes. Forthe benefit of readers a few salient points arementioned below.

Morphological variationsMorphological characters that are often usedto identify adults of anopheline species arelargely confined to scale pattern and colourand their distribution. Characters that areused in the description of immature stagesare sculpture of eggs, setation andpigmentation of larvae, and the forms ofpaddles and trumpets as well as chaetotaxyof pupae. Spermatheca and spiracularmorphology are also used in the identificationof species. In addition to light microscopeexamination for the specific characters,scanning and transmission electronmicroscopes are also used to studymorphological variations. Morphometrics hasproved useful in studying some speciescomplexes when used in conjunction withstatistical analyses. For details see White(1977) and Service (1988).

White (1977) states that morphologicalstudies should not come too early in theprocess of detecting anopheline siblingspecies since it might be misleading tocharacterize taxa which have not beenidentified by trustworthy techniques such ascross-breeding experiments or cytological orbiochemical characterizations.

Crossing experimentsThe assortative mating observed betweensibling species in nature due to pre-matingbarrier (s) generally breaks down in thelaboratory and different sibling species mateat random and produce hybrid progeny.Genetic differences between sibling speciesare expressed in the form of non-viability ofhybrid progeny at immature stages, hybridsterility or both. Hybrid males in one or bothcrosses are sterile and hybrid females aregenerally fertile. Therefore, hybrid sterility isused as the criterion in designating populationsas separate species. Hybrid males exhibitpartial development of reproductive organs(the extent of development ranges fromatrophied testes and vas deferens to fullydeveloped testes but without sperm; accessoryglands and ejaculatory duct are generallynormal) and do not produce progeny whencrossed. For species which do not mate inlaboratory cages, artificial mating methods canbe adopted. Thus, laboratory crossingexperiments demonstrate post-mating barriersand establish the species status of theisomorphic populations. An. gambiae was firstrecognized as a species complex from theresults observed between two strains whichwere crossed to study the genetics of resistanceto an insecticide (Davidson and Jackson,1962). It may be noted that though these post-mating barriers are studied between membersof the complexes, they are not necessarilyrequired to give populations species status.Furthermore, species which exhibit a pre-mating isolating mechanism need notnecessarily have any post-mating barrier, ashas been observed between species B and Cof the Culicifacies Complex (Subbarao,Vasantha and Sharma, 1988). F1 hybrid malesof reciprocal crosses between species B andC are fully fertile. Dobzhansky (1970) reportsthat viable and fertile hybrids may be obtainedin experiments between undoubtedly distinctspecies that maintain complete reproductiveisolation in nature. Therefore, thereproductive status of hybrid males is not


always diagnostic in the recognition of speciescomplexes. While studying post-matingbarriers, a point to be remembered is that forcolonies established from species-specificdiagnostic characters such as fixed inversions,enzyme electromorphs of progeny from singlefemale cultures have to be used. A laboratorycolony established from natural populationsmay be a mixture of two or three sympatricsibling species.

Cytogenetic techniques

Polytene chromosomesAnopheline females in the semi-gravid stagehave the best polytene chromosomes inovarian nurse cells (Coluzzi, 1968). Larvaeat the IV instar stage have polytenechromosomes in salivary glands. For thoseanopheline species which do not have goodovarian polytenes, larval salivarychromosomes can be used ( but salivary glandpolytene chromosomes are not very good inmost anophelines). The advantage with adultfemales is that ovaries can be removed andfixed in modified Carnoy’s fluid (1:3 glacialacetic acid:methanol) and can be used at anytime. Another advantage is that the samefemale can be studied for other parameterssuch as host preference, presence ofsporozoites/sporozoite antigen, susceptibilityto insecticides, etc.

The recommended references for thepreparation of polytene chromosomes are:for ovarian polytene chromosomes from adultfemales, Green and Hunt (1980), and forsalivary gland polytene chromosomes fromIV instar larvae, Kanda (1979). Hunt andCoetzee (1986) describe storing of field-collected mosquitoes in liquid nitrogen forcorrelated cytogenetic, electrophoretic andmorphological studies. The preparation ofpolytene chromosomes from adult femalesis not difficult.

Polytene chromosomes are the result ofrepeated replication of chromosomes atinterphase without nuclear division, theprocess being known as endomitosis.

Chromatids after division remain attached,causing thickening of chromosomes whichresults in the appearance of long ribbon-likestructures with dark and light horizontalportions representing band and interbandregions respectively. The dark and lightregions represent differential condensation ofchromosomes. The banding pattern of eachchromosome is specific in a given species;thus, each species differs from others incharacteristic banding pattern. Any changesin the pattern can be easily detected. In thepolytene chromosome complement, onlyeuchromatic regions are seen and theheterochromatic portions of thechromosomes which are under-replicated arenot seen. Therefore, in anophelines, the shortarm of the X-chromosome and Y-chromosome are not seen in the polytenecomplement. In some species a definitechromocentre is seen. In such cases, all thechromosome arms are seen attached to thechromocentre by their centromeric ends.Generally, this is not the case withanophelines and chromosome arms are seenseparately; occasionally, the two arms of achromosome are seen attached atthe centromeric ends. Homologouschromosomes exhibit high affinity for pairingand, therefore, are seen as a singlechromosome.

In anopheline cytogenetics literature, twotypes of designations are seen for the polytenechromosome arms: (i) the two arms of achromosome are designated as right (R) andleft (L) arms; this system is followed byDrosophila cytogeneticists and is adapted bymany anopheline cytogeneticists; and (ii) thenew nomenclature for arm designation is thatsuggested by Green and Hunt (1980). In this,each arm is given a separate number—2,3,4and 5—for autosomal arms and theeuchromatic arm of the X-chromosome seenin the polytene complement is designated asX. Taking An. gambiae belonging to thePyretophorous series as an arbitrary standard,the two arms of chromosome 2, 2R and 2Lare referred to as 2 and 3 respectively and


those of chromosome 3, 3R and 3L as 4 and5 respectively in this nomenclature.

During evolution in anophelines, whole-arm translocations have occurred. This cameto notice when banding patterns of differentspecies were compared and studied. In An.culicifacies and An. fluviatilis belonging to theMyzomyia series and An. stephensi, An.annularis and An. maculatus belonging to theNeocellia series, the arm association is 2-5and 3-4. In the Myzomyia series, for the An.funestus group of species, the arm associationis 2-4 and 3-5, indicating anothertranslocation event (Green and Hunt, 1980).With this arm- designation system such eventscan be incorporated and it is best suited forstudies on the cladistic analysis of speciesbelonging to a subgroup, group or series.Researchers who use polytene chromosomesin their work are recommended to readGreen and Hunt (1980) for the argumentsand justification for the new arm designations.

Most of the species complexes identifiedso far have been by the examination ofpolytene chromosomes of wild populations.Paracentric inversions are very common inthe natural populations of anophelines. Theadvantage of paracentric inversions in speciesidentification studies is that they act like singlegene loci and the alternate arrangements ascodominant alleles. There are inversions thatare polymorphic within a species(intraspecific) and the three forms of theseinversions, the two homozygotes, standardand inverted, and the heterozygotes, areeasily recognized on polytene chromosomes,while there are inversions that are fixed anddifferent between species (interspecific).

The total absence or significantlydeficient proportion of heterozygotes for aninversion in a population indicatesreproductive isolation within a taxon; hence,the taxon may be considered as a speciescomplex. Thus, the examination of polytenechromosomes of field-collected adult femalesprovides unequivocal evidence for theexistence of different species- specific mate

recognition systems (Peterson, 1980). Inpopulations where heterozygotes for aninversion are absent, the inversion is said tobe fixed and the two banding patterns onpolytene chromosomes, inversion and itsstandard alternate arrangement becomediagnostic tools for the identification ofspecies. The occrrence of a small proportionof heterozygotes can be due to: (i) breakdownof pre-mating barrier(s) between two species(which is rare); and (ii) an inversion which isfixed in one species is polymorphic (floating)in another species and the two species aresympatric (e.g. An. culicifacies species A andD) (Vasantha, Subbarao and Sharma, 1991).

In both cases, the number ofheterozygotes is far less than the numberexpected where an inversion is polymorphicwith random mating. These situations canbe analysed statistically by calculating theexpected number based on Hardy-Weinbergequilibrium and applying a Chi-square test.Thus, the population cytogenetic analysisdemonstrates and distinguishes intraspecificand interspecific occurrence of inversions.

It may be noted that sibling-specieshaving homosequential polytenechromosome banding pattern exists amonganophelinespecies complexes, e.g . An.labranchiae and An. atroparvus, two membersof the Maculipennis Complex (Coluzzi, 1970)and species B and E of the CulicifaciesComplex (Kar et al., 1999). A point to bekept in mind by all those who work withpolytene chromosomes is that inversionfixation is an accidental association withspeciation. As emphasized by Green andBaimai (1984), where variation occurs inpolytene chromosomes due to inversions, itmay provide useful markers for speciation andsubspeciation events, but in its absence onecan say nothing about such events amongindividuals bearing the same chromosomalrearrangements.

In spite of the limitations thathomosequential species exist in anophelinesand that polytene chromosome complements


can only be examined in semi-gravid adultfemales or in salivary glands of IV instar larvae,this is the easiest and cheapest tool nowavailable for the recognition of speciescomplexes and for routine use in theidentification of members of a complex inentomological studies. This tool is, however,more laborious than the DNA- based tools inlarge-scale entomological studies.

Asynapsis in polytene complements inhybrids is used as one of the criteria indetermining species status. The degree ofasynapsis may vary, and thus has to be usedwith caution. In hybrids between membersof the Culicifacies Complex, thechromosomes remain synapsed, except in theinversion heterozygote region where a loopor asynapsis is observed (Subbarao et al.,1983).

Inversions are designated with lower-caseletters of the English alphabet and are specificfor each chromosome arm, that is, aninversion a on chromosome arm 2 bears norelationship to a similarly denoted inversionon another arm, that is, inversion a on arm3. The standard arrangement is designated+ before the letter indicating the inversionconcerned and these may or may not bewritten as a superscript, e. g. 2+a or 2+a.Green (1982a) for the Myzomyia Series andGreen (1982b) for the Neocellia Series havesuggested a unified method for designatinginversions for species belonging to theseSeries, as it provides an efficient means ofstoring data and its subsequent retrieval. Thishas been followed for several membersbelonging to these series (An. fluviatilis, An.culicifacies, An. annularis, An. maculatus, An.philippinensis, etc.).

Mitotic and meiotic karyotypesAll anophelines studied so far have three pairsof chromosomes—two pairs of autosomeswhich are either metacentric or sub-metacentric and a pair of sex chromosomes—which are homomorphic (XX) in females andheteromorphic (XY) in males. X- and Y-

chromosomes have been found astelocentric, acrocentric or subtelocentric, orsubmetacentric (depending on the positionof the centromere in the chromosome) indifferent species of anophelines. The paperof Levan (1964) is recommended forchromosome nomenclature. The best mitoticchromosomes are found in the neurogonialcells of the brain in early IV instar larvae andmeiotic chromosomes in the reproductiveorgans of newly-emerged adults. Therecommended references for the preparationof mitotic and meiotic chromosomes are:Breeland (1961), French, Baker and Kitzmiller(1962) and Baimai (1977).

Structural variations due to the positionof centromere and quantitative variations inheterochromatin blocks are commonlyobserved. The variation in autosomes andX-chromosomes, which are found in thehomozygous state, demonstrate reproductiveisolation between the populations ifheterozygotes for the variation are not foundor are found in deficient numbers. This issimilar to the situations described forparacentric inversions under polytenechromosomes (see above). Unlike thebanding pattern due to paracentric inversions,variations at a given position in thechromosome can exist as more than twoalternatives. Data have to be generated fromsingle female progeny of wild-caught femalesor larvae. Structural variations in the Y-chromosome, though very common inanophelines, do not by themselves reveal thegenetic structure of the population, as thevariations in a population give no indicationwhether they are intra- or inter-specificbecause the Y-chromosome is inherited fromfather to son and is found in hemizygouscondition. However, one can test theassociation of the Y-chromosome with othergenetic variations, where linkagedisequilibrium between Ys and othercharacters would indicate the presence ofdifferent sibling species. Species E in theCulicifacies Complex was identified by


associating sporozoite positivity in femaleswith Y-chromosome variants in their sons (Karet al., 1999).

Heterochromatic variants, revealed byspecial staining techniques using Giemsa,Hoechst, etc., on autosomes and X-chromosomes are distinct and are alsodiagnostic in the identification of siblingspecies. Both structural and heterochromaticvariants in mitotic karyotypes are notconvenient as routine entomological tools forthe identification of sibling species in fieldstudies as progeny of wild-caught femaleshave to be examined for the variants.However, these techniques can be used tostudy the larval ecology of sibling species.

Enzyme electrophoretic variationsEnzyme electrophoresis is extensively usedin the study of species complexes. Thetechnique involves the detection of theprotein bands of an enzyme system withdifferent mobilities as a function of electriccharge and molecular structure. On a gelzymogram of an enzyme system,electrophoretic variations in the form of bandswith different mobilities represent proteinscoded by different alleles (allozymes). Thesealleles, being codominant, behave likeparacentric inversions and the twohomozygotes and heterozygotes can bedifferentiated. Variations at a locus thusenable the detection of the reproductiveisolation between populations resulting frompositive assortative matings within apopulation. Because of the simplicity of theprocedures for the processing andinterpretation of data, this technique permitslarge-scale sampling of natural populationsand is very useful as a diagnostic tool in theroutine identification of species. For detailson techniques, Ayala et al., (1972), Black andMunstermann (1996, 2004) and Steiner andJoslyn (1979) are recommended. However,one has to remember that unlike inversions,where only one inverted arrangement withreference to a standard arrangement exists,more than two electrophoretic forms at a

single locus can exist and in each speciesmore than one allele may be fixed. In An.melanoon and An. sacharovi (two membersof the Maculipennis Complex), Hk-190 and Hk-1100 alleles at the hexokinase locus are fixedin the former sibling species and Hk-195 andHk-197 alleles in the latter. Therefore,electromorphs found to be diagnostic for eachtaxon can be used only to identify individualsfrom populations already sampled (Miles,1981) as geographical variation within aspecies can exist.

If a single fully diagnostic enzyme systemcannot be identified (i.e. without anypolymorphism), several enzyme systemswhich differ in their frequencies of allelesbetween populations can be identified andused in association, which will reduce theerror in identification. For species complexeswith several members, enzyme systems thatare diagnostic for different members of thesame species complex can be identified andused in the form of a biochemical key, as hasbeen developed for the Maculipennis andGambiae Complexes (for details, see Coluzzi,1988).

Electrophoretic variations at enzyme lociare not only useful for the identification ofisomorphic species but can also be used forthe correct identification of morphologicallyidentifiable species. An. minimus, An.aconitus and An. varuna are found sympatricand are morphologically very similar. Hence,errors are made in the identifications. ForThailand populations, alleles at the Malatedehydrogenase-1 (Mdh-1) locus were foundto be diagnostic (Green et al., 1990). Mdh-1100 fixed in An. minimus differentiates it fromAn. aconitus and An. varuna which have theMdh-1157 allele. Less than 0.1 per cent An.minimus had the Mdh-1157 allele and a fewspecimens had the Mdh-1115 allele. Mdh-1115

is diagnostic for An. pampana, but this speciesis very rare in Thailand (Green et al., 1990).

A point to be kept in mind when workingwith electrophoretic variations is that in orderto determine the mobilities of the bands of


samples to be identified, samples of knownreference standards should be run on the samegel. Alternatively, non-enzymatic proteinstandards have to be run along with thesamples. For the identification of the MinimusComplex sibling species, human haemoglobin(homozygous for normal haemoglobin) andknown laboratory colony material were runon the same gel as reference standards (Greenet al., 1990). Mosquitoes collected from thefield if not used immediately should be storedfrozen (£40°) to retain their enzyme activityprior to electrophoresis.

Cuticular hydrocarbon profilesCuticular hydrocarbon analysis for sibling-species identification involves determiningspecies-specific differences in thehydrocarbons contained in the wax layer ofinsect cuticle. The wax layer lies beneaththe outermost cuticular layer. Carlson andService (1979) were the first to use thistechnique to identify An. gambiae s.s. and An.arabiensis (two members of the GambiaeComplex). The review by Philipps et al.(1988) is recommended for details on theprocedure. This technique has now beenused to identify members of several speciescomplexes in mosquitoes and otherhaematophagous insects.

It may be noted that this technique canbe employed to identify members of analready recognized species complex but is notrecommended to be used to recognize newcomplexes. The most important criterion fordesignating different species is reproductiveisolation which is not possible with thistechnique because it is not easy todifferentiate between intra- and inter-specifcvariations in the hydrocarbon profiles ofindividual specimens. Furthermore, this tooluses gas liquid chromatography withexpensive equipment.

DNA methodsAdvancements in DNA recombinanttechnology have facilitated the developmentof simple and rapid molecular tools for the

identification of sibling species. Severalreviews are now available in the literaturedetailing various techniques and theiradvantages in the identification of isomorphicspecies (Besansky, Finnerty and Collins, 1992;Hill and Crampton, 1994; Black andMunsterman, 2004; Collins et al., 2000; andKrzywinski and Besansky, 2003).

The first of the DNA methods used toidentify species was the use of DNA probes.Clones containing specific DNA segments ofthe undefined highly repeated component ofthe genome are identified by differentialscreening of genomic libraries withhomologous and heterologous genomicDNAs. DNA segments from these clones arelabelled and used as probes. The paper ofPost and Crampton (1988) on DNA probesfor the Simulium damnosum Complex andBlack and Munstamann (2004) giveprocedures (with illustrations) used in theisolation of species-specific DNA probes forthe identification of sibling species. Initially,radioactive probes were used. Simple non-radioactive probe assays for squash-blothybridizations have been developed for theidentification of members of the Gambiae(Hill et al., 1991), Punctulatus (Cooper,Cooper and Burkot, 1991) and Dirus (Audthoet al., 1995) Complexes. Johnson, Cockburnand Seawright (1992) have improved theprocedure to clean up the background insquash-blot hybridizations. Non-radioactiveprobe methods remove the hazards ofradioisotopes and make the assays simple andusable under field conditions. The advantagewith DNA probes, as with isozymes, is thatspecies can be identified at all stages of themosquito life-cycle. And if kits aredeveloped, as they have been for theGambiae Complex (Hill, Urwin andCrampton, 1992), probes can be used withmuch more ease in field laboratories. Hill,Urwin and Crampton (1991) have shown thatby producing synthetic probes, the cost ofeach assay can be brought down to betweenUS$ 0.04 and US$ 0.33 depending on thelabelling method used.


With the improved DNA technologiesand reduction in the cost of reagents andequipment, polymerase chain reaction (PCR)assays have become popular for speciesidentification. The PCR assay was developedin 1985 by Saiki et al. (1985). PCR-basedmethods essentially require variation inneucleotide sequence across species. Themajor advantage of this technique is that itrequires only miniscule amounts of DNA.During the reaction period of 2-3 hours, aparticular region of the genome is amplified1-100 million times, and this can then besimply visualized on agarose gel afterelectrophoresis and staining. The mostcommonly used PCR-based methods are:restriction fragment length polymorphism ofPCR-amplified product (PCR-RFLP), single-strand conformational polymorphism (SSCP),Heteroduplex analysis (HDA), and allele-specific PCR (ASPCR). The ASPCR isessentially cheaper and quicker than the othermethods and does not involve specialtreatment following PCR. However, theposition of neucleotide variations is criticalin designing primers for ASPCR so that all theallele-specific amplicons can be distinguishedseparately on a gel. Black and Munstermann(2004) show in detail the steps involved inPCR assay with illustrations.

Basically, there are two types of PCRstrategies: one surveying the genomerandomly and the second targeting specificregions of the genome, such as ribosomalDNA (rDNA) or mitochondrial DNA(mtDNA). The randomly amplifiedpolymorphic DNA PCR (RAPD-PCR) belongsto the first category. This technique does notrequire prior knowledge of the genome, andan added advantage is that commercial kitsare available with large number of randomdecamer primers. Arbitrary regions of thegenome are amplified using a single decamerprimer. Amplified products by each primerare analysed for differences between thespecies concerned. This technique wasdeveloped by Williams et al. (1990). DNAtools are generally developed once the

members of a complex are identified. Thetaxon An. (Nysorhynchus) albitarsis was,however, identified as a complex of foursibling species by the examination of naturalpopulations from Paraguay, Argentina andBrazil, using RAPD-PCR (Wilkerson et al.,1995). Though RAPD-PCR is simple, thistechnique has inconsistent reproducibilityand therefore has been of limited use for theidentification of sibling species.

Allele-specific PCR (ASPCR) assays mostlyexploit variation in the rDNA cistron. Inanophelines this is X-linked (Rai and Black,1999). It consists of tandem repeated arraysof conserved genes (18S, 5.8S and 28S)punctuated by rapidly evolving non-codinginternal transcribed spacers, ITS1 between18S and 5.8S and ITS2 between 5.8S and28S. Each gene cluster is separated byintergenic spacers (IGS). Within interbreedingpopulations the arrays undergo rapidhomogenization through concertedevolution, which drives new sequencevariations to fixation, leading to species-specific differences. For members of manyspecies complexes, differences in ITS2 andvariable regions within 28S rDNA gene havebeen used to develop ASPCR assays formembers of the Culicifacies Complex (Curtisand Townson, 1998; Singh et al., 2004a), forthe Fluviatilis Complex (Manonmani et al.,2001; Singh et al., 2004b), for the DirusComplex (Walton et al., 1999) and for severalother species complexes prevalent in theAfrotropical, and Neotropical regions.Portions of the genes from mitochondrialgenome, COI and COII, are also used todevelop diagnostic PCR assays as has beendone for members of the CulicifaciesComplex (Goswamy et al., 2006). For An.minimus species A and C, Kengue et al. (2001)used the RAPD marker assays of Sucharit andKomalamisra (1997) to develop a robustmultiplex ASPCR. This assay distinguishedother anophelines, An. aconitus, An. varunaand An. pampanai, which are morphologicallyvery close to An. minimus and are foundsympatric with An. minimus.


In PCR-RFLP, the amplified product is cutby certain restriction enzymes which producea different pattern of digested products whenrun on agarose gel. A single nucleotide changemay alter the restriction enzyme site andthereby a different pattern of bands may berevealed. A PCR-RFLP targeted at ITS2 rDNAwas developed by Van Bortel et al. (2000)which distinguishes An. minimus A and C andfour related species, An. aconitus, An.pampanai, An. varuna and An. jeyporiensis.Digestion of the PCR-amplified D3 region of28S rDNA with HPA II endonucleasedistinguished An. funestus from An. veneedeni(Koekemoer, Coetzee and Hunt, 1998), anddigestion of COII-amplified product with DdeI distinguished species E from species B andC of the Culicifacies Complex (Goswamy etal., 2005).

Hiss et al. (1994) developed a techniquebased on single-strand conformationpolymorphism (SSCP) of Orita et al. (1989)as a diagnostic tool. SSCP is a highly sensitivetechnique and detects point mutations withan efficiency of 99% in products with 100-300bp length. As the product lengthincreases, the efficiency is reduced. Thistechnique does not require prior knowledgeof DNA sequence data. In this techniquethe amplified product is denatured to singlestrands at 95 oC for five minutes and thenplaced immediately into an ice bath (0-4 oC)so that single-strand duplexes are formedfrom intrastrand base pairing. Variation inthe confirmation of intrastrand duplexes isvisualized by a gel retardation assay. Sharpeet al., (1999) developed an SSCP assay forthe identification of An. minimus species Aand C, An. aconitus and An. varuna, andKoekmoer et al.(1999) used D3 region ofrDNA to distinguish four members of the An.funestus group, An. funestus, An. vandeeni,An. rivulorum and An. leessoni.

Heteroduplex analysis (HDA) is anothertechnique used in the identification of closely-related species. HDA detects theelectrophoretic retardation of heteroduplexproducts (HDPs) formed between the strands

of a probe and a test DNA molecule. Thenumber and type of mismatched nucleotideswithin a given HDP determine the mobilityof the DNA duplex on an electrophoresis gel(Tang and Unnash, 1997). The target DNAand probe DNA are PCR products of chosenloci, which are hybridized and run on a gel.The probe DNA is chosen from a closelyrelated species. The advantage of thistechnique is that without going through thesequencing of loci, intra- and inter-specificvariation can be detected.

DNA sequences are also being used toestablish the phylogenetic relationship of thespecies between the subgroups and of thosewithin a subgroup/complex. Portions of genesfrom mitochondrial genome, COI, COII,Cytochrome b, etc., and from nuclear rDNAregions, D2, D3, ITS1, ITS2, etc., are used(Chen, Butlin and Harbach, 2003; Dusfouret al., 2004 and Garros, Harbach andManguin, 2005a and b). Sallum et al. (2002)examined the phylogenetic relationship of 32species of the subfamily Anophelinae, whichincluded species from the genera Anopheles,Bironella and Chagasia.

Microsatellite markers are di-, tri- andtetra-nucleotide sequence arrays of variablelength found frequently in the genomes.These sequences are identified from genomiclibraries using labelled repeat probes. Oncethe sequences from the positive clones areselected from unique neucleotide sequencesflanking the repeated sequences, specificprimers for each marker are developed forPCR assays. For An. gambiae s. s., markerswere also developed from genome sequenceanalysis (for this species the entire genomesequence data is now available at http://www.ensemble.org/Anopheles_gambiae)instead of going through the labour-intensivescreening process mentioned above.Microsatellite loci generally have highermutation rates than other regions of thegenome and, therefore, are highlypolymorphic. Beacause these are neutralmarkers, they are preferred for use inpopulation genetic studies. There are a few


anophelines for which these markers havebeen developed— An. gambiae s.s. (Zhenget al., 1993, 1997), An. maculatus(Rongnoporut et al., 1996), An. funestus (Sinkins et al., 2000), An. dirus (Walton et al.,2000a), An. stephensi (Veradi et al., 2002)and An. culicifacies ( Sunil et al., 2004).Microsatellite markers developed for onemember can be used for other members ofthe complex. They can also be used for closelyrelated species as has been done for membersof the Leucosphyrus Complex using thosedeveloped for An. dirus s.s (Walton et al.,2000a).

These markers were initially used for thegenetic mapping of refractory gene(s) andmorphological markers (Zheng et al., 1993,1996) and for developing a fine-scale geneticmap (Zheng et al., 1997) of An. gambiae.Now they are being used in populationgenetic analysis and ecological studies in An.gambiae s.s. and also for other members ofthe Gambiae Complex (Besansky et al., 1997;Kamau et al., 1999 and Donelly andTownson, 2000). Similarly, microsatellites arebeing used for the population genetic analysisof An. maculatus ( Rongnoparut et al., 1999),An. dirus ( Walton et al., 2000b) and An.culicifacies (Subbarao, unpublished).

It is important to highlight the power ofsimple polytene chromosome analysis usingparacentric inversions as a tool in populationgenetic studies and in establishing phylogenticrelationships. This simple tool has brought outthe complexities of population structurewithin the An. gambiae s.s. by identifying fivechromosomal forms — Bamako, Mopti,Savanah, Forest and Bissau in West Africa(Coluzzi, Petrarca and DiDeco, 1985). Now,DNA markers together with cytogenetic toolsare being used to clarify this complexity

(Wondji, Simard and Fontenelle, 2002 andStump et al., 2005). In years to come, someof these forms may be given a species status.Green constructed phylogeny using cladisticanalysis based on paracentric inversions seenin ovarian polytene chromosomes formembers of the Series Myzomyia (Green,1982, 1995) and for the Series Neocellia(Green et al., 1985).

Programs are now available forcalculating gene frequencies, averageheterozyosity, per cent polymorphic lociobserved and expected heterozygote valuesbased on the Hardy-Weinberg equilibrium,Wright’s fixation indices and genetic distanceand identity, and for establishing phylogeneticrelationships. Computer programmes canalso be used for analysing populations thatare polymorphic for many inversions, as hasbeen done for An. gambiae s.s. and An.arabiensis (Garcia et al., 1996) and An.annularis (Atrie et al., 1999).

Currently, more than 240 programmeshave been listed at http://www.nslij-genetics.org/soft. The BIOSYS-1 program inFORTRAN of Swofford and Selander (1981)and its later vesions were used for the analysesof many of the anopheline species. The mostcommonly used programmes are—TFPGA,Arlequin, POPGENE, GDA, GENEPOP,GeneStrut, GeneAlEx, etc. MEGA (MolecularEvolutionary Genetic Analysis) is a softwarefor computational molecular evolutionarygenetics based on nucleotide/proteinsequences. Those interested on populationgenetic analysis may visit http://dorakmt.tripod.com/genetics/popgen.html tolearn about the basics of population genetics,links to freeware, related literatures and otheruseful links.


3.1 The Annularis ComplexAnopheles annularis Van Der Wulp 1984belongs to the subgenus Cellia, AnnularisGroup in the Neocellia Series. The othermembers in this Group which are prevalentin South-East Asia are An. nivipes, An.philippinensis, An. pallidus and An. schueffneri(Harrison 1988, Harbach, 2004).

An. annularis has a wide distribution inthe Oriental region. It is found in Afghanistan,Pakistan, Nepal, India, Bangladesh, Myanmar,Philippines, China and Sri Lanka (Rao 1984;Kondrashin and Rashid, 1987). This speciesis considered to be an important vector inNepal and in certain parts in India (Dash etal., 1982; Rao, 1984; Gunasekaran et al.,1989). It is a secondary vector in certainlocalities though it has a wide distributionand is sometimes found abundantly (Rao,1984). An. annularis has been incriminatedrecently in the Thai-Combodia border area(Baker et al., 1987) and is also a vector inMyanmar (Bang, 1985). In Sri Lanka, thisspecies has been found playing a role in thetransmission of malaria in a new irrigationdevelopment area (Ramasamy et al., 1992).

Evidence for identification of siblingspecies

Two sibling species from Nepal(WHO, 1983)The total absence of heterozygotes for aparacentric inversion and its standardarrangement on the X-chromosome wastaken as evidence for the presence of twosibling species in An. annularis in Nepal. No

3. Species Complexes

further reports on details of these species areavailable.

Species A and B (Atrie et al., 1999)Nine inversions – wI, i1, j1 and k1 onchromosome arm 2; j1 and z on arm 3; h1

and s1 on arm 4; and k on arm 5 – werefound polymorphic in six districts in five statesin India. The total absence of heterozygotesfor inversion j1 and its standard arrangementon chromosome arm 2 in villages inGhaziabad and Shahjahanpur districts in thestate of Uttar Pradesh in India led to therecognition of two sibling species A and B.The deficiency of heterozygotes for threeother inversions – 2i1, 2k and 4h – was alsoobserved. However, the partitioning ofpolymorphic forms of these inversionsbetween the two sibling species characterizedby the +j1 and j1 arrangements indicatedbalanced polymorphisms of the inversions ineach of the presumed sibling species. Thisfurther supported the conclusion that An.annularis is a species complex andheterozygote deficiencies are due to thedifference in the frequencies of theseinversions in the two sibling species.

Techniques available foridentification of sibling species

Polytene chromosomesThis technique is based on the difference inthe banding pattern of polytenechromosomes due to a paracentric inversion.Species A is characterized by the +j1

arrangement and species B by the j1

arrangement on chromosome arm 2. A


photomap of polytene chromosomes with thebreak points for the diagnostic inversion isgiven by Atrie et al. (1999). The X-chromosome and the autosomal arms 3, 4and 5 are homosequential in both species.

Breakpoints of nine polymorphicinversions observed in these species are alsomarked on the photomap. The photomap ispresented in Figure 2. Polytene chromosomemaps for An. annularis are also given by Greenet al. (1985). The 2+j1 arrangement inspecies A is homosequential with the onepresented by Green et al. (1985). Of the nineinversions observed in India, four inversions- 2w, 3z, 4h1 and 5k - were found inpopulations from Taiwan, Philippines,Thailand and Bangladesh (Green et al.,1985).

The mitotic karyotype of both the specieswas found to be the same. Both theautosomes and the sex chromosomes weresubmetacentric (Atrie, 1994).

PCR-RFLPTwo PCR-RFLP assays, one based onendonuclease restriction sites in the ITS2sequence and the other based on those inthe D3 sequence of rDNA, have beendeveloped by Alam et al. (2006). In the ITS2sequence, species A showed three restrictionsites each for MspI, MyaI and Eco24I enzymesand species B showed restriction sites forMspI, HintI and NruI enzymes. In the D3sequence, species A had a unique restrictionsite for Alw26I while species B had the sitefor KpnI. With the D3 sequence two enzymesare needed for the accurate identification oftwo sibling species. MspI restriction sites werefound in both the species in the ITS2sequence and fragments differing in lengthswere produced in the two species followingthe digestion. Therefore, ITS2-MspI could beused as a diagnostic system to identify siblingspecies A and B of the Annularis Complex.However, the two assays have not beencorrelated with the cytological identificationwhich was the basis for the identification of

these two sibling species (Atrie et al., 1999).The assays were developed on the specimenscollected from different areas based on thedistribution pattern reported for siblingspecies A and B by Atrie et al. (1999).

Distribution and biological charactersIn the two districts of Shahjahanpur andGhaziabad in Uttar Pradesh, India, species Aand B were found sympatric. In all the otherstates surveyed, namely, Rajasthan, Haryana,Assam and Orissa, only species A has beenfound. An. annularis samples fromShahjahanpur district, where both species Aand B are prevalent, were found totallyzoophagic when blood meals of these specieswere examined by counter-currentelectrophoresis (Atrie, 1994). Both the specieswere found in the riverine, non-riverine andcanal-irrigated ecotypes found in the villagesin Shahjahanpur. However, in other districtswhere only species A was found, the sameecotypes were observed, and collectionswere also made from hilly-forested areas(Atrie et al., 1999).

In India, An. annularis is considered avector only in certain states. Only species Awas found in Sundergarh, Orissa state, andKamrup in Assam state where An. annularisis considered a vector. However, species Awas also found in Haryana, Rajasthan andUttar Pradesh where An. annularis is notconsidered a vector. Furthermore, species Awas totally zoophagic. Thus, the identificationof An. annularis as a species complex did notexplain why it is a vector only in certain areasin India.

An. annularis is a vector of localimportance in Nepal and Bangladesh, and asecondary vector in India and Sri Lanka. Thestatus of An. annularis as a vector is not knownin Bhutan (Figure1). Recently, in Afghanistan,it was incriminated with a Plasmodium vivaxsporozoite rate of 0.58% (Rowland et al.,2002) in villages with river-irrigated rice fields.The distribution of these two sibling species,A and B, has not been studied so far in any of


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Figure 2: Photomap of polytene chromosomes of An. annularis species A. The break points of inversions aremarked with the letter designations on the right side of chromosome arms. Arrows indicate the centromeric

ends of the chromosome arms (Source: Atrie et al., 1999)


these countries. Studies are required toexamine the biological characters of these twospecies and investigate whether there aremore species in this complex.

3.2. The BarbirostrisComplex

An. barbirostris belongs to the subgenusAnopheles, Barbirostris Subgroup, BarbirostrisGroup in the Myzorhynchus Series (Harbach,2004). There are two Subgroups, Barbirostrisand Vanus, in this Group. The BarbirostrisSubgroup includes barbirostris, campestris,donaldi, franiscoi, hodgkini and pollicaris; andthe Vanus Subgroup includes ahomi,barbumbrosus, reidi, manalangi and vanus(Reid, 1968).

Anopheles barbirostris is reported fromIndia, Pakistan, Bangladesh, Nepal, Myanmar,Thailand, Malaysia, Laos, Cambodia, VietNam, Indonesia, Sri Lanka and South China(Rao, 1984). This species has been suspectedas a malaria/filaria vector in Indonesia andThailand.


Crossing experiments (Choochote,Sucharit and Abeyewickreme, 1983)The evidence showing An. barbirostris to bea species complex came from the progeny ofreciprocal crosses carried out between twolaboratory strains (Choochote, Sucharit andAbeyewickreme, 1983). Two strains, theChumphon strain (CHP) established frommosquitoes collected from Bang LukeCanton, Chumphon Province (southernThailand) and the Chon Buri strain (CHB)from Chan Buri province (central Thailand)were used in this study. The two strains werereared in the laboratory by using the forcedmating technique.

Both the strains differed in average bodyweight and number of eggs deposited. TheCHP strain always weighed more (female —

1.64 + 0.49, male —1.19 + 0.23) and laid143 + 47.54 eggs/female, while the CHBstrain weighed, female — 0.97 + 0.23, male— 0.82 + 0.19, and laid 83.3 + 18.95 eggs/female. An average of 15–20 mosquitoes ofeach category were used in this study.

In the CHB female x CHP male cross,eggs were laid but none hatched, while inthe reciprocal CHP female x CHB male crossthere was 60.8 per cent hatching. The twoparental strains had more than 80 per centhatch. In CHP x CHB cross, there was 47.1per cent of pupation and only 16.7 per centemerged. When F1 females from this crosswere crossed to CHP males, no eggs werelaid in spite of 70.4 per cent insemination.F1 males had abnormal genitalia with veryshort claspers, and atrophied testes andaccessory glands. Polytene chromosomesfrom the salivary glands of the F1 werehomosequential with the maps described byChowdayya et al. (1970), but exhibitedinconsistent asynapsis along the autosomeswhile the X-chromosomes showed completesynapsis. Based on these results, the authorsconsidered An. barbirostris to be a speciescomplex.

Mitotic karyotypes from Thailand andIndonesia (Baimai, Rattanarithikul andKijchalao, 1995)Four forms of metaphase karyotypes wereobserved in mosquitoes collected from wildpopulations. The karyotype consisted of twopairs of autosomes, metacentric chromosome2 and sub-metacentric chromosome 3 andthe X- and Y- chromosomes which differedin size and shape. Three forms, A (X2, X3,Y1), B (X1, X2, X3, Y2) and C (X2, X3, Y3,), wereobserved in Thailand and one form, D (X2,Y4), in Indonesia. The collections made inThailand were extensive and covered theentire country. The D karyotype was notfound in any of these collections and wasrestricted to Indonesia. The authorsconcluded that the X,Y variations found inThailand could be inter- or intra-specificvariations.


Figure 3: Mitotic karyotypes of An. barbirostris found in Indonesia. Plate 1 female, plates 2-4 male of cytologicalform A; plate 5-6 male and plate 7 female of cytological form B; plate 8 female and plate 9 male of cytological

form C; plate 10-11 male and plate 12 female of cytological form D (courtesy of Dr Supratman Sukowati)


Mitotic karyotypes from Indonesia(Sukowati, Andris and Sondakh, 2003)Mitotic karyotypes of the progeny of wild-caught An. barbirostris females from their fourgeographically isolated populations wereexamined. The mitotic karyotypes differed inX and Y- chromosomes, the variation beingin the amount and distribution of constitutiveheterochromatin in giemsa-stainedpreparations. The authors reported that FormA (X1, X2, X3, Y1) is widely distributed inIndonesia, and found sympatric with form B(X1, X2, X3, Y2) and form D (X2, X3, Y4) in Tara-tara 2, North Sulawesi; Konga, Flores andTanjung Bunga, Flores, and form A was foundsympatric with form B and form C (X2, X3, Y3)only in Boru-Boru, Flores. It may be notedthat in Indonesia, Form A included X1variation too. Neither form C nor D werefound with the X1 chromosome inspite ofbeing sympatric with forms A and B. Y3 andY4 chromosomes were associated with the Cand D forms respectively. Mitoticchromosomes of the four forms found inIndonesia are shown in Figure 3.

Thus, there appear to be at least threedistinct cytotypes probably representing inter-specific variation. All three forms werehomosequential in their polytenechromosome banding pattern. S. Sukowati(personal communication) considers these tobe three distinct sibling species in thiscomplex. Species A was found highlyzoophagic and species B and C wereanthropophagic. Species C was found bitingduring daytime. In the areas where theseare found, filariasis due to Wucherariabancrofti and Brugia malayi and malaria areprevalent. An. barbirostris is considered tobe a vector of malaria and filaria. Studies onpolytene chromosome difference, allozymevariation and DNA methods need to beinitiated to give specific taxonomic status tothese populations and establish relationshipswith the prevalent diseases. Studies havebeen initiated to develop a PCR assay for thedifferentiation of these three species(Supratman Sukowati, personal

communication). The CHP and CHB strainsreported by Chuchote, Sucharit andAbeyewickreme (1983), cytotypes decsribedby Baimai, Rattanarithikul and Kijchalo (1995)and three possible species reported bySukowati, Andres and Sondakh (2003) haveto be correlated with each other.

3.3 The CulicifaciesComplex

Anopheles culicifacies Giles belongs to thesubgenus Cellia, Culicifacies Subgroup,Funestus Group in the Myzomyia Series(Harbach, 2004). An. culicifacies sensu lato(s. l.) has a wide distribution in India andextends to Ethiopia, Yemen, Iran, Afghanistanand Pakistan in the west, and Bangladesh,Myanmar, Thailand, Cambodia and Viet Namin the east. It is also found in Nepal andsouthern China to the north and extends toSri Lanka in the south (Rao, 1984). Recently,this species was reported from Cambodia(Van Bortel et al., 2002). An. culicifacies isan important vector of malaria in India andSri Lanka and in the countries west of India.The history of malaria control in thesecountries concerns mainly the control of An.culicifacies.

Significant differences were observed inthe bionomics of An. culicifacies in variousregions, including differences in seasonalabundance, diurnal activity, man-bitingbehaviour and vectorial potential (Rao,1984). As early as 1947, due to such distinctdifferences in biological characters, it wassuggested that the species culicifacies maycover a range of ’biological races’ (SeniorWhite, 1947). An. culicifacies has now beenrecognized as a complex of five siblingspecies, provisionally designated as speciesA, B, C, D and E.

Evidence for identification of siblingspeciesFour sibling species, A, B, C and D in thiscomplex, were identified following the


observation of a total absence or significantdeficiency of heterozygotes in naturalpopulations for the alternate arrangementsobserved in polytene chromosomes due toparacentric inversions. The fifth species,species E, was identified by correlating Y-chromosome polymorphism of sons and thesporozoite positivity of mothers. Laboratorycolonies established from the progeny ofsingle females with species-specific inversionswere used subsequently to study post-zygoticisolation mechanisms between the siblingspecies.

Species A and B (Green and Miles, 1980)Green and Miles (1980) found two distinctpolytene X-chromosomes in the An.culicifacies s. l. population in the village Okhlanear Delhi, India. One X-chromosome waswith two paracentric inversions a and b andthe other with a banding pattern thatresembled the polytene chromosomephotomap described by Saifuddin, Baker andSakai (1978). No heterozygotes for theseinversions were found. The absence ofheterozygotes was taken as an evidence ofreproductive isolation between the twopopulations which were considered as twodistinct species. The population with thestandard arrangement, X+a+b, wasdesignated as species A and that with Xabarrangement as species B. Green and Milesin the same paper (1980), by examininglaboratory colonies of An. culicifacies s. l.,reported species A and B from Pakistan andspecies B from Sri Lanka. Following thisreport, Subbarao, Adak and Sharma (1980)by examining field populations, confirmedthe presence of species A and B within An.culicifacies in the villages of Haryana andUttar Pradesh, both states bordering Delhi.

Species C (Subbarao et al., 1983)The examination of polytene chromosomesof An. culicifacies from villages around Delhiand in the states of Gujarat and MadhyaPradesh, India, revealed the presence of twofixed inversions on chromosome arm 2.Species B was fixed for the g1 inversion in

addition to a and b on the X-chromosome;thus, species B had Xab; 2g1 arrangement(Subbarao et al., 1983). In Gujarat andMadhya Pradesh, two chromosome 2arrangements, g1+h1 and +g1h1, within Xabpopulations were seen. In a large sampleexamined, only four double-inversionheterozygotes (2g1+h1 /+g1h1) wereobserved; therefore, Xab; 2g1+h1 and Xab;2+g1h1 were considered as tworeproductively isolated populations. The newpopulation with Xab; 2+ g1h1 was designatedas species C (Subbarao et al., 1983).

Species D (Subbarao, Vasantha andSharma, 1988a; Suguna et al., 1989;Vasantha, Subbarao and Sharma, 1991)In a few populations in northern India, the i1inversion on chromosome arm 2 was foundpolymorphic in species A, and it was foundfixed in the southern Indian populations ofAn. culicifacies species A (Subbarao, 1984).The latter population with the X+a+b; 2i1

arrangement was found sympatric withspecies B. The evidence for reproductiveisolation between species A (X+a+b;2+g1+h1) and the population with theX+a+b; 2i1 h1 arrangement came from twolocations (inversion i1 includes the g1 inversionregion and the distal breakpoint is the samefor both the inversions). In northern andcentral India, a deficiency of heterozygoteswas found for i1 inversion in the X+a+bpopulations (Subbarao, Vasantha andSharma, 1988a; Vasantha, Subbarao andSharma, 1991) while in southern India a totalabsence of heterozygotes was found betweenspecies A (X+a+b; 2+g1+h1) and thepopulation with the X+a+b; 2i1+h1

arrangement (Suguna et al., 1989). TheX+a+b population with the newchromosome 2 arrangement 2i1+h1 wasdesignated as species D. The explanation forthe heterozygotes (+i1/ i1) of i1 inversionobserved in northern India is that thisinversion is floating in species A with variedfrequencies in different populations and isfixed in species D (Vasantha, Subbarao andSharma, 1991).


Species E (Kar et al., 1999)In Rameshwaram island, Tamil Nadu (a statein southern India), An. culicifacies femaleshad the Xab; 2 g1+h1 inversion arrangementon polytene chromosomes, which isdiagnostic for species B. However, thebiological characters of this population weredifferent than those observed for species Bon the mainland. This prompted a detailedexamination. Because homosequentialspecies exist in nature, Subbarao et al. (1993)investigated the mitotic karyotype variations.The progeny of the field-collected femalesrevealed the Y-chromosome to bepolymorphic with acrocentric andsubmetacentric types. Because the Y-chromosome has patroclinal inheritance, thevariation observed could not be used toindicate reproductive isolation. Correlationwith sporozoite positivity i.e. total absenceof sporozoite positives among females whosesons had acrocentric Y-chromosome andpresence of sporozoite positives amongmothers whose sons had submetacentric Y-chromosome, was taken as evidence forassortative mating between acrocentric andsubmetacentric populations. The twopopulations were considered as twosympatric species. The vector populationwith the submetacentric Y-chromosome wasdesignated as species E and the non-vectorpopulation with acrocentric type Y-chromosome retained the originaldesignation of species B (Kar et al., 1999).

There seem to be at least five specificmate recognition systems operating in An.culicifacies, leading to reproductive isolationand lack of gene flow between thepopulations; hence, there are at least fivesibling species in this complex.

Post mating isolation mechanisms(Mahmood, Sakai and Akhtar, 1984;Subbarao, Vasantha and Sharma, 1988b;Kar et al., 1999)In addition to the pre-mating isolationmechanisms, post-mating isolation

mechanisms were also observed. From oneof the reciprocal crosses between species Aand B, Miles (1981) observed hybrid malesterility. In this study, crosses were carriedout by the forced copulation techniquebetween the progeny of single females.Hybrid males from the species B female x Amale cross were fertile. Mahmood, Sakai andAkhtar (1984) reported fertility of hybridmales varying from 10 per cent to 96 per centdepending on the species B strain used inthe cross between B female and A male.Subbarao, Vasantha and Sharma (1988b)observed almost total sterility with anoccasional hatch of 1 to 5 per cent in the Bfemale x A male cross, in contrast to 90 percent hatch in the A female x B male cross.Crosses by these authors were carried out incloth cages by normal matings. However, incrosses where a low hatch was observed inthe B female x A male cross, hybrid maleswere sterile, having fully developedreproductive organs but without any sperm.In the reciprocal cross (A female x B male),the reproductive organs were partiallydeveloped, testes and vas deferens wereeither totally absent, atrophied or reduced,while the ejaculatory duct and accessoryglands were normal (Miles, 1981; Subbarao,Vasantha and Sharma, 1988b). The resultsfrom the crosses between species A and Cwere similar to those between species A andB, except that in the C female x A male cross,no egg hatch had been observed so far(Subbarao, Vasantha and Sharma, 1988b).Reciprocal crosses between species B and Cproduced fully fertile F1 hybrid males andfemales, suggesting that there is no post-zygotic barrier between B and C. Species Eresembled species B and C in the crosses tospecies A, and there was no post-matingbarrier in the crosses with species B and C(Kar et al., 1999).

Techniques available foridentification of sibling speciesThe techniques available for the identificationof sibling species are summarized in Table 4.


Polytene chromosomesThe use of diagnostic fixed inversionsreadable on the polytene chromosomes(Subbarao, Vasantha and Sharma, 1988a) hasbeen the only technique available untilrecently for the identification of sibling speciesin this complex. This technique has beenextensively used to study the biologicalcharacters and establish the role of the siblingspecies in malaria transmission. The problemsin using this technique are:

(i) Species B and species E arehomosequential, hence cannot bedifferentiated.

(ii) With reference to species A and D andi1 inversion, one encounters any of thefollowing situations in a population:

(a) +i1 and i1 homozygotes without anyheterozygotes, suggesting that speciesA and D are present;

(b) + i1, i1 and + i1/ i1 (heterozygotes) inexpected proportions, suggesting thatonly species A is present and i1 ispolymorphic;

(c) similar to (b) but with a significantdeficiency of heterozygotes,suggesting that species A and D arepresent, and in species A i1 ispolymorphic.

In a situation such as (c), a populationgenetic analysis will indicate the presence ofspecies D but individual specimens cannotbe identified as species D (Vasantha,Subbarao and Sharma 1991).

The photomaps of the polytenechromosome complement of An. culicifacieshave been reported by Saifududdin, Bakerand Sakai (1978). Chromosome maps withbreakpoints of inversions a and b on the X-chromosome are given in Green and Miles(1980), and for those on the X-chromosomeand of g1, h1 and i1 on chromosome arm 2,in Subbarao et al. (1983) and Subbarao,Vasantha and Sharma (1988a). Thephotomaps from the latter paper arereproduced here as Figure 4.

In addition to fixed inversions in thiscomplex, six inversions, five on chromosomearm 2 (j1, k1, l1, i1 and o1) and r on arm 3were found polymorphic in species A, and inspecies B, l1 on arm 2 and r on arm 3 wereseen in natural populations. The breakpointsof all these inversions are mapped onphotomaps by Vasantha, Subbarao andSharma (1991).

Mitotic karyotypesInitial studies carried out on limited samplesfor mitotic karyotypes in sibling speciesrevealed Y-chromosome characters to be

Table 4: Techniques for the identification of An. culicifacies sibling species*

Polytene Mitotic LDH Cuticular Species- PCR-RFLP ASPCRSpecies chromosome karyotype- enzyme hydro- specific

inversion Y-chromosome alleles carbon DNAgenotypes profile probes

A X+a+b; 2+g1+h1; Submetacentric Fast Identified Yes Yes Yes +i1/i1

B Xab; 2g1+h1 Acrocentric Slow Identified Yes Yes YesSubmetacentric

C Xab; 2+g1h1 Acrocentric Slow Identified as B as B YesSubmetacentric

D X+a+b; 2i1+h1 Submetacentric Fast Not done Not tested as A Yes

E Xab; 2g1+h1 Submetacentric Slow Not done Not tested Yes Yes


diagnostic for the identification.Submetacentric Y-chromosomes in species Aand C and acrocentric in species B wereobserved by Vasantha et al. (1982; 1983).Suguna et al. (1983) also observedcorresponding differences in species A and Bpopulations from southern India. Adak et al.(1997) reported both acrocentric andsubmetacentric Y-chromosome poly-morphism in species B and C populations inseveral areas surveyed. Species E has asubmetacentric Y-chromosome (Kar et al.,1999). Thus, the Y-chromosome cannot beused as a diagnostic tool for the identificationof members of the complex except forspecies E. Mitotic karyotypes of specie B andE are given in Figure 5.

Electrophoretic variationsOut of the nine enzyme systems studied,electrophoretic variation in lactatedehydrogenase (LDH) was found to bediagnostic (Adak et al., 1994). Twoelectromorphs, fast (F) and slow (S), wererepresented at the Ldh locus. The frequencyof LdhF in species A and D varied between0.94 and 1.00, as against 0.0 and 0.19 inspecies B and C. Because of the low-levelpolymorphism observed within each species,the power of this technique in theidentification of sibling species was evaluatedby the authors using three indicators:sensitivity, specificity and predictive value.Overall, the probability of correct separationof species pairs by LDH enzyme wasdetermined as 94.6 per cent. In species E,LdhS was found (Kar et al., 1999), thus, thisspecies falls into the same category as speciesB and C. As Ldh is autosomal and is expressedat all stages of the life-cycle, Ldh allozymemethod is useful in the identification of siblingspecies in certain sympatric associations.

Cuticular hydrocarbon profilesCuticular hydrocarbon profiles wereexamined in species A, B and C (Milligan etal., 1986). Cuticular wax extracted fromsingle specimens from pure stocks was

analysed by gas liquid chromatography. Thethree species were found to be significantlydifferent in their cuticular hydrocarboncomposition by multi-variate analysis ofvariance. The best separation between thespecies was obtained using 27 peaks in adiscriminant analysis. Using thechromatographic characteristics of thesepeaks, each specimen analysed was assignedto the group to which its probability ofmembership was the greatest. With this, theaverage correct identification was 78 per cent.Analysis of a small number of field samplesalso exhibited intraspecific variability similarto that observed in laboratory cage samples.

DNA probesThree highly repetitive DNA sequences, Rp36, Rp 217 and Rp 234, were selected froma genomic library of species B (Gunasekeraet al., 1995). Radio-labelled fragments of Rp36, Rp 217 and Rp 234 gave positive signalsin dot-blot hybridization assays with siblingspecies A, B and C. The hybridization signalgiven by species A was much less than thatgiven by species B and C. Species A can bedistinguished from species B and C whensingle-mosquito extracts are diluted 200-foldand assayed by dot-blot hybridization withany of the three probes, which then give anegative signal for species A. These probeshave not been evaluated in differentgeographical regions.

PCR-RFLPThe use of restriction endonclease Rsa I forthe ITS2 amplicon and Alu I for mitochondrialcytochrome oxidase (CO) subunit II groupedspecies A and D in one category and B, Cand E into another (Goswamy et al., 2005).The COII amplicon digested with Dde 1distinguishes species E from species B and C.

ASPCRThree PCR assays from the rDNA cistron weredeveloped for the identification of siblingspecies of the Culicifacies Complex. One wasdeveloped from the variable D2 domain


(Cornel et al.,unpublished) and the secondfrom the D3 variable domain (Singh et al.,2004) of the 28S rDNA cistron. Both theseassays distinguish species A and D fromspecies B, C and E but fail to distinguishspecies within each of these groups. Primersfor both these have been evaluated againstfield-collected and cytotaxonomically-identified specimens. A third assay reportedby Curtis and Townson (1998) was developedfrom the ITS2 region of the 28S rDNA cistron.The assay distinguishes species A from speciesB. Other species have not been tested withthese primers and this method has not beenevaluated on field samples. Recently,sequence analysis of ITS2 region revealed thatspecies D is similar to species A and speciesC and E to species B (Goswami et al., 2005).

Two allele-specific PCR assays, AD-PCRand BCE-PCR, were developed from the COIIregion of mitochondrial DNA to distinguish thespecies. The strategy in this assay is that oncethe two groups, i.e. species A and D andspecies B, C and E are identified either byallele-specific PCR of the D3 or D2 regions orby the PCR-RFLP assays of the ITS2 and COIIregions (Goswamy et al., 2005), one can usespecific assays developed from the COII regioni.e. the AD-PCR assay to distinguish species Aand D and the BCE-PCR assay to distinguishspecies B, C and E (Goswamy et al., 2006).This assay was evaluated on An. culicifaciescollected from different areas of India, andsimultaneously, identifications were correlatedwith cytological identification based onspecies-specific diagnostic inversions.

Microsatellite markersAbout 31 microsatellite markers weredeveloped from the An. culicifacies speciesA. Some of the markers tested were foundpolymorphic in species A, B and C. The allelenumber varied from 2-12 (Sunil et al., 2004)

Distribution and biologicalcharacteristicsIn India all five species of the CulicifaciesComplex have been identified. Species Aidentified in Yemen (Akoh, Beidas and White,1984) and Iran (Zaim et al., 1993) has beenfound sympatric with species B in Pakistan(Mahmood, Sakai and Akhtar, 1984).Recently, An. culicifacies has beenincriminated as one of the eight speciesresponsible for malaria transmission inAfghanistan (Rowland et al., 2002). A fewspecimens from Afghanistan werecytotaxonomically identified several years agoas species A. Larger samples from differentregions of the country need to be examinedfor sibling species composition. Extensivesurveys carried out in Sri Lanka using polytenechromosomes (Abhayawardena et al., 1996)and DNA probe (de Silva et al., 1998)identified only species B. Surendran et al.(2000) in Sri Lanka found both acrocentricand sub-metacentric Y-chromosome types inAn. culicifacies population and by analogywith the situation in Rameshwaram island asproposed by Kar et al. (1999) they assumedthat the acrocentric population was speciesB and sub-metacentric type was species E.Recently, cytologically-identified An.culicifacies sub-metacentric and acrocentricand unidentified sensu lato specimens fromSri Lanka were assayed by the PCR methodof Goswami et al. (2006). Specimensbelonging to the sub-metacentric andacrocentric categories were identified asspecies E and B respectively. Among thecytologically-unidentified specimens, bothspecies B and E were also found (Surendran,Sri Lanka, and K. Raghavendra, MRC, India,personal communication). This establishesthat in Sri Lanka, species B and E aresympatric as in Rameshwaram island in India.Baimai, Kijchalao and Rattanarithikul (1996)


reported species A and B from the ChiangMai province of Thailand. Theseidentifications were based on mitotickaryotypes described by Vasantha et al.(1982; 1983). Species A was identified asthe karyotype which had a submetacentricY-chromosome and species B as that whichhad an acrocentric Y-chromosome. Later, itwas pointed out that Y-chromosomepolymorphism had been observed in speciesB and also in species C (Adak et al., 1997).Taking into consideration that species B,which is found in the eastern districts of thestate of Uttar Pradesh and in the state ofAssam in India, the population in Thailandmay be species B with Y-chromosomepolymorphism. In Cambodia, Van Bortel etal. (2002) reported the presence of species Bbased on the ITS2 sequence analysis.Following this, the authors also suggest thatAn. culicifacies from the Sichuan province inChina is also species B. This information onthe distribuition of sibling species clearlyindicates that the distribution of species Aextends to countries to the west of India whilethat of species B to the east of India.

In India, where all the five sibling speciesare prevalent, species B was found almosteverywhere throughout the country whereverAn. culicifacies was encountered. Species Bwas found exclusively in some areas, whereasin other areas it was found sympatric with Aor C or D or E alone or in combination(Subbarao, Vasantha, Sharma, 1988a;Subbarao, 1991, Kar et al., 1999). Species Aand B are sympatric in northern and southernIndia, with the predominance of species A inthe north and species B in the south.However, in the eastern states of north India,species B predominates or is the only speciespresent. Species B and C were predominantin the western and eastern regions, whilespecies D was found in sympatric associationwith A and B in the north-western region,and with A, B and C in central India and in afew places in the state of Tamil Nadu in thesouth, where, in Rameshwaram island andalso in a few blocks on the mainland in

Ramanathapuram district, species B and Ewere found sympatric. The distribution ofspecies E in other areas is yet to be mapped.A map showing the distribution of siblingspecies in India is reproduced from Subbarao(1991) as Figure 6. In this map, the recentlyfound species E is also shown.

The sibling species were not only foundto have definite distribution patterns but, ina given area, the prevalence of a speciesvaried according to the seasonalenvironmental changes. Species A was foundpredominant throughout the year. Anincrease was observed in the proportion ofspecies B in the post-monsoon months invillages around Delhi, where A and B weresympatric (Subbarao et al., 1987). Similarobservations were made in the district ofSurat, in Gujarat state, where species B andC were found sympatric. Species C of Gujaratbehaved like species A did in and aroundDelhi (Subbarao, unpublished), and also inthe district of Sundergarh, Orissa state (Nanda et al., 2000).

Biological variations such as host-specificity, susceptibility to malarial parasitesand response to insecticides have also beennoticed between the species. An. culicifaciess.l. is predominantly zoophagic and feeds onman only when the cattle population is low(Rao, 1984). The four species, A, B, C and D,were found to be predominantly zoophagic.However, species A was found with arelatively higher degree of anthropophagy(about 3.5 per cent), compared to species Bin several areas (Joshi et al., 1988). SpeciesC and D were also found to have lowanthropophagy (<1 per cent). InRameshwaram island, where species B andE are sympatric, An. culicifacies s.l. was foundto be much more anthropophagic than inother areas (Jambulingam et al., 1984, HemaJoshi, personal communication). By analogy,as species B has low anthropophagy, speciesE can be considered to be contributing to highanthropophagy in Rameshwaram island.


The biting rhythms were distinct amongspecies A, B, C and D (Satyanarayan, 1996).The biting activity of species A, B and C wasfound all through the night, while no bitingactivity of species D was observed aftermidnight. The peak biting activity of speciesA and B was in the second quarter of thenight, between 2200 hours and 2300 hours;while the peak biting activity of species C wasfound to be in the first quarter of the night,between 1800 and 2100 hrs., in the monthof April. However, the peak biting activity ofspecies C shifted to the second quarter of thenight in December in one of the study areas.Whether this is true in all areas where speciesC is prevalent needs to be examined. Noseasonal change was observed for species Aand B. Though the peak activity of species Dwas between 2100 hours and 2200 hours,30 per cent to 40 per cent of this species bitein the first quarter (this is significantly higherthan that observed for species A and B).

In northern India, where species A andB are sympatric, in the cytologically-identifiedspecimens of species A, sporozoites werefound (Subbarao, Adak and Sharma, 1980).Using the two-site immunoradiometric assaytechnique of Zavala et al. (1982), species A,C and D were found to be vectors of P. vivaxand P. falciparum malaria and species B a poorvector if at all (Subbarao et al., 1988b; 1992).The cumulative sporozoite rates for speciesA, C and D are 0.51 per cent, 0.3 per centand 0.4 per cent respectively in India(Subbarao and Sharma, 1997). In Pakistan,where species A and B are sympatric, speciesA was incriminated and is considered themajor vector (Mahmood, Sakai and Akhtar,1984). In the same paper, the authorsreported that though species B could not beincriminated under field conditions, inlaboratory feeding experiments both speciesA and B supported sporogony of P. vivax andP. falciparum. Species B used in this study wassupplied by the Malaria Research Centre,Delhi, India. This colony was established fromAn. culicifacies s.l. collected from an areawhere both A and B are sympatric. The

colony was identified as species B severalgenerations after its establishment. Inlaboratory feeding experiments on P. vivax-infected blood (Adak, Kaur and Singh, 1999)and P. vincei petteri and P. yoelii yoelii-infectedmice (Kaur, Singh and Adak, 2000), speciesA had significantly higher oocyst ratecompared to species B and C and species Bwas found the least susceptible. Recently, astrain totally refractory to P. vivax and partiallyto P. falciparum was isolated from species B(Adak et al., 2006). In this strain malariaparasites are encapsulated very early in theoocyst development leading to the death ofthe parasites. The low vectorial potential ofspecies B in malaria transmission was alsoconfirmed from our observation where thereis no malaria, or its incidence is low, as in theeastern districts of Uttar Pradesh and innorthern Bihar in India, where species B ispredominant (Subbarao et al., 1988a). Theidentification of species E, a vector species,now explains and resolves theepidemiological paradox that sporozoite-positive specimens were found in An.culicifacies s. l. in Rameshwaram island(Sebesan et al., 1984), and specimens fromthe same area were identified as species Bby polytene chromosome examination.Similarly, in Sri Lanka, only species B (Greenand Miles, 1980; Subbarao 1988;Abhayawardena, Dilrukshi and Wijesuriya,1996) and several sporozoite-positivespecimens were found (Amerasinghe et al.,1991 & 1999). The recent molecularidentification of An. culicifacies from Sri Lankaas species B and E (mentioned earlier in thissection) also resolved the epidemiologicalparadox confronted with the cytotaxonomicidentifications of this species. In Iran, whereonly species A is prevalent, it was incriminatedfor P. vivax sporozoites antigen in May andAn. pulcherrimus for P. vivax antigen in theSeptember and October collections byimmunoradiometric assay in the same study(Zaim et al., 1993). In Afghanistan, in river-irrigated rice-growing villages, of the eightspecies incriminated, An. culicifacies had the


lowest sporozoite rate of 0.20 per cent (onlyAn. splendidus had lower rate than An.culicifacies) (Rowland et al., 2002). An.culicifacies s.l. is resistant to DDT and HCHin most parts of India, and in a few areas tomalathion as well (Subbarao, 1988). Recently,this species was reported to be showingresistance to pyrethroids in Gujarat (Singh etal., 2002) and in Tamil Nadu (Mittal et al.,2002).

Species A remains more susceptible toDDT than species B in areas where both Aand B are sympatric and DDT has beenwithdrawn for long periods (Subbarao,Vasantha and Sharma, 1988c). In areas withspecies B and C sympatric association, speciesC developed resistance to malathion at afaster rate than did species B (Raghavendraet al., 1991), and species A at a slower ratethan species B (Raghavendra et al., 1992).In Gujarat, where An. culicifacies recentlydeveloped resistance to pyrethroids,sympatric species B and C exhibited a similarpattern in the resistance levels (Singh et al.,2002).

Surendran et al. (2002a & b; 2003)studied the biological variations between Y-chromosome polymorphic types. Though theauthors refer in their reports to thesepolymorphic types as species B and E, thesestudies were carried out before the two formswere correlated either with sporozoitepositivity, as has been done by Kar et al.(1999), or with molecular identifications ofGoswami et al. (2005 & 2006). As mentionedearlier in this section, the two Y-chromosomepolymorphic forms in Sri Lanka have recentlybeen identified as species B and E bymolecular assays. Surendran et al. (2002a)reported that species E larvae were found ina wide variety of breeding sites and thisspecies developed oocysts when fed on P.vivax and P. falciparum-infected patients(Surendran et al., 2002b). In species B,oocysts were not developed and it was found

breeding in rock pools and sand pools alongriver margins. These studies need to beextended to more areas, and sporozoitepositivity has to be examined in both speciesB and E in field collections too. Species Ewas observed to be more resistant tomalathion than species B (Surendran et al.,2003). Both species B and E were foundequally susceptible to deltamethrin andlambdacyhalothrin. The Sri Lankan researchgroups are now planning to carry out thesestudies in detail with field-collectedspecimens and identifications by molecularmethods of Goswami et al. (2005 & 2006)(N. Surendran, personal communication).

Taking into consideration the biologicaldifferences observed among sibling species,an insecticide spray strategy for the controlof An. culicifacies in India has been proposed(Subbarao and Sharma, 1997). Later,Subbarao, Nanda and Raghavendra (1999)stratified the country into seven majordivisions based on the prevalence of An.culicifacies sibling species. An. fluviatilis siblingspecies’ prevalence was also taken intoconsideration while proposing specific controlstrategies in each of these divisions. It isconsidered that the implementation of thisstrategy will lead to more efficient use ofinsecticides and this will reduce theexpenditure on insecticides and their impacton the environment. In Karnataka state,southern India (Ghosh et al., 2005),selectively-introduced larvivorous fish intotanks and wells brought down the densitiesof An. culicifacies, the major vector, andconsequently malaria incidence in severalvillages. The concentration on tanks and wellsand omission of streams was based on thefinding that species A, the vector species,breeds predominantly in wells and tanks andspecies B, the non-vector species, in streams.This study emphasizes the advantage ofsibling species identification and takingaccount of their biological characters whileimplementing vector control strategies.


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Figure 4: Schematic representation of polytene chromosomes of Anopheles culicifacies sibling species


Figure 5: Mitotic karyotypes of Anophles culicifacies; Plates a-c are chromosome plates prepared from larval braintissue. Plate a–Species B female, plate b–Species E male with submetacentric Y–chromosome; plate c–Species

B male with acrocentric Y–chromosome. ( Source : Kar et al., 1999)

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Figure 6: Map showing the distribution of members of the Culicifacies Complex in India (Source: Subbarao, 1991)


3.4 The Dirus ComplexThe Dirus Complex belongs to the subgenusCellia, Leucosphyrus Group in theNeomyzomyia Series (Harbach, 2004), andoccurs in the Oriental region. There has beena considerable interest in the LeucosphyrusGroup as three members of this group,Anopheles dirus, An. balabacensis and An.leucosphyrus, are important malaria vectorsin South and South-East Asia. However, therehas been much confusion, in the literatureregarding the taxonomy of this group. Peyton(1989) resolved the confusion and included20 species and two forms belonging to theNeomyzomyia series in the LeucosphyrusGroup based on the following morphologicalcharacteristics. In the adult stage they possessa very broad conspicuous white-scaled bandcovering the apex of the hind tibia and thebase of hind tarsomere 1 and speckled legs.The wings have many discrete pale- and dark-scaled spots on all veins and with four or moredark spots present on vein Cu-A and terminalabdominal segments always with some scales.

Following a detailed morphological study,Sallum, Peyton and Wilkerson (2005)reclassified the species belonging to theLeucosphyrus Group under three Subgroups:Leucosphyrus, Hackeri (earlier referred to asElegans) and Riparis (new). Leucosphyrus andDirus are the two Complexes within theLeucosphyrus Subgroup (Table 5).

The name, An. balabacensis, has in thepast been frequently used in a misleading wayuntil Peyton and Ramalingam (1988) andPeyton (1989) amended the LeucosphyrusSubgroup as follows:

(i) It now includes what was described asAn. balabacensis in the South-East Asianmainland but is now referred to as An.dirus (Peyton and Harrison 1979).

(ii) The Balabacensis Complex referred to byBaimai, Harrison and Somchit (1981),Baimai et al. (1984) and Hii (1985) nolonger exists.

(iii) An. balabacensis Perlis form is nowspecies B and An. balabacensis Fraser’sHill form is now species F of the DirusComplex.

(iv) An. balabacensis Taiwan form has beenraised to the species status by Peyton andHarrison (1980) and has been designatedas An. takasagoensis Morishita. This is nowincluded as a member in the Dirus Complex.

(v) The mosquitoes now given the name An.balabacensis and included as one of themembers of the Leucosphyrus Complex(Table 5) are those belonging to the taxondescribed from Sabah, east Malaysia.

(vi) An. elegans earlier described under theElegans Subgroup has been redescribedand is now placed in the Dirus Complex(Sallum, Peyton and Wilkerson, 2005)(Table 5).

Table 5: Species now (2005) * included in theLeucosphyrus Group

Leucosphyrus subgroup1. An. baisasi2. Con Son island, Viet Nam FormLeucosphyrus Complex3. An. balabacensis Baisas4. An. introlatus Colless5. An. leutens (leucosphyrus A)6. An. leucosphyrus s.s. (leucosphyrus B)Dirus Complex7. An. dirus (species A)8. An. cracens (species B)9. An. scanloni (species C)10. An. baimaii (species D)11. An. elegans (species E)12. An. nemophilus (species F)13. An. takasagoensis

Hackeri (elegans) subgroup1. An. hackeri Edwards2. An. pujutensis Colless3. An. sulawasi Waktoedi4. An. leucosphyrus Sumatra form

Riparis subgroup, new subgroup1. An. cristatus King & Baisas2. An. macarthuri Colless3. An. riparis King & Baisas4. Negros form

Source: Peyton (1989) and *Sallum, Peyton and Wilkerson(2005)


An. dirus has a wide distribution. It isfound in India, Nepal, Bangladesh, Myanmar,Thailand, Indonesia, Malaysia, Viet Nam,Cambodia, south China, and Taiwan. Thiscomplex includes seven members recognizedand two more suspected (details in thefollowing sections). Peyton and Ramalingam(1988) provided the morphological andgeographical descriptions of the DirusComplex for the first time. They describedthe morphological characters of this complexwhich distinguish it from other members ofthe Leucosphyrus Group.

The most significant adult character is:accessory sector pale (ASP) spot on the costaand usually also on the subcosta (anoccasional male in some species belongingto the Leucosphyrus Complex exhibits an ASPspot on the costa, but always in less than 6per cent of specimens in any population).

Additional adult characters that serve tocharacterize this complex are: presector dark(PSD) spot on wing vein R with one or morepale spots on at least one wing (exceptoccasional specimens of An. nemophilous)and hind tarsomere 4 with a distinct basalpale band or dorsal patch.

Evidence for identification of siblingspeciesThe cytological (mitotic karyotype) analysesof natural and laboratory colonizedpopulations, crossing experiments betweenpopulations and morphological variationsobserved in natural populations have led tothe recognition of members of this complex.

Species A and B (Hii, 1985)The unidirectional F1 hybrid male sterilityobserved between the Bangkok strainidentified as An. dirus by Peyton and Harrison(1979) and the Perlis form strain was the firstevidence that An. dirus is a complex of twosibling species (Hii, 1985). The Bangkok strainwas designated as species A and the Perlis

form as species B. A cross between B femaleand A male produced sterile hybrid males.Mitotic karyotypes of these two strains weredescribed by Baimai, Harrison and Somchit(1981).

Species C and D (Baimai et al., 1987)A strain from Kanchanaburi, Thailand,derived from a single female culture andmitotically different from species A and B(Wibow, Baimai and Andre, 1984) wasdesignated as species C as males in onedirection in crosses with species A, and incrosses with species B it produced noprogeny. Species A female and C male crossproduced sterile F1 males. The strain fromRanong and Phangna, Thailand, with a mitotickaryotype different from those of species A,B and C was designated as species D as itproduced no progeny in reciprocal crosseswith species A, B and C.

Natural populations comprising mixturesof the chromosomally identified species A,B, C and D were examined forelectrophoretic variations of six enzymesystems to associate chromosomal forms withallozyme genotypes (Green et al., 1992).Data were analysed by GENESTAT of Blackand Krafsur (1985). The mean value of FIS forAn. dirus sensu lato (sympatric species) was+0.28 (SD 0.02). The partitioning ofelectromorph data for the chromosomalforms reduced the mean FIS to +0.03 (SD0.01), suggesting that positive assortativemating is a characteristic of each form (valuesof FIS range from +1.0 for total absence ofheterozygotes in mixtures of two or morehomozygotes to -1.0 for total absence ofhomozygotes). Furthermore, significantdeviations observed by chi-square analysiswere removed by partitioning. This indicatesthat different electromorphs at each locus areassociated with one or the other of thechromosomal forms, which supports thefinding that chromosomal forms arereproductively isolated populations.


Species E (Sawadipanich, Baimai andHarrison, 1990)A colony established from mosquitoescollected from south-western India, andwhich produced sterile F1 hybrid males in allreciprocal crosses with species A, B, C andD, was designated as species E. The mitotickaryotype was described as different fromthose of species A and B, and resembling thatof species D.

Sallum, Peyton and Wilkerson (2005)examined morphologically more than 8000specimens from seven countries fortaxonomic revision of the LeucosphyrusGroup. After describing larval, pupal andadult morphological characters of theprovisionally designated members of theDirus Complex, they have given formaldesignations to sibling species A, B, C, D andE (see Table 5 for the formal designations).

Species F (Baimai, Harbach andKijchalao, 1988)From the mosquitoes collected from the Thai-Malaysia border, an isofemale line selecteddue to its new cytotype was designated asspecies F as it produced hybrid males withatrophied testis lobes in crosses with speciesA, B, C and D and also with An. balabacensisfrom Sabah. Morphologically, it resembledthe Fraser’s Hill form of An. balabacensisrecognized by Colless (1956; 1957). Themitotic karyotype resembled that of speciesB in having acrocentric sex chromosomes(Table 6 and Figure 7) but theheterochromatic short arm is smaller than thatof species B. Autosome III is metacentricbecause of a large block of heterochromatinwhile this chromosome is submetacentric inspecies B and also in other species.

Peyton and Ramalingam (1988) haveformally described species F (Fraser’s hillform) as An. nemophilus after comparing thevoucher specimens with the cytotype ofspecies F. This paper has a detailedmorphological description of An. nemophiluswhich is compared with An. dirus (Species A)

and An. introlatus (a member of the An.leucosphyrus complex).

An. takasagoensis MorishitaAn. balabacensis Taiwan form was elevatedto species status by Peyton and Harrison(1980) and designated as An. takasagoensis.The mitotic karyotype of this species wasdescribed by Baimai, Harrison and Somchit(1981). At that time it was described as amember of the An. balabacensis complex.This species is not sympatric with othermembers of the An. dirus complex and isfound only in Taiwan (Peyton andRamalingam, 1988). This species is includedin the An. dirus complex because of itspossession of diagnostic morphologicalcharacters of An. dirus.

In addition to taking structural andheterochromatin variations in mitotickaryotypes and hybrid sterility in interspecificcrosses as evidence for designating species,the asynapsis observed in polytenechromosomes of F1 progeny of interspecificcrosses was also taken into consideration (Hii,1985; Baimai et al., 1987; Baimai and Green,1988; Sawadipanich, Baimai and Harrison,1990).

Two suspected new speciesThe ITS2 sequence analysis revealed twotypes of sequences of species C, fromnorthern Thailand (site 12) and another ofspecies C from southern Thailand (site 14)(Walton et al, 1999). The population geneticanalysis using 11 microsatellite markers ofnorthern and southern populations of speciesC in Thailand, showed significant differencesin the genetic structure (Walton et al., 1999& 2001). Because of the geographicalseparation of these populations, it is notpossible to distinguish the hypothesis of theexistence of two species from that ofgeographical variation within species.

From the sequence analysis ofamplifications from PCR of the ITS2 region,Walton et al., (1999) report that species D


from Thailand (with 493 bps) is different fromspecies D from China as reported by Xu, Xuand Qu (1998). The authors suggest thatspecies D from China may represent yetanother species in this complex.

Techniques available foridentification of sibling speciesThe techniques available and their diagnosticfeatures for the identification of sibling speciesare given in Table 6. Additional information,for which there was no space in the table,and the relevant references are given below.

Mitotic karyotypesBaimai (1989) gives diagrams of the mitotickaryotypes of all sibling species and theirdistribution in South-East Asia (this figure isreproduced as Figure 7). All species have twopairs of autosomes, metacentric autosome IIand submetacentric autosome III, and X andY sex chromosomes. Variations in thestructure and blocks of heterochromatin inchromosomes are used in the identification.In addition to the variations shown in Table6, the following features can be used in thediagnosis:

(i) Species B has centromericheterochromatin in the autosomes(Baimai and Traipakvasin, 1987).Observation of five types of X-chromosome and four types of Y-chromosome suggests extensivepolymorphism in this species.

(ii) Species F has a block of centromericheterochromatin in autosome III whichmakes the chromosome resemble themetacentric type (Baimai, Harbach andKijchalao, 1988). The short arms of thesex chromosomes are smaller than thoseof species B.

Polytene chromosomesA standard map of polytene chromosomesof speices A has been prepared and has beenused in comparative studies of thechromosomes of other species (Baimai,Poopittayasataporn and Kijchalao, 1988).

The tips of chromosomes X, 2R and 2Lcan be used as diagnostic characters for theidentification of species. Species B has a fan-shaped tip on the X-chromosome, which isquite distinct (Hii, 1985; Baimai,Poopittayasataporn and Kijchalao, 1988).Inversion a on the X-chromosome in speciesA is polymorphic with the inversion in aminority compared with the standardarrangement. Species D is highly polymorphicwith at least a single paracentric inversion ineach autosome (break points are given inBaimai, Poopittayasataporn and Kijchalao,1988). In species D and E hybrids, the X-chromosome was found totally synapsed;therefore, it is inferred that it is also fixed forthe a inversion of species D (Sawadipanich,Baimai and Harrison, 1990). The same paperalso reports that species E possesses floatinginversions in at least two autosomes, but nodetails are given of these inversions.

Though initially mitotic karyotypes wereused in the identification of sibling species,Baimai, Poopittayasataporn and Kijchalao(1988) report that polytene chromosomes arenow being extensively used, and find thismethod more convenient.

Electrophoretic variationsGreen et al. (1992) reported thatelectrophoretic phenotypes were suitable forthe identification of sibling species. In thisstudy, two sets of samples were used: (i)chromosomally-identified samples whichincluded species A, B, C and D; and (ii)specimens identified by DNA probes whichincluded sympatric populations of species Aand D. Species A and D could be identifiedwith an accuracy of 99 per cent using GCDand AAT enzyme systems. Species B couldbe identified by slow electromorph(s) of ACP,while in species A, C and D, a fastelectromorph was observed. As a marker,human whole blood with normalhaemoglobin was used (photographs of gelswith a scale were presented to show theactual distances moved by alleles of differentenzyme systems and haemoglobin).


Egg morphology under light and scanningelectron microscopes (Damron-gphol andBaimai, 1989)The egg size of species A (length 0.524mm xwidth 0.124 mm) and C (0.548mm x0.143mm) was intermediate between that ofspecies B (0.570mm x 0.146mm) and speciesD (0.515mm x 0.144mm). The number ofrows of cells formed by tubercles in the outerchorionic membrane between the frill andthe float was found to be species-specific(Table 6). Size variation and cells formed bytubercles could be observed under the lightmicroscope. The tubercles that formedaggregates on deck surface were species-specific (Table 6) and were examined underthe scanning electron microscope.

Pupal setae under scanning electronmicroscopeAn. dirus Bangkok strain (now identified asspecies A) and An. balabacensis Perlis form(now identified as species B) weredistinguished with 100 per cent accuracy byexamining pupal setae under the scanningelectronmicroscope (Choochote et al., 1987).An. dirus species A has a stout and simplepupal seta 9-IV on both right and left sides,while in species B it is long and slender andusually has side branches. There is no reporton the evaluation of this observation in thefield population.

DNA probesPanyim, Yasothornsrikul and Baimai (1988)report four species-specific DNA sequences,and Audtho et al. (1995) have developed asimple system for the use of DNA probes,horse-radish peroxidase - labelled DNAprobes - and a chemiluminscent detectionsystem. The sensitivity of the system was suchthat it could detect 1-5 ng of target DNA,which was comparable to 32P labelled probes.This technique successfully identified speciesA, B, C and D from field collections.

RFLP profilesTwenty restriction enzymes were used tostudy restriction fragment length

polymorphism (Yasothormsrikul, Panyim andRosenberg, 1988) in species A, B, C and D.Seven enzymes (Ava II, Alu I, Bgl II, Hae III,Hinf 1, Mbob and Sau 3A I) produced uniquepatterns for each species while other enzymesproduced unique patterns for one or twospecies.

Allele-specific PCR (ASPCR) assayXu, Xu and Qu (1998) reported a PCR assayusing three primers, one derived from highlyconservative 5.8S coding sequences and twofrom the internal transcribed spacer (ITS2)region of ribosomal DNA. This assaydistinguished species A from species D with374 base pairs and 663 base pair lengthamplicons respectively.

Walton et al. (1999) reported a multiplexPCR assay with primers designed fromribosomal DNA internal transcribed spacer-2 (ITS2) sequences of different species. Thisassay identifies species A (An. dirus s.s.), B,C, D or F (An. nimophilous) found in Thailandin a single reaction. Under the assayconditions developed, no products weregenerated with DNA from individuals of An.leucosphyrus, An. macarthuri or An. pujuteasisfound in Sarawak. An. hackeri belonging tothe Elegans (now Hackeri) Subgroup gave twofaint bands of intermediate length betweenthose produced by An. dirus B and C, andthus would not result in a false positiveidentification. Using this assay, 179 field-collected An. dirus s.l. from 15 sites distributedthroughout Thailand and Malaysia wereidentified as one of the five species expected.The results were in agreement with thechromosomal identification of specimens orwith known distributions of the species. Thefragment size of species D from Thailand inthis assay was 306 bases while the sizes forspecies A, B, C and An. nemophilus were 562,514, 349 (or 353) and 223 bp respectively.The authors further state that species D fromThailand differs from that described asspecies D by Xu, Xu and Qu (1998) fromChina.


Manguin et al. (2001) and Huong et al.(2001) also developed PCR assays whichdistinguished species A, B, C and D of theDirus Complex. The method of Huong et al.(2001) used a cocktail of four primer sets foridentificaton, and the authors report that it isa sensitive method requiring a small amountof DNA. They also report a short method,i.e. homogenizing the mosquito in 1x PCRbuffer and use of the supernatant directly forPCR identification. It is a simple and rapidmethod for the identification of field-collected specimens.

Microsatellite markers for populationanalysisWalton et al. (2000a) isolated 11microsatellite markers from An. dirus speciesA and tested for polymrophism in species Cand D. Walton et al. (2000b) used thesemarkers to study populations of species A, Cand D to address the issues of gene flowwithin each species. All the three species werefound well differentiated from each other atthe microsatellite loci used. The populationgenetic analysis using microsatellite markersalso showed mtDNA introgression betweenspecies A and D (Walton et al., 2001).

Distribution and biologicalcharacteristicsThe association of An. dirus with malariatransmission in forests and forest-fringe areasis well established in Bangladesh (Rosenbergand Maheshwari, 1982), India (Rao, 1984),and Thailand (Rosenberg , Andre andSomachit, 1990).

Baimai et al. (1988) report that thedistinct differences exhibited by these speciessuggest some implications for Plasmodiumtransmission, and therefore recommend theidentification of these sibling species duringmalariometric studies.

Approximate information on thedistribution of sibling species in Thailand isgiven below (for details see Baimai 1988, andBaimai et al., 1988). The map from Baimai

(1989) is reproduced as Figure 7. For thedistinct pattern observed in the distributionof sibling species in Thailand, Baimai et al.(1988) do not suggest any geographical/topographical reasons.

Species A is widespread throughoutThailand except in the south. It occursexclusively in the central and north-easternregions. Species B and C have a restricteddistribution. Species B is found only in thesouthern peninsular region of Thailand andextends into peninsular Malaysia. Species Bis also reported from Sumatra island,Indonesia. Species C is reported only fromThailand (Kanchanaburi, Nakhon SiThammarat and Phat-thalung). Species D iscommonly found on the north-western sideof Thailand. Along the Thai-Myanmar borderit is found in sympatric association withspecies A. It is exclusively found in Myanmarand Bangladesh (Baimai et al., 1988) andnorth-eastern states of India (Baimai, 1989).The distribution of species D in India waspredicted based on its distribution in Thailandand Myanmar. Recently, in four north-easternstates of India (Assam, Arunachal Pradesh,Meghalaya and Nagaland) using Walton etal. (1999) PCR assay, Prakash et al. (2006)confirmed the presence of species D. SpeciesD is reported from Yunan province, southernChina, but as mentioned earlier, this may bedifferent from the one reported from Thailandand the other neighbouring countries. SpeciesE is exclusively found in south-western Indiain the Kyasanur area of Shimoga hills inKarnataka state (Tiwari, Hiriyan and Reuben,1987; Bhat, 1988). Species F (An. nimophilus,Peyton and Ramalingam, 1988) is found onthe Thai-Malaysia border and is also reportedfrom the monsoon forests of the mountainareas of south-eastern, southern and westernThailand and peninsular Malaysia(Rosenberg, Andre and Somachit, 1990). An.takasagoensis is exclusively found in Taiwan.

Distinct seasonal variations in relativefrequency were observed in sympatric species(Baimai et al., 1988). In areas where species


B and C are sympatric, species B was foundin abundance at the beginning of the rainyseason (May to October) while species C wasabundant at the end of the season. In speciesA and D sympatric areas, species A is moreabundant at the end of the wet season,compared to species D which is abundant inthe middle of the wet season. All-nighthuman-bait catches indicated that all the fourspecies bite throughout the night with thepeak biting times being strikingly different inthe four species studied (for details on studysites and other information, see Baimai et al.,1988). The peak biting activities were:Species A — 2100-2300 hours; Species B —1900-2100 hours; Species C — 1800-2000hours; and Species D — 0100-0300 hours.

A study was carried out at Mae-tao-keenear Maesod in north-western Thailand(Green et al., 1991) to examine the vectorialpotential of the members of the complex. Inthis area, An. dirus species A and D weresympatric and were found in association withfour members of the Maculatus Complex andAn. minimus species A. The man-biting ratesof species A and D were 1.26 and 0.61 (thesewere lower than those observed for An.pseudowillmori, a member of the MaculatusComplex, and An. minimus species A), buthad the highest sporozoite rates of 6.4 percent and 2 per cent respectively. The onlyother species which was found positive forsporozoites was An. pseudowillmori (0.5 percent) in this study.

Baimai (1989), in a review on themembers of the Dirus Complex, reported thata study carried out at Mae Sot, Tak province,and Tung Song, Thailand, revealed thatspecies A, C and D were efficient vectors ofP. vivax and P. falciparum malaria. (It is notmentioned in the review whether thisconclusion was drawn from theepidemiological investigation in the area orwhether they were incriminated.) In the samereview it is mentioned that there was noevidence to show that species B was a vectorunder field condition and species F was notknown as a vector of human malaria in

Thailand. In Binh Thuan province, south-central Viet Nam, several An. dirus specimenswere found positive for P. falciparumsporozoite antigen by ELISA (Van Bortel etal., 2001). The authors considered An. dirusto be the main vector in the area andsuggested that despite the fact that othersecondary vectors were found in the area,vector control should target against An. dirus.They further proposed that one round of bed-net treatment preceding the transmissionseason of An. dirus would be sufficient tocontrol malaria in the area.

In India, in the north-eastern states, An.dirus D is abundant and implicatedepidemiologically in malaria, but there is noinformation or report to say that species E isinvolved in malaria transmission. In the stateof Assam in the north-eastern part of India,An. dirus (species D) was found to be highlyanthropophagic, biting both indoors andoutdoors equally (Dutta et al., 1996). In theforest areas of Assam, the vectorial capacityof An. dirus was the highest, 0.779 and 0.649for P. vivax and P. falciparum respectively,during the hot monsoon season (June-September) and about 10-fold lower valueswere seen during the cool-dry season (March-May) (Prakash et al., 2001). Prakash et al.(2005) calculated the effective entomologicalinoculation rate (EEIR) for An. baimaii(formerly An. dirus species D) in a forest-fringevillage in Dibrugarh district, Assam state, indifferent seasons. With an overall sporozoiterate of 1.9 per cent, in the monsoon season(June-September), 0.39 infective bites/person/night and in the post-monsoon season(October-November), 1.47 infective bites/person/night were observed for this species.Though biting was observed all night, themaximum infective bites were in the secondquarter of the night. As 21 per cent of thetotal infective bites were recorded before2100 hours, the authors suggested thatappropriate protective measures are neededto supplement the impact of insecticide-treated nets (ITN) against An. baimaii in north-east India. The national malaria control


Table 6: Diagnostic characters for the identification of sibling species of the Dirus Complex

Figure 7: Map showing the distribution and diagrammatic representation of mitotic karyotypes of members ofthe Dirus and Leucosphyrus Complexes in South-East Asia (Source: Baimai, 1989)

Egg morphologyTubercles RFLP PCR

Species

Mitotic

appearance ofX- and Y-

chromosomes

Inversion‘a’ on

polyteneX- chromo-

somes

Enzymesystems

DNAprobes

Rows of cellsbetween frill

and float

Aggregateson decksurfaces

An. dirus s.s(Species A)

Telocentric X+a/a Gcd 100Aat 100

pMU-A40,1#5

2.5-3.5 rowsbroad

Moderatelylarge and

widely spaced

� �

An. cracens(species B)

Acrocentric X+a Acp (slow) pMU-B5 3.5-4.5 rowslong

and narrow

Same as Aand closely

spaced

� �

An. scanloni(species C)

Telocentric X+a - pMU-C19.2

2-3 rows longand narrow

Large andcloselyspaced

� �

An. baimaii(species D)

Telocentric Xa Gcd 128Aat 123 or

100

pMU-D9 2.5-4 rowsbroad andirregularshaped

Small andcloselyspaced

� �

An. elegans(species E)

Telocentric(Y-small rod or

dot like)

Xa - - - - - -

An. nemophilus(Species F)

Acrocentric - - - - - - �

An.takasagoensis

Telocentric - - - - - -

Note: Kampuchea is now known as Cambodia.


programme is planning to launch a large-scaleITN programme in the north-eastern statesof India.

In a study carried out to examine thebehavioural heterogeneity of Anophelesspecies in six different localities in South-EastAsia, Trung et al. (2005) found An. dirus s. s.in the central province of China and inCambodia. This species was found to behighly anthropophagic, exophilic, exophagicand an early biter. Based on thesecharactersistics the authors state that thisspecies is not suitable for control either bytreated nets or by indoor residual insecticidesprays. A detailed review of literature on thedistribution and bionomics of all membersof this complex is given by Sallum, Peytonand Wilkerson (2005).

Prakash et al. (2002) also studied the larvalecology of An. dirus in the rain forest area ofAssam. An. dirus-positive shady ground poolsshowed higher mean values of total alkalinity,hardness and chloride content, while stream-side pools showed lower pH and dissolvedoxygen and higher total alkalinity andhardness, compared to negative breeding sitesof similar type. In Mudon, a coastal area inMon state in south Myanmar, An. dirus wasfound breeding in wells, a situation notencountered in other parts of Myanmar (Oo,Storch and Becker, 2002) and from any othercountry in South-East Asia so far. Shadevegetation and debris on the surface of well-water were important factors influencing theabundance of larval and pupal density. InMudon area, the malaria incidence is high,with slide positivity ranging between 9.9 percent and 34.28 per cent throughout the year(Oo, Storch and Becker, 2003).

In Myanmar, Bangladesh and north-eastern parts of India, only species D has beenreported. An. dirus breeding in wells insouthern Myanmar calls for a detailedexamination of the population for siblingspecies composition. Now that effectivemolecular tools are available, a study shouldbe initiated at the earliest.

Somboon et al. (1999) isolated lines fullyrefractory and fully susceptible to Plasmodiumyoelii nigerriensis (an African rodent malariaparasite) after 17 generations of massselection. Most of the mosquitoes of therefractory line inhibited the parasitedevelopment by encapsulating oocysts, whichappeared as melasized spots in the gut. Butthis line showed normal susceptibility tohuman malaria parasites, P. falciparum and P.vivax.

3.5 The Fluviatilis ComplexAnopheles fluviatilis James belongs to thesubgenus Cellia, the Minimus Subgroup andthe Funestus Group in the series Myzomyia(Harbach, 2004). The Fluviatilis and MinimusComplexes, An. flavirostris and An. lessoni,are members in the Minimus Subgroup. An.fluviatilis has a wide distribution in theOriental region and parts of the West Asiasubregion (Rao, 1984). In the Oriental region,it is found in Pakistan, Afghanistan, India,Nepal, Bangladesh, Myanmar, Thailand andsouth China, and in the West Asia region it isfound in Iran, Iraq, eastern and southernArabia, Oman and Bahrain. It is consideredan important vector only in India, Pakistanand Nepal (Rao, 1984). Distinct differencesobserved in densities, preference to feed ona host and infection rates between thepopulations led Rao (1984) and several earlierworkers, Senior White (1946), Viswanathan(1950), Brooke Worth and Sitaraman (1952),and Bhombore, Sitaraman and Achutan(1956), to suggest that this species may havetwo biological races. Subbarao et al. (1994)recognized An. fluviatilis as a species complexin India.

Evidence for recognition of siblingspeciesThe evidence for the three sibling speciescame from the absence of heterozygotes forthe three polytene chromosomearrangements observed due to two fixed


paracentric inversions q1 and r1 onchromosome arm 2. Chromosome arms 3,4 and 5 and X-chromosome werehomosequential in all the three species. Thethree sibling species were designated asspecies S, T and U (Subbarao et al., 1994).

A population differing from the otherthree sibling species in polytene chromosomearrangement on chromosome arms 2 and 3was recently recognized in district Hardwar,Uttaranchal state (a new state carved out ofthe erstwhile Uttar Pradesh state in northernIndia). In the same area species T and Uwere found sympatric. The absence ofheterozygotes for the two new inversions wastaken as evidence to designate this populationas a new species (Nanda et al., unpublished).An. fluviatilis now is a comlex comprising foursibling species, S, T, U and new species V.

Recently, based on the homology of D3domain of 28S rDNA, Harbach (2004),Garros, Harbach & Manguin (2005) and Chenet al. (2006) considered An. fluviatilis Sconspecific with An. minimus C.Consequently, Harbach (2004) removed An.fluviatilis S from the Fluviatilis Complex in histaxonomic update. Furthermore, all theseauthors said that the Fluviatilis Complexconsists of only two sibling species, species Tand U. However, recent analysis of Singh etal. (2006) on the sequences belonging to theinternal transcribed spacer 2 (ITS-2) regionand D2-D3 domain of ribosomal DNA of An.fluviatilis S and An. minimus C showed thatthe two species are appreciably different withpair-wise distance (Kimura-2-Paramtetremodel) of 3.6 per cent and 0.7 per centrespectively for ITS-2 and 28SD2-D3 loci.Based on this analysis, Singh et al. (2006)concluded that An. fluviatilis S and An.minimus C were not conspecific. Further,from pair-wise and phylogenetic analysis,these authors showed that An. fluviatilis S wasmore closely related to other members of theFluviatilis Complex than to An. minimus C.


Ploytene chromosomesParacentric inversions, q1 and r1, onchromosome arm 2, are diagnostic: +q1+r1

is the standard arrangement diagnostic forspecies S, q1+r1 for species T and +q1r1

arrangement for species U (Subbarao et al.,1994). Other chromosome arms arehomosequential in all three species. Aphotomap of polytene chromosomes ofspecies S, with inversion break points, ispresented by Subbarao et al. (1994).Chromosome arm 2, marked withbreakpoints q1 and r1, is shown in Figure 8.Nanda et al. (unpublished) designated newinversions as s1 on chromosome 2 and s onchromosome 3 and the new inversionkaryotype 2s1; 3s is diagnostic for species V.

Mitotic karyotypesNo variations were observed in the mitotickaryotypes of any of these sibling species.

ASPCR assaysA PCR assay developed from rDNA ITS2region by Manonmani et al. (2001) was latercorrelated with cytological identifications(Manonmani et al., 2003). This assayaccurately distinguished species S fromspecies T in about 94 per cent of thespecimens. Lack of 100 per cent correlationwas probably due to the presence of q1inversion polymorphism. This assay does notdistinguish species T from species U(Manonmani, personal communication).

An allele-specific PCR assay developed bySingh et al. (2004), based on differences inthe D3 variable region of 28S ribosomal rDNA,differentiates all three species S, T and U. Thismultiplex assay has four primers, two universalprimers – D3A forward and D3B reverse andtwo allele-specific primers – AFS and AFT –which are specific for species S and Trespectively. The specimens not reacting to


S- and T-specific primers were regarded asspecies U. Cytological correlation was donewith specimens collected from different partsof India, and in these areas the three specieswere found in different sympatric associations.PCR identifications were in full agreement withthe cytological identifications. In Mandya andGulbarga districts in Karnataka state (southIndia), q1 inversion is polymorphic with thekaryotypes +q1/+q1, +q1/q1 and q1/q1 inHardy-Weinberg equilibrium. The questionthat arose was whether these belonged tospecies S or T. The allele-specific PCR assayidentified all three karyotypes as species T(Singh et al., 2004).

Singh et al. (2006), by examining ITS-2sequences of species X reported by Manonmaniet al. (2003) and of species S, conluded thatspecies X was synonymous with species S.

The five sibling species of the CulicifaciesComplex which are sympatric with An.fluviatilis in many areas did not react withspecies S or T primers. However, they reactedwith the universal primers, giving an ampliconof 380 base pairs like species U. The universalprimers may react with other anophelines aswell. Therefore, care must be taken toidentify mosquitoes morphologically prior tothe use of this assay.

PCR-RFLPA slight modification of the above assay, byincluding a restriction enzyme, distinguishes thenew species V (O. P. Singh et al., unpublished).

Distribution and biologicalcharacteristicsFollowing the discovery of sibling specieswithin An. fluviatilis, studies were carried outto map the distribution of the sibling speciesand study their bionomics. The biologicaldifferences observed are summarized in Table7. Species S was found either alone or insympatric association with species T, andspecies U was more frequently found withspecies T. In Kamrup district, Assam state,only species U was found but the number of

samples examined were very few. Allopatricpopulations of T were also observed (Figure9). From Table 7, it can be seen that speciesS is distinctly different from species T and Uin several biological characters.

A longitudinal study was carried out onthe bionomics of An. fluviatilis in the foothillsof the Shivalik range of the Himalayas inHardwar and Dehradun districts ofUttaranchal state (which is the newlydemarcated state that was a part of the formerUttar Pradesh state in north India) (Sharmaet al., 1995). It was found throughout theyear with high densities in October/November and low densities in May toAugust, densities in peak months beingrecorded as more than 200 caught per man-hour of collection (“man-hour density”). An.fluviatilis breeding in these villages was foundin slow-running streams, irrigation channelsand subsoil seepage water with grassymargins, preferably under some shade. Nobreeding was observed in fast-flowing steamsor in rice fields. In both the districts, speciesT and U were sympatric. In Dehradun,which is at an altitude of 640 m, species Twas predominant (>95 per cent), while inHardwar (294.7 m altitude), species U waspredominant (>65 per cent). The biologicalcharacteristics of the two species were asreported in Table 7. The new species foundin a few villages in district Hardwar wassympatric with species U and T (Nanda etal., unpublished). In Nainital district ofUttaranchal state, species T and U were alsosympatric (Shukla et al., 1998).

In Malkangiri and Koraput districts inOrissa state (in south-eastern part of India),where species S and T are prevalent(Manonmani et al., 2003), the breeding ofthese species was observed in terraced paddyfields, streams and stream channels (Sahu etal., 1990). Species S was found to be highlyanthropophagic (~91 per cent) while speciesT and U were almost totally zoophagic(Nanda et al., 1996). From the district ofHardwar a small number of species T and U


(a total of 189) and 294 specimens of An.fluviatilis s.l. processed by immuno-radiometric assay for sporozoite antigen werefound not positive (Sharma et al., 1995). Incontrast, in the districts of Malkangiri andKoraput where species S is predominant,earlier in 1989 An. fluviatilis s.l. wasincriminated for sporozoites (Gunasekaran etal., 1989) and later species S was incriminated(MRC, 1995). In areas where species S hasbeen found, malaria is hyperendemic, andthe prevalence of P. falciparum and deathsdue to malaria are reported.

In Sundergarh, a district in the north-western part of Orissa, a longitudinal studywas carried out to examine the bionomicsand to delineate the role of An. fluviatilis andAn. culicifacies sibling species in malariatransmission (Nanda et al., 2000). In Birkerablock in a village in the forest, mainly An.fluviatilis species S and of the two An.culicifacies sympatric species B and C, speciesC maintained high transmission. The annualparasite incidence (API) was 269 cases/1000population with 83.5 per cent P. falciparum.And in a deforested village, only An.culicifacies was found with species B and Csympatric (of these two, only species C isvector). Malaria incidence in this village wasrelatively low (API 39 cases/1000 population)and P. falciparum cases accounted for 57.9per cent; P. vivax was the other Plasmodiumspecies in these villages. Another detailedlongitudinal epidemiological study is beingcarried out in the same district in 13 villages,eight in forest and five in the plain areas. Thisstudy is being conducted in order to developa field site for vaccine trial. The observationsare similar to the above-mentioned study with347.9 API in the forest area and 61.0 in theplain area (Sharma et al., 2004a). Thecontribution of An. fluviatilis species S andAn. culicifacies species C to entomologicalinoculation rate (EIR) in the forest area was0.395 and 0.009 infective bites per personper night respectively, while in the plain area,only An. culicifacies species B and C were

found and the EIR was 0.014 (Sharma et al.,2006). In spite of continuous exposure toinsecticides, An. fluviatilis still remainssusceptible to all insecticides—DDT, HCH,malathion and pyrethroids in Orissa (Sharmaet al., 2004b) and also in other areas wherethis species was tested in India (K.Raghavendra, personal communication).

About 0.5% of An. fluviatilis s.l. fromAfghanistan was found infective with both P.falciparum and P. vivax (CSP 210) CSP antigens(Rowland et al., 2002). The villages wherethis species was incriminated had river-irrigated rice fields. In the mountainous areasof the Hormozgan province, south Iran, An.fluviatilis is considered an efficient vector ofmalaria. It is exophilic and exophagic anddensities start to build up in September andtwo peaks, one in January and another in May,are observed. Using the PCR assay ofManonmani et al. (2001), An. fluviatilis in thearea was identified as sibling species T(Vatandoost et al., 2005). Specimens fromIran were also cytologically identified asspecies T (H. Vatandoost and N. Nanda,personal communication). Species T in Iran,unlike in India, apprears to be a vector.Recently, Adak et al. (2005) reported thatsibling species T from India was susceptibleto P. vivax infection in laboratory feedingexperiments.

This suggests that species T in India isgenetically susceptible to Plasmodiuminfection, but because of its preference tofeed on animals and the environmentalconditions in addition may be making it anon-vector.

Now that well-established cytotaxo-nomic and molecular methods are availablefor identification, populations from all thecountries where An. fluviatilis is reported needto be examined for sibling speciescomposition and their bionomics need to bestudied to find intra- and inter-specificvariations, if any.


Figure 8: Photomap of the polytene chromosomes of An. fluviatilis from ovarian nurse cells. When the blocks areinverted with respect to standard arrangenents (of An. funestus), a dot is placed over the block designation. Break points

of paracentric inversions q1 and r1 on chromosome arm 2 are shown (source: Subbarao et al., 1994)

Table 7: Biological differences and diagnostic characters of An. fluviatilis sibling species observed in India*

* For details, see "Distribution and biological characteristics" in this section.

SpeciesInversion

karyotypes ASPCRMan-hourdensities

Feedingpreference

Sporozoitepositives

Preferedadult

habitat

Ecotype Endemicity

S q1 r1 Yes Low(1-40)

Anthropophagic(>90%)

Found Humandwellings

Hillyforests &foothills

Hyper-endemic

T

U

q1+r1

+q1 r1

Yes High(up to 200)

Almost totallyzoophagic(~99%)

Not found Cattlesheds

Foothills &plains

Hypo-endemic


Kulu

Hardwar

Nainital

Allahabad

Sonapur

Kangra

Alwar

Kheda

Bahraich

Jabalpur

Gulbarga

Tumkur

Kolar

Mandya

Malkangiri

Koraput

Balangir

Sundergarh

Phulbani

Kalahandi

Koenjahar

Mayurbhanj

Purulia

Sambalpur

Species S

Species T

Species U

Figure 9: Map showing the distribution of members of the Fluviatilis Complex in India (Source: The map is courtesy of Dr Nutan Nanda, NIMR, Delhi)

3.6 The LeucosphyrusComplex

The Leucosphyrus Complex consists ofAnopheles balabacensis Baisas, 1936, An.introlatus Colless, 1957, and An.leutens(leucosphyrus A) and An. leucosphyrus s. s.(leucosphyrus B) (see Table 5).

Evidence for recognition of siblingspeciesTwo isomorphic species, An. leucosphyrus Aand B, were identified following thecytological examination of mitotic karyotypesand reproductive isolation observed in crossesbetween allopatric populations (Baimai,Harbach and Sukowati, 1988). The threeallopatric populations of An. leucosphyruswere collected on human bait from twoislands of Indonesia: (i) Sumatra (Bukit Baru,


near Muarabungo, Bungo Tebo regency,Jambi province); (ii) South Kalimantan(Salaman, near Kintap, Tanah Laut regency);and (iii) Southern Thailand (Pedang Besar,Songkla province). There was no evidenceof reproductive isolation between the Thaiand South Kalimantan populations, as the F1progeny were fertile from both reciprocalcrosses. In contrast, from both reciprocalcrosses between the Sumatra population andthe populations from South Kalimantan andThailand, F1 males were sterile, indicatingreproductive isolation. Based on these results,the authors concluded that An. leucosphyrusincludes two allopatric species, the oneinhabiting Borneo, west Malaysia, andsouthern Thailand designated as species A,and the population confined to the island ofSumatra designated as species B. Species Bis An. leucosphyrus Doenitz, the nomino-typical member of the widely distributedLeucosphyrus Group.

Baimai, Harbach and Sukowati (1988)presented photographs and diagrams of themitotic karyotype of species A and B (seeFigure 7). The differences between the twospecies were due to major blocks ofheterochromatin. In species A, the X- and Y-chromosomes of specimens from SouthKalimantan possess a very small segment ofextra heterochromatin at the centromericregion, which gives the chromosomes asubtelocentric configuration. In the Thaipopulation, because of the absence of thisblock, the sex chromosomes appeartelocentric (as shown in Figure 7). TheSumatra population of species B possessessubmetacentric X- and Y-chromosomes. Theshort arm of the X and the whole of the Y-chromosome is heterochromatic. The shortarm of the X has a secondary constriction inthe middle which is not found in any of themembers of the Dirus Complex.

On the X-chromosome of both thespecies, the presence of a distal block ofheterochromatin is very conspicuous and

appears to be a character which distinguishesthese two species from An. balabacensis. Thisdiagnostic character also distinguishes theabove-mentioned species A and B from themembers of the Dirus Complex. Two typesof Xs due to size variation in the long arm ofthe X were observed in each species. Aconspicuous pericentric heterochromaticsegment was seen in both the autosomes.In the Sumatra population (species B), thiswas more prominent in autosome III.

Hii (1985) reported, for the first time,that mosquitoes from Sabah, which hereferred to as An. balabacensi s. s., weredistinct from An. dirus A and An. dirus B (Perlisform). An. balabacensis s.s.produced sterilehybrid males (partially-developedreproductive organs) when crossed with An.dirus A and B.

Peyton (1989) has assigned An.balabacensis for the first time to theLeucosphyrus Complex (see Table 5) becuaseit is morphologically closely similar to An.leucosphyrus. A diagram of the mitotickaryotype of An. balabacensis is presentedby Baimai, Harbach and Kijchalao (1988)(see Figure 7). This species has acrocentricX- and Y-chromosomes. The Y-chromosomeis totally heterochromatic and autosome IIIhas a large block of heterochromatin. Theauthors also report that An. balabacensis, incrosses with species F of An. dirus, producedF1 males with atrophied testes without sperm,and the polytene chromosomes were totallyasynapsed in the F1 progeny. No informationon the fourth species, An. introlatus, has beenfound.

Formal designationsSallum, Peyton and Wilkerson (2005), aftermorphological examination of severalthousand specimens belonging to thiscomplex, formally designated An.leucosphyrus species A as An. leutens andspecies B as An. leucosphyrus s. s.


Techniques available foridentification of sibling speciesStructural variations in mitotic karyotypes andthe banding pattern associated withherochromatin variations are the onlyavailable methods for the identification ofspecies.

Distribution and biological charactersAn. leucosphyrus species A is widelydistributed in southern Thailand, westMalaysia, and Sarawak, east Malaysia, andKalimantan in Indonesia, while species B isprobably confined to the island of Sumatra,Indonesia (Baimai 1988; Baimai, Harbachand Sukowati, 1988). An. balabacensis isconfined to the locality from which the typespecimen came in Balbac island andneighbouring areas, i.e. Polawan island,Sabah and north-east Kalimanatan (Peytonand Harrison, 1979; Hii, 1985; Peyton,1989). All species of this complex breed inshaded temporary pools in forests.

An. balabacensis was found positive forP. falciparum sporozoite antigen by IRMAalong with another species An. flavirostris onBanggi island, Sabah, Malaysia (Hii et al.,1988). Based on the human landing rate andsporozoite positives, the EIR/Year wascalculated as 160. The vectorial capacitycalculated for March was 1.44-7.44 and forNovember 9.97-19.7. On Banggi island,malaria is holoendemic.

In south Kalimantan where An.balabacensis and An. leucosphyrus species Aare sympatric, these two species togethercomprised 97.7% of the total anophelinescollected (Harbach, Baimai and Sukowati,1987). Large numbers of these specimenswere collected from within the village thanin the forest. The P. falciparum sporozoiteantigen detection rate was 1.0% for An.leucosphyrus species A and 1.3% for An.balabacensis. After more than 50 years ofeffective management of malaria, in the sub-districts of Menoreh Hills and Dieng Plateu,

Java, Indonesia, a sharp increase in malariaoccurred in the year 2000. Two importantvectors, An. maculatus and An. balabacensis,which favour forested hill sides in Java, wereconsidered responsible for the transmission(Barcus et al., 2002).

An. leutens (species A) is consideredhighly anthropophilic and an importantvector of human malaria, both in villages andforest areas of Sarawak, east Malaysia (Changet al., 1995). This species is also considereda vector of Wuchereria bancrofti to humansin Sarawak (from Sallum, Peyton andWilkerson, 2005). There is no report on theincrimination of An. leucosphyrus species B.

3.7 The MaculatusComplex

Anopheles maculatus Theobald belongs to thesubgenus Cellia and the Maculatus Group inthe Neocellia Series (Harbach, 2004). Thisspecies is recognized as an important vectorof human malaria parasites in Thailand,Indonesia and peninsular Malaysia.

To facilitate the understanding of theMaculatus Complex, information on eightnominal forms described in the literature(Christophers, 1931; Rattanarithikul andGreen, 1986) is given below. Christophersregarded maculatus as a single species basedon studies of the morphological variation inadults and recognized two vectorial forms:one with reduced abdominal scaling, thenominotypical form (maculatus), and theother with heavy abdominal scaling, var.willmori. He considered pseudowillmori,dravidacus and hanabusai as synonyms of thenominotypical form and considereddudgeonii, indicus and maculopsa assynonyms for var. willmori. Rattanarithikuland Green (1986) further state that in spiteof extensive studies by Puri (1931),Christophers (1933), Crawford (1938), Reid,Wattal and Peters (1966), and Reid (1968),the morphological concept and formal


taxonomy of this group have remainedunchanged.

The cytotaxonomy and morphologicalstudies together have now unequivocallyidentified eight biological species, and theDNA analysis has recently identified a newspecies corresponding to the chromosomalform K in this complex. Nine biologicalspecies are described and discussed below.

Evidence for recognition of siblingspeciesAll the rearrangements in An. maculatus arereferred to the An. stephensi map (Green,1982) which serves as an arbitrary standard.(This is in contrast to other species complexeswhere chromosome maps prepared for anunspecified member of the complex or oneof the sibling species are used as standard).

The first evidence that An. maculatus is aspecies complex came from two largelyindependent studies on chromosomes–polytene and mitotic chromosomes fromnatural populations of An. maculatus. WithinAn. maculatus polytene chromosomes, novariation has been seen in chromosome arms3, 4 or 5 while fixed and floating inversionswere seen on the X and arm 2. A total of 18paracentric inversions were observed andthese inversions were observed in six distinctchromosomal forms in Thailand. Thesechromosomal forms were found in differentsympatric associations in different areas. Thesix chromosomal forms were given the speciesstatus based on the population genetic data.The absence of heterozygotes for the alternatearrangements of the inversions in a given areawas taken as evidence for the reproductiveisolation.

Species A, B and C (Green and Baimai,1984, Green et al., 1985)In addition to the differences in paracentricinversions (as mentioned above),heterochromatin variation in X- and Y-chromosomes was also observed (Green andBaimai, 1984). Three types of X-

chromosomes (X1, X2 and X3) and four typesof Y-chromosomes were observed. Noheterozygotes were found of X1 either withX2 or X3. And X1 was considered indicativeof species A in contrast to X2 and X3 in speciesB, which were found in heterozygouscondition. Y-chromosome polymorphicforms were not found associated with anyparticular species.

The three species, A, B and C, werefound sympatric in three localities, two nearKanchanaburi, west of Bangkok, and one innorthern Thailand, near Chiang Mai and noheterozygotes were found for the diagnosticinversions (Green and Baimai, 1984).

Two allopatric populations that are closeto species B but differ from it in the frequencyof two paracentric inversions on X-chromosome and four on chromosome arm2 were identified as the E and F forms. Theobservation of heterozygotes for all theinversions that distinguish between speciesB and the E form in Petchaburi (nearKanchanaburi, Thailand) suggested that theform E is a chromosomal race of species B(Green and Baimai, 1984). The authors,however, caution that the sample size wassmall, hence the conclusions drawn are notfinal. Furthermore, F1 progeny from bothreciprocal crosses between the B and E formswere fertile, supporting the concept thatthe form E may be a chromosomal race ofspecies B. The status of form F could not beconfirmed as B/E and F are separated by theChao Phraya river basin from which An.maculatus s.l. is absent (Green et al., 1985),and larger samples of the F form arerequired.

Cuticular hydrocarbon analysis(Kittayapong et al., 1990) differentiatedspecies B from E form. There is also indirectevidence from cuticular hydrocarbon analysisthat the two forms coexist in peninsularMalaysia (Kittayapong et al., 1993).Microsatellite marker analysis suggested thatthere was restricted gene flow between the


northern populations of An. maculatusextending from latitude 11

o to 16

oN and the

southern populations from latitude 7o to 6

o N

(Rongnoparut et al., 1999). Race B (speciesB) extends northwards from latitude 13

o N,

while race E extends southwards from latitude12

oN into peninsular Malaysia (Green et al.,

1985). Based on microsatellite data analysis,the authors suggest that the southernpopulations may become distinct species(details in the next section). Rongnoparut etal. (1999) report that unpublished data of R.Rattanaritikul shows that inversion karyotypesshow little indication of hybridization betweenB and E.

Green et al. (1985) noted that B and Eforms are either two distinct sibling speciesor they represent geographical variationwithin An. maculatus. Keeping in view thatdistinct cuticular hydrocarbon profiles wereobserved for the two forms, Harbach (2004)and Walton et al. (2005) suggest that theymay be distinct species.

Species G (Green and Baimai, 1984)A population fixed for inversions x and y onchromosome arm 2, and for d and e on X-chromosome, was found along with speciesA and B in a locality near Petchaburi in thenorthern part of peninsular Thailand. Noheterozygotes were observed for theseinversions and the inversions were unique forthe population. This population wasdesignated as species G.

The results from reciprocal crossesbetween species A, B, C and G producedsterile F1 males and fertile females, whichprovided support for the designation ofseparate species based on cytogeneticevidence.

Species D and J (Rattanarithikul andHarbach, 1990)An. maculatus form D was identified on thebasis of chromosomal differences notedduring the comparative cytologicalexamination of populations from thePhilippines, Thailand and Malaysia (Green et

al., 1985). Another population was collectedfrom Subic Bay, to the west of Manila(Philippines), and the cytological examinationof this population revealed that this wasdifferent from form D collected from thenorth-east of Manila. These two cytologicalforms, though very different, could not bedesignated as separate species because thetwo forms were identified from allopatricpopulations.

Rattanarithikul and Harbach (1990)correlated distinctive morphological traits toeach of the cytotypes. The observation oftwo morphotypes from single localities in thePhilippines led them to state that there isreproductive isolation between An. maculatusform D and the new (J) cytotype, and, hence,these are distinct species. An. maculatus formD described in Green et al. (1985) was giventhe formal name An. greeni, and the newcytotype, J form, the name An. dispar(Rattanarithikul and Harbach, 1990). This isthe first case where the morphological andcytological differences taken togetherdemonstrated reproductive isolation.

Species H and I (Green, Rattanarithikuland Charoensub, 1992)An. pseudowillmori and An. willmori weredescribed morphologically by Rattanarithikuland Green (1986) and they were alsoidentified as distinct cytological forms (I andH respectively) (Green, Rattanarithikul andCharoensub, 1992). The evidence for theirspecies status came from the cytotaxonomicexamination of populations from fourlocalities in north-western Thailand. In theselocalities, An. willmori, cytologicallydesignated as form H, and An. pseudowillmorias form I, were found in sympatric associationwith species A and B, with a total absence ofheterozygotes for the inversions fixed in them.

Enzyme polymorphism at 6-Pgd (6-phosphogluconate dehydrogenase) alsosupported the specific status of An.pseudowillmori . Three allozymes orelectromorphs were found associated with


three species: An. pseudowillmori — 6-Pgd70, An. maculatus (species B) — 6-Pgd100, and An. sawadwongporni (species A)— 6-Pgd130, with a total absence ofheterozygotes.

In Thailand, six species (A, B, C, G, Hand I) were found, while forms D and J wereconfined to the Philippines. The six forms inThailand were found in the following differentsympatric associations (Rattanarithikul andGreen, 1986; Green, Rattanarithikul andCharoensub, 1992):

A, B and C — from several widelyseparated localities inwestern Thailand;

A, B and I — from Mae Sariang, MaeHong Son province;

B, H and I — from Doi Inthanon,Chiang Mai province;

B and H — from Mae Sa, Chiang;

A, B, C and G — in a single locality nearPhet Chong , NikhonRatchasma province;

A, B and G — in one locality near PhetBuri in the northern partof peninsular Thailand;

A and F — in a single locality nearNokhorn Nayak, north-east of Bangkok;

A, B, H and I — in four localities in north-western Thailand.

In all these localities no heterozygoteswere seen for the fixed inversions identifiedfor these forms, which strongly supports thespecies status given to these cytologicallydistinct populations, and establishes thepresence of six species specific materecognition systems in the An. maculatusComplex in Thailand.

New species, putative species K (Waltonet al., 2007)The ITS2 sequence analysis of the specimenscollected from eastern Thailand revealed that

they have a unique sequence and is 3.7 percent divergent from the next closely relatedtaxon An. sawadwongporni in the group(Walton et al., 2007). The authors considerthat this corresponds to the chromosomalform K reported by Baimai (1989). Based onthe negligible intra-specific variationsobserved in the specimens analyzed, theauthors designated the chromosomal form Kas a new species in the complex.

The biological species that wereprovisionally designated with the letters of theEnglish alphabet were formally recognized bystudying the morphological variations inprogeny broods from wild-caught femalesthat were identified by chromosomalrearrangements observed in their ovarianpolytene chromosomes. Papers byRattanarithikul and Green (1986) andRattanarithikul and Harbach (1990) arestrongly recommended for details regardingthe formal recognition of these species. Theprovisional letter designations and theirspecies names are given in Table 8. SpeciesK does not have a formal designation yet.


Polytene chromosomesThe members of the Maculatus Complex canbe identified unequivocally from each otherand from An. stephensi (which is used as anarbitrary standard) by examining polytenechromosomes for paracentric inversions onautosomes. Sixteen inversions onchromosome arm 2, one on arm 3 and oneon arm 5 are unique to this complex andhence diagnostic. Though differences in thebanding pattern in the X-chromosomes arereported, the homologies have not beenworked out well. The diagnostic inversionsgiven in Table 8 are from Green,Rattanarithikul and Charoensub (1992). Thispaper gives the photomap of An. stephensichromosome arm 2 with breakpoints of theinversions marked, and the diagnosticinversions summarized in a figure. For


photomaps of chromosome arms 3, 4 and 5,the readers are referred to Green (1982).

Morphological keyA morphological key for the identification ofall eight members of the Maculatus Complexis provided by Rattanarithikul and Green(1986). Upatham et al. (1988) found a smallpercentage of errors in the identification ofspecies A, F form of species B and species B,using a morphological key (identificationswere confirmed by cytological examination).

Electrophoretic variations/Cuticularhydrocarbon profiles6-Pgd allelic variations (Table 8) can also beused to distinguish An. sawadwongporni(species A), An. maculatus (species B) and An.pseudowillmori (species I) (Green,Rattanarithikul and Charoensub, 1992). Thetwo forms of species B, E and F could bedistinguished by gas-liquid chromatographicanalysis of cuticular lipids in association witha multivariate principal component analysis(Kittayapong et al., 1990).

PCR-RFLPAn. greeni and An. dispar occur sympatricallyin the Philippinnes. In order to develop asimple and reliable method of identification,Torres, Foley and Saul (2000) carried out ananalysis of two regions of rDNA, ITS2 andD3 domain of the 28S gene of An. maculatuss.l. specimens from localities throughout thePhilippines. Two distinct sequence groupswere observed, one corresponding to An.greeni and the other to An. dispar. Digestionof the ITS2 amplicon with Hae II restrictionenzyme yielded distinct fragments, and thetwo species could be identified with ease andaccuracy.

PCR assayBased on interspecific variation in the ITS2region, a diagnostic PCR assay thatdistinguishes five members of the complexfound in China, An. sawadwongporni, An.maculatus, An. willmori, An. dravidacus and

An. pseudowillmori, was developed (Ma, Liand Xu, 2006). Another PCR-based diagnosticassay has been developed to facilitate fieldresearch in northern Thailand, whichdistinguishes An. maculatus, An. dravidacus,An. pseudowillmori, An. sawadwongporni andspecies K (Walton et al. 2007).

Microsatellite markersAbout 23 microsatellite markers wereidentified from An. maculatus s.s. (species B)(Rangnoparut et al., 1996). Seven of thesemicrosatellites were used to study the geneticvariation in eight widely dispersed localitiesin the western and peninsular Thailand(Rangnoparut et al., 1999). The datasuggested that the populations could begrouped into two clusters: one including theupper and lower northern populations(extending from latitude 11o to 16o N) andthe other including the southern population(extending from latitude 7o to 6o N). Amongthe populations, within each cluster, extensivegene flow was observed, while restricted geneflow was observed between the northern andsouthern populations. Geographical orgenetic barriers could be limiting the geneflow between these populations.

Distribution and biological charactersThe distribution of the species given below isfrom Rattanarithikul and Green (1986),Baimai (1989), Ma, Li and Xu (2006) andWalton et al (2007):

An. sawadwongporni (species A) — Myanmar,China, Cambodia, Thailand and Viet Nam.It is found at low and high altitudes inassociation with all other members of thegroup

An. maculatus s.s. (species B) — Bangladesh,Myanmar, China, India, Indonesia,Cambodia, Malaysia, Nepal, Pakistan, SriLanka, Taiwan, Thailand and Viet Nam

An. dravidicus (species C) — Myanmar (Kaleyvalley), India, China and Thailand

An. notanandai (species G)—Thailand


An. willmori (species H) — India, Nepal,Pakistan, China and Thailand (ChiangMai)

An. pseudowillmori (species I)— China, India,Nepal, Thailand and Viet Nam

Species K — Thailand

An. greeni (species D) and An. dispar (speciesJ) are indigenous to the Philippines.

The distribution of members of this complexin Thailand is given in Baimai (1989) and themap is reproduced in this document as Figure10.

An. greeni is widely distributed both inthe low land and hilly areas of the Philippines(Rattanarithikul and Harbach, 1990). An.dispar appears to be more common,particularly at higher elevations than An.greeni (Rattanarithikul and Harbach, 1990).No sporozoite-positive specimens were foundin An. greeni. However, Rattanrithikul andHarbach consider that this may be the samespecies which Ejercito (1934) found infectedwith oocysts and sporozoites of P. falciparumin the Bulacan province of Luzon island,Philippines.

The first study conducted on thebionomics and vector potential of membersof the Maculatus Complex was by Upathamet al. (1988). An. maculatus s.l. werecollected from a village in the Pakchongdistrict, Nakhon Ratchasima province, centralThailand, and another village in the Sadaodistrict, Songkhla province, southernThailand. In the central Thailand, speciesA, B (form F) and C, and in southernThailand, only form E of species B, wereidentified.

In Pakchong, An. maculatus species Awas the most dominant species, followed byspecies B (form F) and species C, which wasrare. The densities of species A and speciesB (form F) were high between July andNovember, with their peaks in October. Thebiting activities of both the species occurredthroughout the night, with a major peak

during the first quarter of the night in allseasons. In the Sadao district, only An.maculatus species B (form E) was detectedwith peak densities between February andJune. The biting activities of this speciesvaried according to the season. Theprevalence of mosquitoes was influenced bymonthly rainfall, relative humidity and airtemperature.

All species identified in the study werefound to be predominantly zoophagic andpreferred to bite humans outdoors ratherthan indoors. The life expectancy recordedwas the highest for species B (E form) — 0.7days to 21.2 days; the maximum recordedfor species A was 6.6 days and for the F form8.1 days. No sporozoite-positive glands werefound in any species (out of 4472 dissected)but 0.23 per cent oocyst-positives werefound. In both the areas in the same study,An. dirus were found positive for P. falciparumsporozoites. This indicates that species A, B(the E and F forms) and C do not play anyrole in the transmission of malaria inThailand. The E form was incriminated asthe primary vector of human malaria in thepeninsular Malaysia.

Another study was carried out todetermine the vector potential of themembers of the Maculatus Complex at Mae-Tao-Kee near Maesod in north-westernThailand (Green et al., 1991). Four species,An. maculatus, An. dravidacus, An.sawadpongpornii and An. pseudowillmori,were found sympatric. One specimen of An.pseudowillmori was found infected with P.vivax and one with P. falciparum sporozoites(0.5 per cent sporozoite rate). Thesespecimens were collected on human baits.The man-biting rate of An. pseudowillmoriwas 4.41/night while that of An. maculatusand An. sawadwongporni were 0.89/nightand 0.41/night respectively. The man-bitingrate of An. pseudowillmori was almostequivalent to that of An. minimus species Acollected during the same study. Nosporozoite-positives were found in An.


Species name

Cytotaxo-nomicdesig-nation

Diagnosticinversiongenotypeson arm 2*

6-Pgdelectro-morphs

PCR-RFLP

ITS2-basedPCR assay

An.sawadwongporni Rattanarithikul &Green 1986

A pt1u1v1w1 130 - �

An. maculatus s.s. Theobald 1901 B,E,F j 100 - �

An. dravidicus Christophers 1924 C x1y1z1 - - �

An. greeni Rattanarithikul & Harbach D q � -

An. notanandai Rattanarithikul & Green1986

G xy - - -

An. willmori James 1903 H - - - �

An. pseudowillmori Theobald 1910 I o1p1q1 70 - �

An. dispar Rattanarithikul & Harbach1990

J r1 - � -

Putative species K �**

minimus, but An. dirus A and D in the samearea had the sporozoite rates of 6.4 per centand 2 per cent respectively. These resultsclearly suggest that An. pseudowillmori iscapable of maintaining a low-gradetransmission in the absence of efficientvectors like An. dirus A and D. According toGreen, Rattanarithikul and Charoensub(1992), An. pseudowillmori is at the limit ofits distribution in Thailand, and theytherefore suggest that its role in malaria maybe different in Myanmar and north-eastIndia, which are the centre of its distribution.Sporozoite-positive mosquitoes were earlierreported from Assam state in north-eastIndia, Myanmar and Nepal (Rao, 1984). Twostudies by Upatham et al. (1988) and Greenet al. (1991) have shown that An.sawadpongpornii (species A), An. maculatuss.s. (species B) and An. dravidacus (speciesC) may not be playing a role in thetransmission of malaria in Thailand.However, it should be noted that An.maculatus s.s. in penninsular Malaysia

(chromosomal form E at present assigned tospecies B) is a major vector of malaria (Greenet al., 1991). An. willmori from the higheraltitudes in Nepal is considered responsiblefor malaria transmission (Pradhan et al.,1970). Since An. willmori is found at highaltitudes in Thailand, Green, Rattanarithikuland Charoensub (1992) suggest that its roleshould be studied. In river-irrigated, rice-growing districts of eastern Afghanistan, An.maculatus s.l. was incriminated for malariatransmission during a study carried out fromMay 1995 to December 1996 (Rowland etal., 2002). One specimen was found positivefor P. vivax CSP210 from the 30 tested. Aftermore than 50 years of effective managementof malaria, in sub-districts of Menoreh Hillsand Dieng Plateu, Java, Indonesia, a sharpincrease in malaria occurred in the year2000. Two important vectors, An. maculatusand An. balabacensis, which favour forestedhill sides in Java, were considered responsiblefor the transmission (Barcus et al., 2002).

Table 8: Diagnostic inversion genotypes and other methods available for the identificationof Maculatus Complex members

* These inversions are used to distinguish An. maculatus sibling species from An. stephensi. In addition to these, in all species of theMaculatus Complex, inversions a on arm 3, x on arm 4, and c and d on arm 5 are fixed, an exception is An. pseudowillmori in whichthe +x arrangement on arm 4 is seen as in An. stephensi. An. willmori is distinguished from An. stephensi only by 3a, 4x and 5cd.

** ITS2 sequence of this species was different from other species but the ITS2 assay developed by Ma, Li and Xu (2006) was nottested on this species.


20

18

16

14

o

o

o

o

12

10

8

6

o

o

o

o

95 97 99 101 103 105o o o o o o

MYANMAR

LAOS

THAILAND

VIETNAM

COMBODIA

GULF OF THAILAND

MALAYSIA

Figure 10: Map showing the distribution of members of the Maculatus Complex in Thailand(Source: Baimai and Green (1988))

3.8 The Minimus ComplexAnopheles minimus Theobald belongs to thesubgenus Cellia, the Minimus Subgroup, theFunestus Group in the Myzomyia Series(Harbach, 2004). This species has a widedistribution in the Oriental region, andthroughout the range of its distribution it isconsidered an important vector of malaria.It is found in India, Nepal, Sri Lanka,Bangladesh, Myanmar, Thailand, Malaysia,Indonesia, south China, Hong Kong, Taiwanand the Ryuku Archipelago, Japan (Rao,1984). Harrison (1980) reported 12morphological variants in An. minimus

populations from Thailand. Suthas et al.(1986a) observed significant differences inparous rates between females fed on bovidsand those on humans. Also, a significanttendency was observed in females to returnto the type of host upon which they werefirst caught (Suthas et al., 1986b). To explainthe genetic heterogeneity observed in An.minimus populations in Thailand, the authorssuggest the possibility that the taxoncomprises morphologically cryptic species.

Yuan (1987) reported two morphologicalforms, A and B, from the hilly regions ofChina. Sucharit et al. (1988) identified three


morphologically variant forms in the Thaipopulation: Typical minimus form (M),Varuna form (V) and Pampanai form (P).Among the morphological differencesdescribed, the characters that are distinct inthe three forms are:

M form - wing with presector pale spot(PSP) on costa;

V form - wing with completely darkprehumeral and humeral bandson costa;

P form - with two pale spots, presectoral(PSP) and humeral (HP), on costa.

An. minimus has now been recognizedas a complex of 5 sibling species, A, B, C, Dand E. Though species B (Sucharit et al.,1988) and species D (Baimai, 1989) havebeen reported, no further information isavailable on these species.

Evidence for recognition of siblingspecies

Species A, B and C (Sucharit et al., 1988)Sucharit et al. (1988) examined wildpopulations in Thailand for electrophoreticvariations in seven enzyme systems in relationto the morphological forms. The enzymesystems studied were: esterases (EST), malicenzyme (ME), leucine aminopeptidase (LA),lactate dehydrogenase (LDH), malatedehydrogenase (MDH), xanthinedehydrogenase (XDH) and aldehyde oxidase(ALDOX). Of these, ALDOX and MDH weremonomorphic. Although for such studiesmore enzyme systems are required to drawreliable conclusions, a clear-cut relationshipwas nevertheless seen. In the Pu Toeipopulation, the P form was predominant(frequency 0.947), in other populations theM form was predominant (frequency 0.963to 1.00), and in Sattahip, the M formfrequency was 0.794, and that of the V formwas 0.206. This was the highest frequencyrecorded for the V form; in other populationsthe frequency ranged from 0-0.033.Furthermore, the authors reported that,

among the five alleles observed at the Est-2locus, Est-298 was predominant (frequency0.833) in the Pu Toei population, while Est-2100 allele was common in all otherpopulations (frequency 0.357 to 0.604).Based on these results, the authors, for thefirst time, concluded that An. minimus is aspecies complex consisting of three species.Typical An. minimus (M form) withpredominant Est-2100 or 102 electromorph wasdesignated as species A, the P formpredominant in Pu Toei with Est-298

electromorph was designated as species C,and the V form found in low frequency inThailand was designated as species B.

Specis A and C (Green et al., 1990)The population genetic evidencedemonstrating a lack of gene flow within An.minimus in Thailand came from the study ofwild populations for electrophoretic variationsin six enzyme systems by Green et al. (1990).Sympatric occurrence of two electromorphs,134 and 100, at the Octanol dehydrogenase(Odh) locus, and the absence ofheterozygotes in two localities, was taken asevidence that An. minimus is a complexcomprising two sibling species. Thisconclusion was further supported by therelative deficiency of heterozygotes observedat both the Manose phosphate isomerase(Mpi) and the Glycerol dehydrogenase (Gcd)loci, and the disappearance of the deficiencywhen the genotypes of these loci are classifiedinto the two Odh classes. In the other enzymesystems—phosphoglucomutase (Pgm),hydroxyacid dehydrogenase (Had), lactatedehydrogenase (Ldh) and malatedehydrogenase (Mdh)—studied, the relativefrequencies of alleles were different in thetwo populations. The authors ruled outpossibilities such as immigration/emigrationand close linkage between loci which couldcause such associations. The evidence forthis was based on genetic analyses whichestablished linkage relationship of these loci(Thanaphum et al., 1990) and consistentabsence of heterozygotes over a four-year


study period at Ban Phu Rat. Following thenomenclature given by Sucharit et al. (1988)to sibling species in this taxon, Green et al.(1990) equated Odh100 to species A andOdh134 to species C.

Van Bortel (1999) studied An. minimuspopulations from Viet Nam usingelectrophoretic variations in 14 enzymesystems. Significantly high positive Fis valuesat the Odh locus gave a clear indication ofnon-random mating within this species, andthe deficiency of heterozygotes indicatedreproductive isolation between two sympatricpopulations, species A and C, as in Thailand.

Species E (Somboon et al., 2001)An. minimus specimens collected fromIshigahi island, Ryuku Archipelago, Japan,were identified as species E by Somboon etal. (2001). Based on morphologicalcharacters and results from genetic crosses,the population from Ishigahi island wasrecognized as a new species. 99.5% of thespecimens from Ishigahi island resembledspecies A in having a pale spot on the costa,but differed with species A and C in having apale fringe spot at the tip of vein A (a characterrarely found in An. minimus populations fromother countries). In scanning micrographs,cibarial armamature showed cone filamentsdiffering in shape from those of species A andC. In a cross between ISG males (strainestablished from An. minimus collected fromIshigahi island) and species A (CM strain)females, the hybrid males were sterile due toatrophied testes or presence of abnormalspermatozoa. The hybrid females, when backcrossed to the parental males, laid very feweggs and there was low hatch. No F1 progenywere produced from the reciprocal cross.Metaphase karyotype of ISG differed fromCM strain (species A) in having differentpolymorphic Y-chromosomes. Polytenechromosomes of F1 hybrids exhibited noasynapsis. These data conclusively suggestedthat species A and E are distinct species.Recently, Somboon et al., (2005), based onresults from genetic crosses between species

C laboratory colony and provisionallydesignated species E ISG strain, showed thatspecies C and E are distinct species. In boththe crosses, F1 males were sterile and polytenechromosomes of F1 hybrid larvae exhibitedpartial asynapsis. In all the chromosomepreparations an inversion heterozygote in 3Rchromosomal arm was observed.

Suspected new species (Sharpe, Harbachand Butlin, 2000; Somboon et al., 2001)From the DNA sequences at a mitochondriallocus (Cytochrome Oxidase II) and threenuclear loci (ITS2 and D3 regions of rDNAand Guanylate cyclase), Sharpe, Harbach andButlin (2000) confirmed the presence ofspecies A and C within An. minimus fromThailand and also reported the possiblepresence of another species. The specimensuspected to be a new species (specimen no.157) morphologically resembled species C(humeral pale spots on both wings), and hadunique D3 and ITS2 sequences. But the COIIsequence was identical to that of somespecimens belonging to species C; thus, itresembled species C more than species A.Since only one specimen was observed, aspecies status was not assigned to thisspecimen. The authors also reported apersonal communication from C. A. Greenwhich stated that allozyme data from thesame locality supported the presence of anew species.

Somboon et al. (2001) observedvariations in D3 sequences in the specimensfrom Viet Nam. Sequences from two of thefour specimens were similar to those ofspecies A and C, while two sequencesdiffered from each other and also from thoseof species A and C. These sequences alsodiffered from that of species E. The authorsargue that novel sequences might representnew species because each specimen ishomozygous for a particular sequence, andsuggest that this represents reproductiveisolation. Based on this evidence the authorsconsider that four species are present in VietNam. Van Bortel and Coosemans (2003)


argue that this evidence is unconvincing.According to them, the two novel sequencesprobably are due to intra-specific variation.And this has been insufficiently ruled out bySomboon et al. (2001) and considered thatthe sequences represent new species.


Mitotic karyotypesSucharit et al. (1988) briefly described thesex chromosomes and a fluorescent band inthe X-chromosome of the M and P forms.Baimai, Kijchalao and Rattanarithikul (1996)described mitotic karyotypes of species A andC from Thailand. Species A can bedistinguished from species C, as species C hasprominent pericentric heterochromatin in theautosomes and the short arm of the X.Furthermore, two types of X-chromosomesdiffering in the length of the long arm inspecies A and a third type of X in species Cwere described. Both microphotographs andkaryotype diagrams are given in Baimai,Kijchalao and Rattanarithikul (1996).

Polytene chromosomesKanda et al. (1984) reported photmaps andline-drawing maps of salivary gland polytenechromosomes of An. minimus. The authorsconcluded that the polytene chromosomesof ISG strain and of two strains isolated fromKanchanburi, Thailand, have homosequentialbanding pattern as no asynapsis was observedin F1 progeny from the crosses between thestrains. Although the species status of the twostrains from Kanchanburi is not known, theISG strain has recently been identified bySomboon et al. (2001) as species E. Somboonet al. (2005) reported that species A andspecies E have homosequential bandingpattern, while species C differs from speciesE by a fixed inversion in chromosome arm3L and one in arm 3R, and partial asynapsiswas observed on chromosome arms 2R and3L in F1 hybrid larvae.

Diagnostic Electrophoretic variationsAlleles at the Odh locus, allele 100 in speciesA and 134 in species C, are diagnostic forthe Thai populations (Green et al., 1990)(Table 9). In the absence of known referencestandards, human AA haemoglobin has to berun on each gel to estimate the mobility ofthe bands of unknown specimens. Therelative mobility of haemoglobin AA wasmore useful in the TEB (Tris-boric acid EDTAbuffer system) gels than in the TC (Tris-citricacid buffer system) gels. Relative mobilitiesof a single human Had electromorph andthree intense bands of human Ldh were moreuseful on TC gels. Green et al. (1990) alsoreported Mdh-1 electromorphs to bediagnostic for distinguishing An. minimus s.l.,An. aconitus and An. pampanai. Van Bortelet al. (1999), following the Green et al. (1990)technique, could distinguish An. minimus s.l. from the closely related species An. aconitusand An. jeyporiensis at the Odh locus innorthern Viet Nam. The authors furtherreported that in Viet Nam An. minimus A wasmonomorphic for Odh100 allele but in contrastto Green et al. (1990) finding in Thailand,An. minimus C in Viet Nam was polymorphicfor the Odh locus. However, Van Bortel et al.(1999) in Viet Nam could not separateunambiguously An. minimus s.l. from An.aconitus using Mdh-1 variation. Est-2 allelesreported by Sucharit et al. (1988) should betested for their specificity against diagnosticOdh alleles. Yuan (1987) also reported thattheir A form and B form exhibit distinctesterase banding patterns.

Molecular assaysNow there are molecular assays availablewhich not only distinguish species A and C(Table 9) but also other sympatric anophelinesfound with An. minimus. Sucharit andKomalamisra (1997) used RAPD-PCR todistinguish species A and C. Fifteen differentcommercially available primers (Operonoligonucleotide kit M from OperonTechnologies, Inc.) were used. Six primerswere found diagnostic on the basis of the


Species SEMMitotickaryotypes

Electrophoretic

Variation

RAPD-

PCR

PCR-RFLP

SSCP ASPCR2

A Yes Yes Odh100 Yes Yes Yes Yes

C Yes Yes Odh134 Yes Yes Yes Yes

E Yes - - - - - -

presence or absence of amplified fragmentsin species A and C. However, these markershave not been validated on the field samples.

Sharpe et al. (1999) developed an allele-specific amplification assay (ASPCR) from theD3 variable region of the 28S rDNA gene,which distinguished species A and C. A singlestrand confirmation polymorphism (SSCP)assay of the D3 amplified region was alsodeveloped, which distinguished species Afrom C and also An. aconitus and An. varuna.Van Bortel et al. (2000) developed a PCR-RFLP technique which distinguished speciesA and C and An. pampanai Buttiker andBeales, An. aconitus, An. varuna and An.jeyporiensis, which are sympatric. ITS2 rDNAamplification followed by digestion with BisZl enzyme accurately distinguished six speciesbelonging to the Myzomyia Series (Van Bortelet al., 2000). The technique was evaluatedon specimens collected from Viet Nam,Thailand, Cambodia and Laos. Garros et al.(2004b) extended the above PCR-RFLP assayby including more species from the FunestusGroup and An. gambiae as outgroup in theevaluation. The assay distinguished An.minmus A and C, hybrids between A and Cand 11 other species.

In an effort to provide a simple androbust technique for the identification ofmembers of this complex, Kengue et al.(2001) developed a multiplex PCR methodbased on RAPD markers. From the sequencesof the RAPD markers, sequence characterizedamplified regions (SCARs) specific primers of20-24mer were designed. In the assay, one

primer set for distinguishing species A and Cand one each for An. pampanai, An. varunaand An. aconitus were combined. Themethod was validated on a large number ofspecimens collected from Viet Nam,Thailand, Cambodia and Laos. In northernViet Nam and north-western Thailand wherespecies A and C are sympatric, <1% ofhybrids were observed by the isoenzymetechnique (Van Bortel et al., 1999), the PCR-RFLP method (Van Bortel et al., 2000) andby the multiplex PCR of Kengue et al. (2001).This suggests that all techniques are equallysensitive and specific.

Studies correlating the morphologicalcharacters with the electrophoretic variationby Van Bortel et al. (1999) and with moleculardiagnostic markers by Sharpe (1999) andChen, Harbach and Butlin (2002) haveestablished that the morphological charactersmentioned by Sucharit et al. (1988) areunreliable in distinguishing species A fromspecies C. Furthermore, it has been clearlyshown that form A and form B described inChina by Yuan (1987) should not be equatedto species, and these forms have notaxonomic status. The An. minimus describedby Harrison (1980) has been identified asspecies A.

Another multiplex PCR assay has beendeveloped for the identification of An.minimus and four other anopheline speciesbelonging to the Minimus Group (Phuc et al.,2003). Allele- specific primers weredeveloped from the rDNA ITS2 region.Species A and C and An. varuna, An. aconitus,

Table 9: Techniques1 available for the identification of sibling species of An. minimus

1 Details in the text.2 Several PCR assays are available which not only distinguish species A and C, but also distinguish other sympatric

anophelines (details in the text).


An. pampanai and An. jeyporiensis could beeasily differentiated by this assay. The authorsclaim that their assay results can beinterpreted more readily than previous assays.Garros et al. (2004a) developed a multiplexallele-specific PCR which not onlydistinguishes members of the MinimusGroup, An. minimus A and C, An. aconitus,An. varuna, An. leesoni and An. pampanai,but also four members of the Funestus Group,An. funestus, An. vandeeni, An. rivulorum andAn. parensis, in a single step. The assay wasdeveloped based on the variation seen in theITS2 region.

Distribution and biological charactersBaimai and Green (1988) give a map showingthe distribution of the sibling species of An.minimus in Thailand (see Figure 11). SpeciesA is widespread in Thailand, in the Thai-Myanmar border areas species C is present,and in one locality species D is also seen.Species A has now been reported from VietNam, Laos and Cambodia and is foundsympatric with species C in Viet Nam (VanBortel et al., 2000, 2001; Kengne et al., 2001;Garros et al., 2005), Laos (S. Manguin,personal communication) and China (Zhouet al., 2002; Chen, Harbach and Butlin,2002). Though Sucharit et al. (1988) reportedspecies B from Thailand and China, recentmolecular studies identified only species Aand C from these countries.

Both species A and C from Thailand werereported to be predominantly zoophilic,feeding on humans more outdoors thanindoors. At MaeTao-Kee near Masod in north-western Thailand where members of theMaculatus, Dirus and Minimus Complexeswere prevalent, An. pseudowillmori andspecies A and D of the Dirus Complex werefound positive for sporozoites, but nosporozoite-positive An. minimus species Awere found, even though the man-biting rate/night (4.41) of this species was almostequivalent to that of An. pseudowillmori(Green et al., 1991). Furthermore, species A

and D of the An. dirus complex had man-biting rates of only 1.26 and 0.61. Green etal. (1991) suggest that An. minimus species Ain this area is not as efficient as members ofthe Dirus Complex in transmitting malaria.An. minimus populations in India are highlyanthropophagic (Rao, 1984). In Sonapur,Assam state, this species has been foundresting indoors, highly anthropophagic andwith sporozoite rates ranging between 2.3and 3.35 (Wazihullah, Jana and Sharma,1992). It is from this area, based on Odhelectromorphs An.minimus specimens wereidentified as species A (T. Adak, personalcommunication). Recently, from four north-eastern states of India–Assam, ArunachalPradesh, Meghalaya and NagalandAn.minimus specimens were identified asspecies A using Phuc et al. (2003) PCR assay(Prakash et al., 2006). In Orissa state, An.minimus species A was collected from hilltopvillages in Singhbhum hills of Koenjhar districtafter 42 years of its disappearance followingDDT spraying (Jambulingam et al., 2005).These findings suggest that there is only onemember of the Minimus Complex, species Ain India. The introduction of insecticide-treated bednets in Assam, reduced the slidepositivity rate considerably (Jana-Kara et al.,1995). The An. minimus species A from Assamappears to be different from that found inThailand with respect to host-feedingpreference and sporozoite positivity.

In northern Viet Nam, Van Bortel et al.(1999) observed species C to be moreexophagic and zoophilic than species A.Species A was found to be highly endophagicand five times more abundant in indoorcollections than species C. This behaviouraldifference is considered significant, becausecattle are kept inside the houses. Theseobservations suggest that An. minimus C maynot be a vector of importance in Viet Nam.In China this species is considered a vector(Chen, Harbach and Butlin, 2002). Van Bortelet al. (2001), using the PCR-RFLP methoddeveloped by Van Bortel et al. (2000) for theMinimus Group, correctly identified a


majority of the specimens from a village insouth-central Viet Nam as An. varuna, whichis predominantly a zoophagic species. Earlier,following morphological identifications, amajority of the specimens from this area werewrongly identified as An. minimus s. l.Because it is considered an important vector,the control strategies in the area were focusedagainst two vectors, An. minimus and An.dirus. Now, it has been established that An.

dirus is the main vector in the area, severalspecimens of An. dirus being foundsporozoite-positive by ELISA (Van Bortel etal., 2001).

A study by Van Bortel et al. (2003) foundspecies A breeding along the banks of slow-moving rural streams, while in the suburbanareas of Hanoi it was found breeding in watertanks. This demonstrates the adaptability of

Figure 11: Map showing the distribution of members of the Minimus Complex in Thailand(Source: Baimai and Green (1988))

MYANMAR

LAOS

THAILAND

VIETNAM

COMBODIA

GULF OF THAILAND

MALAYSIA


this species to different breedingenvironments. In a recent study carried outby Van Bortel et al. (2004), intra-specificbehavioural differences among thepopulations of species A collected from sixdifferent localities were observed. Thisspecies was found more anthropophagic incentral Viet Nam, Laos and Cambodia wherecattle were scarce, than in northern Viet Namwhere cattle were in good numbers. A similarobservation that the anthropophagy of speciesA was dependent on cattle was made byTrung et al. (2005). These authors observedthat species A is a late biter and it bitesthroughout the night. These observations ledTrung et al. (2005) to suggest that insecticide-treated bednets was a suitable strategy for thecontrol of this species. Garros et al. (2005)reported that with the introduction ofpermethrin-treated bednets from 1999 to2001 in central Viet Nam, species Apopulation was drastically reduced, andduring the same priod species C which wasfound in low numbers in the area, increasedsignificantly. In this study, molecularidentifications were done with AS multiplexassay developed by Garros et al. (2004a).

3.9 The Philippinensis-Nivipes Complex

An. philippinensis and An. nivipes belong tothe Annularis Group of mosquitoes in theNeocellia series. (Harbach, 2004) An.philippinensis and An. nivipes aremorphologically very similar to the onlydiagnostic wing character being: presectordark mark on vein 1 does not reach as farback as the distal end of the humeral darkmark on the costa in An. philippinensis, andthe presector dark mark overlaps the humeraldark mark on the costa in An. nivipes.However, this character is equivocal (Reid,1968).

Because of the overlap in themorphological characters, An. nivipes wasconsidered as a synonym for An.

philippinensis. Reid (1967) reported thatthere is little or no overlap in distinguishingthese two species if the characters on thepaddle at the pupal stage are used, and heelevated nivipes to species level. An.philippinensis is reported to be distributedfrom India to the Philippines, and northwardto China. Because of the overlapping adultwing characters with An. nivipes, accuratedistributions of these two species cannot bedefined. Klein et al. (1984) reported thatmalaria workers in Thailand refused to acceptAn. nivipes as a separate species from An.philippinensis because of the overlap in theadult morphological characters.

In Bangladesh, An. philippinensis isconsidered to be the principal vector ofmalaria in the vast plains of its central, westernand northern regions. After the withdrawalof the large-scale DDT spraying in the late1970s, An. philippinensis reappeared in someof the areas (Elias et al., 1987). In India, An.philippinensis was found abundantly in WestBengal, Assam and the neighbouring states(Rao, 1984). For the first time, Nagpal andSharma (1987) have reported An. nivipes,along with An. philippinensis, from Assam andMeghalaya states. In Thailand An.philippinensis/nivipes are consideredsecondary vectors of malaria.


An. philippinensis and An. nivipes(Klein et al., 1984)Two laboratory colonies were established,based on the distinguishing characters on thepaddle at the pupal stage (Klein et al., 1982).The genetic incompatibility, observed inreciprocal crosses between An. philippinensisand An. nivipes laboratory colonies in theform of hybrid mortality at egg, larval andpupal stages, and hybrid sterility in thesurviving males, established that these twoare distinct species and justified the decisionof Reid (1967) to raise An. nivipes from thesynonym to species level (Klein et al., 1984).


An. nivipes species A and B (Green et al.,1985)Two allopatric populations fixed for thealternative banding pattern on the X-chromosome due to a paracentric inversion,b, were observed in Thailand (Green et al.,1985), which suggested that there wasanother species in this complex. An. nivipeswith X+b is designated as species A, and Xbas species B.


Morphological variationsThe differences between An. nivipes and An.philippinensis are clear at the pupal stage onthe paddle (Reid, 1967). In An. nivipes, thepaddle has a longer refractile border withdistinctly numerous teeth and shorter paddlehair. In An. philippinensis, the paddle has lessthan ten fringe teeth on the refractile borderand has longer paddle hair, theunstraightened length being about one thirdthat of the paddle.

Polytene chromosomesBy examining polytene chromosomes of An.nivipes and An. philippinensis colonies (Kleinet al., 1982), two autosomal inversions wereidentified that could unequivocally distinguishAn. nivipes from An. philippinensis at the adultstage by Green et al. (1985). The twoinversions were t on the chromosome arm 2,and l on arm 5. The X-chromosome andchromosome arms 3 and 4 arehomosequential in An. philippinensis and An.nivipes. The b inversion on the X-chromosomedifferentiates An. nivipes A from B.

The diagnostic inversion genotypes of thespecies are:

An. philippinensis — X+b; 2+t; 5+l

An. nivipes species A — X+b; 2t; 5l

An. nivipes species B — Xb; 2t; 5l

The photomaps of the polytenechromosomes of An. philippiensis with the

break-points of inversions found in An. nivipeswere presented by Green et al. (1985), andthe photomaps of An. nivipes are shown inFigure 12.

Molecular AssaysThere are now two assays, an allele-specificPCR assay of Walton et al., (2007) and a PCR-RFLP assay of Alam et al., (2007), developedto distinguish members of the An. annularisGroup which can be used to accuratelyidentify An. nivipes and An. philippinensis.

The allele specific PCR assay wasdeveloped based on differences seen in ITS2sequences of five species—An. annularis, An.nivipes, An. philippinensis, An. pallidus andAn. schueffneri—of the An. annularis Group,which are morphologically very close (Waltonet al. al., 2006). While the An. schueffneri isrestricted to Java and Sumatra islands ofIndonesia, An. pallidus to Sri Lanka, India andMyanmar, the other species are widespreadin the region. The authors report that theassay developed will be a rapid and reliableepidemiology tool, which should work fromnortheastern India through Myanmar andThialand to Laos and Cambodia.

Prakash et al. (2006) examined ITS2sequences of morphologically confirmed An.nivipes and An. philippinensis specimens fromAssam and Nagaland states in the northeastIndia to assess the applicability of Walton etal. (2006) PCR assay. The sequences of An.phillipinensis shared 99.2% and An. nivipesshared 99.3% respective similarity to thosefrom the Thialand.

The PCR-RFLP assay was developed basedon endonuclease restriction sites in the D3sequence to differentiate four members in theAn. annularis Group—An. annularis, An.nivipes, An. philippinensis, and An. pallidus—(Alam et al., 2007). Restriction digestion of D3fragment individually with SmaI, ApaI and NcoIproduced distinct pattern of all four specieson 3. 5 per cent agarose-gel. D3-SmaI systemcan be used to distinguish An. nivipes fromAn. philippinensis.


X +b

b

b

c

c

c

c

c

t

l

l

t

2 t5 l3 4

Distribution and biological charactersSubbarao, Vasantha and Sharma (1988) andSubbarao et al. (2000) carried out extensivecytotaxonomic examinations of specimenscollected from five states in north-east Indiafollowing the report of Green et al. (1985).

Collections were made from Kamrup districtin Assam, Umbling in Meghalaya, Bhalapurin Arunachal Pradesh, Imphal in Manipur andDimapur in Nagaland. All the specimens werefound to be An. nivipes A. (All thesespecimens had been examined for the

Figure 12: Photomap of polytene chromosome of An nivipes. The break points for inversions b,t and l on chromosomearms, X,2 and 5 respectively are marked. C indicates the centromere end of each chromosome arm

(Source: Subbarao et al., 2000).


characters on the wing which distinguish An.nivipes and An. philippinensis). Some of theseareas were the same as those surveyed byNagpal and Sharma (1987) who reported thepresence of both the species based on theirwing characters. Thus, the wing character wasnot found diagnostic for Indian populationsas concluded by Reid (1967) for the Thaipopulations. However, the presence of An.philippinensis cannot be ruled out in thesefive states. Prakash et al. (2000) reported An.nivipes and An. philippinensis, based on larvaland pupal diagnostic characters, from theChanglang district of Arunachal Pradesh andrecently (2004) from Dibrugarh district inAssam, and also from the neighbouring states,Arunachal Pradesh and Meghalaya. InDibrugarh, all the nine females from human-landing collections were identified as An.philippinensis, while all the 16 females fromthe resting collections made near cattle shedswere An. nivipes.

An. philippinensis, once dominant in thedeltaic regions of West Bengal andresponsible for the high incidence of malariain this area, is almost absent today (Rao,1984). The earlier dissection records showedsporozoite-positive specimens from WestBengal, but not from Assam, in spite of a largenumber of dissections (Krishnan, 1961).However, An. philippinensis, identified basedon its wing character, was incriminated as avector of malaria in Burnihat, which is nowin Meghalaya state (Rajgopal, 1976). Thecytotaxonomic examination (Subbarao et al.,2000) has revealed only An. nivipes inMeghalaya, and from pupal paddle charactersrecently by Prakash et al. (2004). Among An.philippinensis-nivipes s. l. collected from CDClight traps placed in human dwellings indistricts Dibrugarh and Jorhat, Assam state,specimens positive for P. vivax and P.falciparum sporozoite antigens were foundby ELISA (Prakash et al., 2005). Extensivecytotaxonomic (Subbarao et al., 2000) ormolecular identifications (Prakash et al.,2006) along with ELISA tests are needed toestablish unequivocally the relative role of An.

philippinensis and An. nivipes in malariatransmission in the north-eastern states ofIndia.

Following the discovery of the diagnosticinversions, the Thai populations wereexamined for the presence of two species.An. nivipes was found to be more common,but An. philippinensis was found to be morewidespread than was earlier thought (Baimaiet al., 1984). In the light of the findings fromIndia, the populations from Bangladesh needto be examined.

3.10 The PunctulatusComplex

Anopheles punctulatus belongs to thesubgenus Cellia, and the Punctulatus Groupin the Neomyzomyia Series. This taxon hasa wide distribution from the Moluccas in thewest through New Guinea and the SolomonIslands to Vanuatu in the east, and as far southas northern Australia. Rozeboom and Knight(1946) identified four species in thePunctulatus Group, based on themorphological characters of the proboscis(Bryan, 1973a; Foley et al., 1993).

(i) An. farauti Laveran — totally darkproboscis, except for a pale subapical ring(based on this character, An. moluccensisfrom the Moluccas and Irian Jaya,Indonesia, was synonymized with An.farauti).

(ii) An. punctulatus Doenitz — with theapical third to half of the proboscis pale,the remainder dark.

(iii) An. koliensis Owen — white scaling onthe proboscis variable but more oftenrestricted to a small discrete patch on theapical third of the proboscis.

(iv) An. clowi (Rozeboom and Knight) —proboscis is similar to that of An. koliensis,and the legs are yellowish instead ofhaving dark areas on at least some or allof the fore- and mid-tarsomers. This


species was described from Irian Jaya,Indonesia (Rozeboom and Knight, 1946).This species was rediscovered by Cooperet al. (2000) in Papua New Guinea.

Marked differences observed in the An.farauti s.l. populations in peak biting timesand breeding habitats suggested the presenceof more than one species within this taxon.Cross-mating experiments and cyto- andbiochemical-taxonomic studies haveidentified 11 species so far in the PunctulatusComplex. In the South-East Asia region,members of this complex occur only inIndonesia. Kondrashin and Rashid (1987)report An. punctulatus, An. koliensis and An.farauti as important vectors in Irian Jaya/Maluku islands.


An. farauti No. 1 and An. farauti No. 2An. farauti (Bryan, 1970; 1973a and b) fromtwo colonies established from (1) Rabraul,New Britain, Papua New Guinea, and (2)Innisfail, northern Queensland, Australia,were crossed. Both female and male progenyfrom the reciprocal crosses were sterile. Thiswas taken as evidence for the existence oftwo species within An. farauti; the colonyfrom Papua New Guinea was designated asAn. farauti No. 1 and the colony from Australiaas An. faruati No. 2. Bryan and Coluzzi (1971)observed two paracentric inversions, one onthe left arm and another on the right arm ofchromosome arm 2 in An. farauti No. 2. Inhybrids between An. farauti No. 1 and An.farauti No. 2, inversion heterozygotes wereobserved in these regions. This suggests thatthe two populations differ by fixedparacentric inversions.

An. koliensis (Bryan, 1973a and b)Doubts about whether An. koliensis is a hybridbetween An. punctulatus and An. farauti(because of the variable pale scales on theproboscis) were removed by cross-matingexperiments carried out by Bryan (1973a and

b). There was either reduced hatchability ortotal sterility in crosses between An. koliensisand An. farauti and An. punctulatus. Theadults which emerged had reduced orpartially developed reproductive organs.These results proved that An. koliensis is aseparate species.

An. farauti No. 3 (Mahon and Miethke,1982)A population found sympatric with An. farautiNo. 1 and An. farauti No. 2 in Australiaproduced sterile progeny in reciprocal crosseswith An. farauti No. 1 and No. 2. A fixedparacentric inversion was found in thispopulation, and the population wasconsidered a new species and was designatedas An. farauti No. 3.

An. farauti No. 4, No. 5 and No. 6Bryan et al. (1990) studied variations at 14enzyme loci in the populations from PapuaNew Guinea and the colony material of An.farauti No. 1, No. 2 and No. 3. Thephenogram revealed nine clusters for whichmore than 20 per cent loci were fixed fordifferent alleles. The authors considered thatthere may be nine species in this complex,as so many fixed differences could not bemaintained within a population which sharesthe same gene pool.

Subsequently, Foley et al. (1993)analysed cellulose acetate gels of populationsfrom 19 localities in Papua New Guinea forelectrophoretic variations at 35 loci. Basedon fixed allele differences, six species wereidentified. Of these, An. punctulatus, An.koliensis and An. farauti No. 1 had alreadybeen reported; three were new species,which were provisionally designated as An.farauti No. 4, No. 5 and No. 6. Thepopulation designated as An. farauti No. 4from Gonoa produced sterile F1 progenywhen crossed with An. farauti No. 1, andasynapsis in polytene chromosomes of F1progeny (Bryan et al., 1990).


An. farauti No. 7 (Foley, Meek and Bryan,1994)The specimens collected either as larvae oradults from 33 localities in the Solomon Islandsand six localities in Vanuatu were analysed forelectrophoretic variations. The same enzymesystems that were used for the Papua NewGuinea population (Foley et al., 1993) wereused in this study, except that β-galactosidasewas added and the Ak-1, Ao, Fdp-1 and Mpiloci were excluded because of unreliablestaining. Enzyme analysis revealed the threealready identified sibling species, An. farautiNo. 1 and No. 2 and An. punctulatus, and apopulation which did not correspond with anyof the earlier identified species was found intwo localities on northern Guadalcanal,Solomon Islands. Fifteen specimens belongingto the new population were examined and allhad dark probosces as described for An. farautis. l. This group of mosuqitoes was consideredas a new species and was designated as An.farauti No. 7.

An. sp. near punctulatus (Foley, Cooperand Bryan, 1995)Within the specimens identifiedmorphologically as An. punctulatus, twogenetically distinct groups were found whenexamined for enzyme electrophoretic variationsand analysed for genetic distance. The newgroup differed from An. punctulatus populationswith 34-39 per cent fixed genetic differencesand Nei’s D ranging between 0.5 to 0.61. Themaximum genetic divergence recorded withinAn. punctulatus collected from widely separatedlocalities in Papua New Guinea was 18 per cent(Foley et al., 1993) and 6 per cent for specimenscollected in the Solomon Islands (Foley, Meekand Bryan, 1994). This was taken as evidenceto show that in the western province of PapuaNew Guinea there is a population conspecificwith An. punctulatus as described earlier and apopulation that was not described earlier, i.e. anew species designated as An. sp. nearpunctulatus.

Formal namesSchmidt et al. (2001) studied themorphological variations and identifiedmarkers for three species. An. farauti No. 1,No. 2 and No. 3 in the adult female, IV instarin larvae and pupae. On this basis formalnames were given:

An. farauti No. 1 (conspecific with An.farauti sensu stricto as described by Foley andBryan, 1993, and Foley, Meek and Bryan,1994) designated as An. farauti Laveran.

An. farauti No. 2 designated as An.hinesorum Schmidt sp. n.

An. farauti No. 3 designated as An.torresiensis Schmidt sp. n.


Crossing experimentsAn. punctulatus, An. koliensis and An. farautiNo. 1, No. 2, No. 3 and No. 4 producedprogeny in reduced numbers, and withreduced reproductive organs in reciprocalcrosses between the above-mentionedspecies (Bryan, 1973a and b; Mahon andMiethke, 1982; Foley et al., 1993).

Polytene chromosomesPolytene chromosomes from IV instar larvalsalivary glands were used for theidentifications, since polytene chromosomesfrom ovarian nurse cells were not readablein contrast to those in other anophelinespecies. Bryan and Coluzzi (1971) identifiedparacentric inversions diagnostic for An.farauti No. 1, No. 2 and No. 3.

Salinity tolerance Test (STT)An. farauti No. 1 showed a higher toleranceof salinity than An. farauti No. 2 and No. 3(Sweeney, 1987). Tests carried out with 0-3.4 per cent salinity showed completemortality of An. farauti No. 2 and An. farautiNo. 3 at 1.7 per cent. At the sameconcentration, An. farauti No. 1 from field


and Rabaul colony showed about 92 per centsurvival, while Port Moresby colony showed73 per cent survival. An. farauti No. 7 wasthought to be an obligatory fresh-waterspecies which would not tolerate salinity.However, Foley and Bryan (2000) found An.farauti No. 7 could survive the STT and thuscould not be efficiently differentiated fromAn. farauti s.s. which is a vector onGuadalcanal, Solomon Islands. The authors,therefore, recommend alternatives to the STTfor the vector species on Guadalcanal.

Biochemical taxonomy andelectrophoretic keysBryan et al. (1990) and Foley et al. (1993)analysed populations from Papua NewGuinea for electrophoretic variations anddeveloped electrophoretic keys. Foley andBryan (1993) give the keys for six species, An.farauti No. 1, No. 4, No. 5 and No. 6, An.punctulatus and An. kolinesis, from PapuaNew Guinea, and for An. farauti No. 2 andNo. 3 from Australia. Alternative keys aregiven depending on the availability ofdifferent species for standards.

Foley, Meek and Bryan (1994) presenteda key for distinguishing An. farauti s.s., An.farauti No. 2 and No. 7, and An. punctulatuswhich are prevalent in the Solomon Islandsand Vanuatu. These species can bedistinguished by staining for the enzyme LDHonly. The authors also discuss combinationsof standards and diagnostic enzymes requiredto identify the species.

Specimens of new species An. sp. nearpunctulatus were found homozygous for aunique allele at Pep D-1 (Foley, Cooper andBryan, 1995). The authors provisionallyreported this allele to be diagnostic for thisspecies based on the small number ofspecimens examined. Additional alleles thatdiscriminate An. punctulatus and An. sp. nearpunctulatus are at the Hk, Me-1 and Hbd IIloci.

DNA probesThree DNA probes, pAf1, pAf2 and pAf3,were selected by screening partial genomiclibraries with radio-labelled total genomicDNA from each species (Booth, Mahon andSriprakash, 1991). pAf1 hybridized with bothAn. farauti No. 1 and No. 2, pAf2 hybridizedstrongly to An. farauti No. 3, less to An. farautiNo. 1 and faintly to An. farauti No. 2,suggesting that there is cross-hybridization.Hartas et al. (1992) sequenced these probesand identified oligonucleotides of 25-26 basepairs specific for each probe. Radio-labelledoligonucleotides, oAf1, oAf2 and oAf2,hybridized respectively to An. farauti No. 1,No. 2 and No. 3. These oligonucleotideprobes identified both dot-blots and squash-blots of larvae, pupae and adults veryefficiently.

Cooper, Cooper and Burkot (1991)independently identified three DNA probesspecific for An. farauti No. 1, No. 2 and No.3. Total genomic libraries of the three specieswere screened by hybridizing withradiolabelled homologous and heterologoustotal genomic DNA of species. Three probes,1/1, 9/2 and 5/3, specific for An. farauti No.1, No. 2 and No. 3 respectively, wereidentified. Probes labelled with P32 or horse-radish peroxidase with enhancedchemiluminescence detection system orbiotinylated streptavidin-conjugated alkalinephosphatase with the chromogenic detectionsystem were all found equally sensitive inidentifying the species both in dot-blots andsquash-blots.

Beebe et al. (1994) made DNA probesfor the identification of five members of thePunctulatus Complex namely, An.punctulatus, An. koliensis and An. farauti No.4, No. 5 and No. 6 by differentially screeninggenomic libraries with P32-labelledhomologous and heterologous DNA. Thesizes of the probes that were selected rangedbetween 273 and 630 bp. The probesidentified specimens from DNA dot-blots and


squash-blots. These probes are sensitiveenough to identify fragments of mosquitoes.

Beebe et al. (1996) developed two DNAprobes specific for An. farauti No. 7 and An.sp. near punctulatus, by screening genomiclibraries. A probe which hybridizes to allmembers of the Punctulatus Complex has alsobeen developed. These probes have beenfield-tested.

All members of this complex can beidentified with DNA probes.

PCR-RFLPBeebe and Saul (1995) differentiated 10species, An. farauti No. 1 to No. 7, An.koliensis, An. punctulatus and An. sp nearpunctulatus, by restriction enzyme digestionof rDNA ITS2 product (750bp amplicon) withMsp-I.

rDNA-ITS2 sequence analysisBeebe et al. (1999) analysed sequencevariations in the rDNA ITS2 region among10 species of this complex. The length ofITS2 ranged from 549 to 565bp. Sequencevariation among the 10 species studiedranged from 2.3 per cent to 2.4 per centmainly due to indels of simple sequences.

Distribution and biological charactersInformation on the distribution of membersof this complex in Papua New Guina,Solomon Islands and Australia is avilable fromrecent investigations using allozymes (Bryan,Reardan and Spark, 1990; Foley et al., 1993and Foley, Meek and Bryan, 1994). From IrianJaya Islands of Indonesia, An. punctulatus, An.koliensis and An. farauti s.l. have beenreported (Kondrashin and Rashid, 1987), andAn. clowi (from an earlier report).

An. farauti No. 2 and No. 7 were foundto be zoophilic while An. farauti No.1 (nowAn. farauti Levaran) was highlyanthropophilic. Differences in anthropophilywere observed on north Guadalcanal in theSolomon Islands by Foley, Meek and Bryan

(1994). In Gonoa, Papua New Guinea, An.farauti No. 4 and An. koliensis were caughton opposum, pig and human bait, while An.punctulatus was caught on human and pigbaits (Foley et al., 1993). On human baitfour out of six An. farauti No. 4 were caughtbefore 2000 hours, suggesting early bitingactivity of this species. Now that elevenmembers have been identified in thiscomplex and techniques are available fortheir identification, studies should beundertaken to map their distribution and toestablish their role in malaria transmission.

3.11 The Sinensis ComplexAnopheles sinensis Wiedman, 1828 belongsto the subgenus Anopheles, the HyrcanusGroup in the Myzorhynchus Series (Harbach,2004). An. sinensis is an important vector ofmalaria throughout the Republic of Korea(Chow, 1970). This species is considered tobe the main vector responsible for malaria incentral China. Malaria in this area is unstableand outbreaks have regularly been reportedsince the 1950s. As recently as in 1995,Honan province in China experienced anoutbreak of P. vivax (Sleigh et al., 1998). Thisspecies is also considered to be a vector inJapan, Indonesia and Thailand (O’Connor1980, Kanda et al., 1981). However, inThailand, it is not considered an importantvector.


Sibling species in China— An. sinensisand An. lesteri syn. anthropophagusTwo morphological variants based on the sizeof the deck of the egg were reported in the1950s (cited in Reid, 1953). Later, it wasfound that the narrow deck egg variety wasmore anthropophilic, endophagic andendophilic than the wider deck variety whichfed outdoors and predominantly on cattle.Because the narrow deck egg varietyresembled An. lesteri, which also has a narrow


deck, it was initially considered as asubspecies of An. lesteri and was called An.lesteri anthropophagus. Later, Ma (1981) (asmentioned in Gao, Beebe and Cooper, 2004)raised anthropophagus to the species statusand designated it as An. anthropophagusbased on its morphology, distribution andvectorial capacity. The wider deck variety isdesignated as An. sinensis. An.anthropophagus and An. sinensis areisomorphic species at the adult stage.Wilkerson et al. (2003), however, reportedthat An. anthrophagus and An. lesteri areidentical in ITS2 sequences and are,therefore, synonymous. This was based onthe examination of An. lesteri from the typelocality in Laguna province from thePhillippines and An. anthropophagus fromJhangsu Province, China. Ma and Xu (2005)supported this conclusion and said that An.anthrophagus is a junior synonym of An.lesteri. Wilkerson et al. (2003) clarified thestatus of An. lesteri present in China by statingthat it is another unknown species and is alsofound in Korea. This is supported by Gao,Beebe and Cooper (2004) observation that itis different from An. anthrophagus and An.sinensis based on ITS2 sequences.

Sibling species in Japan — An. sinensis,An. engarensis and An. sineroidesSuccessful colonization of An. sinensis usingan induced copulation method devised byOguma and Kanda (1976) led to crossingstudies. Strains collected from Koniya,Kanoya, Karurizawa, Yomogita, Yakumo andEngaru ranging from north to south in Japanwere used in the crosses (Oguma, 1978). Intwo crosses, Engaru with Kanoyo and Engaruwith Yakumo, F1 males were sterile andfemales were fertile. In the other crosses (ofthe 36 possible crosses, 19 were carried out)where Engaru was not involved, the progenywere fertile. Therefore, it was concluded thatthe Engaru strain is distinct and was givenspecies status, while all the other strains wereconsidered to belong to one species, An.sinensis. The Engaru strain was also found to

be different physiologically from An. sinensis,in the mean frequencies of the claspermovements during induced copulation(Kanda and Oguma, 1978). Based on thisevidence the Engaru strain was considered adistinct sibling species, morphologicallysimilar to An. sinensis and designated as An.engarensis.

Kanda et al. (1981) reported from thepolytene chromosomal examination of about2000 specimens from Engaru, where both thespecies exist sympatrically, that noheterozygotes for 2RB inversion were found.The absence of heterozygotes furthersupported the view that there are two distinctspecies in the area, and also suggested thatthere is pre-mating isolation in addition tothe post-mating barrier already reported(Oguma, 1978).

An. sineroides Yamada 1924 has arestricted distribution, being found fromKokkido to Kyushu in Japan, in China and inKorea. An. sineroides and An. sinensis aremorphologically similar, hence hybridizationexperiments were conducted to establish thetaxonomic status (Kanda and Oguma, 1977).In crosses between the strains of An. sinensisand An. sineroides collected from Japan,heavy mortality in F1 larvae and pupae, andasynapsis in polytene chromosomes of F1progeny were observed. Based on theseresults, the authors concluded that An.sinensis and An. sineroides are two distinctsibling species. The X-chromosome of An.sineroides was found similar to that of An.koreicus rather than to that of An. sinensis(Oguma and Kanda, 1970).

Cytotypes of An. sinensis in Thailand andRepublic of KoreaTwo forms designated as form A and form Bwere identified within An. sinensis, based onstructural difference in the Y-chromosome(Baimai, Rattanarithikul and Kijchalao, 1993).Form A has a telocentric or acrocentric Y-chromosome (short arm being very small) andform B has a sub-metacentric Y-chromosome.


Choochote et al. (1998) carried out crossingexperiments between form A and form Busing induced copulation. In all crosses viableprogeny were observed and no geneticincompatibility was observed in crosses withthe F1 progeny. Polytene chromosomes of4th instar larvae of F1 progeny showedcomplete synapsis. In another study (Min etal., 2002), form A from Thailand was crossedwith form B isolines from Thailand and Korea.In the reciprocal crosses, and correspondingback crosses, no genetic incompatibility wasobserved affecting hatchability, viability ofimmature stages, sex ratio or adultemergence. Thus, both cytotypes, form A andform B, were considered as intra-specific Y-chromosome variations within An. sinensis.This was further supported from sequenceanalysis of ITS2 and COII regions of form Aand B. The sequences were near identicalwith a sequence variability of 0.0-0.6% (Minet al., 2002).

Thus, it appears that An. sinensis is acomplex of at least four species, An. sinensiss.s., An. lesteri (syn. anthropophagus), An.sineroides and An. engarensis.

Techniques for identification ofsibling species

Morphological variationsAt the adult stage all four species, An. sinensis,An. engarensis, An. sineroides and An. lesterisyn. anthropophagus, are isomorphic. Thesize of the deck of the egg varies betweenthe two species, i.e. it is narrow in An. lesterisyn. anthropophagus and is wider in An.sinensis. An. lesteri (an earlier designationand needs formal redesignation followingWilkerson et al., 2003) also has a narrow deckand thus resembles An.lesteri syn.anthropophagus. Both An. lesteri and An.yatsushiroensis (another species in theHyrcanus Complex which is sympatric withthese species) can be distinguishedmorphologically at the adult stage from An.sinensis and An. lesteri syn. anthropophagus.

Molecular methodsRestriction fragment length differences ofgenomic repetitive DNA (Li et al., 1991 fromAbstract)

High molecular weight DNA of five species,An. sinensis, An. lesteri syn. anthropophagus,An. liangshanensis, An. crawfordi and An.xiaokuanus, belonging to the HyrcanusGroup, was cut with three restrictionendonucleases. All three enzymes, Bgl II, HaeIII and Pst I, produced diagnostic fragmentsdiscernable on agarose gel for all species.Thus, all five species could be distinguishedby this method.

An allele-specific PCR assay (Ma Qu and Xu,1998 cited in Gao, Beebe and Cooper, 2004)

This assay was developed from ribosomalDNA to differentiate An. sinensis from An.lesteri syn. anthropophagus. With this assayonly 78.9% of field-collected samples couldbe identified, i.e. many remainedunidentified because of failure to producePCR products. According to the authors, thiswas because unidentified samples belongedto another yet unknown cryptic species.Another possible reason as pointed out byGao, Beebe and Cooper (2004) could be dueto intra-specific sequence variation within theprimer region which inhibited annealing.

PCR-RFLP assay (Gao, Beebe and Cooper, 2004)

Universal primers from the rDNA ITS2 regionwere used to amplify, and the amplicons weredigested with Hind I and Rsa I restrictionenzymes. After digestion the patterns onacrylamide gel were distinct with both theenzymes. To validate the assay, field-collectedspecimens from seven areas in China and An.sinensis specimens from an establishedlaboratory colony were used. All thespecimens identified morphologically basedon egg deck size as An.lesteri syn.anthropophagus were also identified by thePCR as the same species except for 4 of 8specimens from Guangdong province whichwere identified as An. sinensis. The sequenceanalysis also confirmed that these specimens


were An. sinensis. An. sinensis from Liaoningprovince mostly had narrow decks typical ofAn.lesteri syn. anthropophagus, with a fewwith wide decks typical of An. sinensis. Butall the specimens were identified as An. lesterisyn. anthropophagus by molecular assay. Thissuggests that the egg deck size is not reliableto distinguish these two species as it wasthought earlier.

Distribution and biological charactersAn. sinensis forms, A (XY1) and B (XY2),identified in Thailand by Baimai,Rattanaritikul and Kijchalao (1993) were alsoreported to be found in Taiwan (China) andChina. Recently, Ma and Xu (2005), basedon ITS2 sequences reported that in Chinaonly B form haplotype was found.Throughout the Republic of Korea, An.sinensis has been considered for a long timeto be an important vector. Recently, Colemanet al. (2002) collected anopheline mosquitoesfrom 29 locations distributed throughout thecountry. Three species, An. sinensis/lesteri (asthese two species cannot be identifiedaccurately, no effort was made to separatethem), An. yatsushiroensis Miyazaki and An.sineroides Yamada, were found. 89.7% wereAn. sinensis/lesteri, and of these two pools,one of 10, and another of nine (screened byELISA) were found positive for P. vivax (CSP247).

For An. sinensis, the anthropophilic index(AI) and average probability of daily survivalrate respectively were 0.7 per cent and 0.89in the Guonggi–do (Ree et al., 2001) and 0.8per cent and 0.79 per cent in the Paju (Leeet al., 2001) areas of the Republic of Korea.In both publications the investigators suggestthat in spite of their tendency to zoophily andwith moderate survival rate, this species isresponsible for malaria transmission becauseof high population densities. In Thailand, An.sinensis is not considered an important vector.In a laboratory-feeding experiment, twocytologically different An. sinensis strains (formA-XY1, and form B-XY2) were found to betotally refractory to P. falciparum with none

developing oocysts or sporozoites while bothexhibited low susceptibility to P. vivax with0.00– 0.571 per cent and 0.00-.588 per centoocyst and sporozoite rates respectively. Boththe strains were refractory to P. yoelii whenfed directly on infected rodents (Rongstrriyamet al., 1998).

Malaria is endemic in central China butit is unstable and with regular occurrence ofoutbreaks (Li et al., 1995). Gao, Beebe andCooper (2004) report An.lesteri syn.anthropophagus to be widespreadthroughout central China and it occurssympatrically with An. sinensis. However,they report that the distribution of An.lesterisyn. anthropophagus is patchy compared toAn. sinensis. An. sinensis is predominantly acattle-feeder in China. Throughout centralChina, An.lesteri syn. anthropophagus isconsidered a primary vector and An. sinensisa secondary vector. Molecular identifications(Gao, Beebe and Cooper, 2004) haveextended the distribution of An. lesteri syn.anthropophagus northward by 1200 km (420N, 1200 E). This region is north of the Koreanpeninsula. A small sample examined usingmolecular techniques by Ma et al. (2000)cited in Gao, Beebe and Cooper (2004)identified only An. sinensis, An. lesteri andAn. yatsushiroensis. Along the North/SouthKorea border, Stricman et al. (2000) reportedthat, based on morphological identifications,An. sinensis was the most abundant mosquitobiting humans, constituting more than 80 percent of the anopheline human-landingcollections. These mosquitoes were collectedduring an outbreak of P. vivax malaria. Nowthat a molecular assay is available (Gao,Beebe and Cooper, 2004) for accurateidentification of An.lesteri syn.anthropophagus and An. sinensis, largesamples from the Republic of Korea are tobe identified to establish whether only An.sinensis is prevalent as is being reported. Thismay resolve the anomalies reported in thefeeding behaviour of the mosquito hithertodescribed as An. sinensis.


3.12 The Subpictus ComplexAnopheles subpictus Grassi belongs to thesubgenus Cellia, and the Pyretophorus Series(Harbach, 2004). An. subpictus is very widelydistributed and found in abundance in theOriental region. It is found to the west ofIndia in Pakistan, Afghanistan, and Iran, andin the east as far as Papua New Guinea andthe Marina island, Malaysia. It is also foundin Sri Lanka in the south and China in thenorth. In India, it is found throughout themainland and in Lakshadweep islands but notin the Andaman and Nicobar islands (Rao,1984).

In Indonesia, it is considered as asecondary vector (Van Hell, 1952;Sundararaman, Soeroto and Siran, 1957). InIndia, sporozoite-positive specimens werereported from a coastal village in the state ofTamil Nadu (Panicker et al., 1981) and in thedistrict of Bastar in Madhya Pradesh (Kulkarni,1983). Amerasinghe et al. (1991; 1992)reported this species to be a vector in SriLanka.

Based on the morphological differencesin the eggs of An. subpictus, Reid (1966)suggested that this might be a speciescomplex, and Suguna (1982) for the first timeprovided clear evidence that this taxon is acomplex of two sibling species, A and B. Later,this taxon was found to be a complex of foursibling species, A, B, C and D (Suguna et al.,1994).

Evidence for recognition of siblingspeciesDifferences in egg morphology and in thebanding pattern of polytene X-chromosomedue to a paracentric inversion were the basisfor the identification of species A and B.Species A with the X+a karyotype (standardarrangement) was predominant in inlandvillages while in the coastal villages species Bwith the Xa inversion arrangement was found

together with species A in the Union Territoryof Pondicherry in India (Suguna,1982).

The four sibling species, provisionallydesignated as A, B, C and D, were identifiedby examining polytene chromosomes fromthe ovaries of adult females collected fromthe field and those from the salivary glandsof larvae collected from breeding sites. Twoinversions on the X-chromosome, a, a smallinversion towards the tip of the chromosome,and b, an inversion in the middle of thechromosome (this same inversion wasdesignated as a in the earlier publication ofSuguna, 1982), and their combinations+a+b, ab, a+b and +ab were found with atotal absence of heterozygotes in the naturalpopulations examined. This was taken as anevidence for the recognition of four species(Suguna et al., 1994).

Techniques available foridentification of sibling speciesInversions on polytene X-chromosome arediagnostic characters in the identification ofsibling species. The four inversionarrangements observed were: species A –X+a+b, species B – Xab, species C – Xa+band species D – X+ab. A photomap ofpolytene chromosomes complemented withbreak-points marked for inversions a and bare given in Figure 13.

Reuben and Suguna (1983) reportedmorphological differences in eggs, larvae,pupae and adults between sibling species Aand B. Now that species C and D have alsobeen reported within the fresh water-breeding populations in Pondicherry (Sugunaet al., 1994), the differences reported bySuguna (1982), and Reuben and Suguna(1983) between species A and B, are notdiscussed here as they are no longer valid forthe identification of all the sibling species.

Differences in egg, larval, pupal and adultmorphological characters among the fourspecies were also observed (Suguna et al.,1994) and these are summarized in Table 10.


Distribution and biological charactersAn. subpictus population from villages aroundDelhi, India, were identified as species A(Subbarao, Vasantha and Sharma, 1988) byexamining the ovarian polytenechromosomes of adult females collected fromthe field, following the report of Suguna(1982). The ridge number on the egg floatranged between 21 to 30, and this did notcorrespond with the characters of species Awhich has, on an average, 33 ridges (Suguna,1982), but it corresponds to those of species

C of Suguna et al. (1994). Atrie (1994)reported that in the Delhi population, theapical pale band in adult females was longerthan the sub-apical dark band, which is thecharacteristic feature of species A (Table 10).Thus, it was felt that there was a need for adetailed study to correctly identify the specifictaxonomic status of the Delhi population.Recently, Singh et al. (2004) conducted adetailed study on An. subpictus collected fromSonepat district, Haryana (a stateneighbouring Delhi). In this study, field-collected adult females, and from their

Figure 13: Polytene chromosome complement of An. subpictus species A. Break-points of inversions, a and b, reportedby Suguna et al. (1994) are shown on the X-chromosome. (Source: Subbarao, Vasantha and Sharma, 1988)


isofemale lines, eggs, larvae, pupae andadults, were examined for morphologicalcharacters following Suguna et al. (1994).Species A, C and D were found in almostequal proportions, and no variation wasobserved in the proportion of the three siblingspecies from field-collected adults andisofemale lines. An. subpictus breeding inthe villages was found in the river-bed pools.Thus, in northern India, all three fresh water-breeding species are sympatric. In Sri Lanka,both species A and B were identified(Abhayawardana, Wijesuriya and Dilrukshi,1996) based on species-specific diagnosticinversion genotypes reported by Suguna(1982). In Sri Lanka, species A was found tobe more endophilic and seasonally moreabundant than species B (Abhayawardana,Wijesuriya and Dilrukshi, 1996).

No studies have been reported so far onthe biological characteristics of these foursibling species in India. Detection ofsporozoite-positive specimens in coastal

villages of Pondicherry by Panicker et al.(1981) suggests that species B may be a vector.Data of Kulkarni (1983) in Bastar district,Madhya Pradesh state in India, andAmerasinghe et al. (1991 and 1992) in anirrigation development area of the Mahaweliproject in Sri Lanka suggested that fresh water-breeding sibling species may also be playinga role in malaria transmission. Fresh water-breeding An. subpictus from Delhi, when fedon Plasmodium vivax-infected blooddeveloped oocysts and sporozoites (Nandaet al., 1987). This suggests that fresh waterbreeding An. subpictus is geneticallysusceptible to plasmodial infection. Generally,the densities of this species are relatively high.The species may not be playing a role inmalaria transmission probably due to highzoophagy and poor longevity, which arerelevant for the species to be a vector in thefield.

Resistance to malathion and fenitrothionwas observed in An. subpictus predominant

Table 10: Morphological, biological and cytological differences among An. subpictus sibling species(Source: Suguna et al. (1994)

Egg Larva Pupa Adult Breeding habitats

Species Inversionson X-

chromo-some

Meanridge No.(range)

Frill Seta 4M Seta 7-1 Length ofapical pale

band onfemalepalpi

(Salinity range %)

A +a+b 35(31-36)

Opaque 2-branched(rarely 3)

Simple; aslong as

hairs 6&9

Longer thansub apical dark

band

Paddy fields(0.0054-0.2636),

Riverine pools(0.0247-0.7827) and

Back waters(0.5574-5.3554)

B ab 18(16-20)

Transp-arent

2-branched(rarely 3)

Branched4-5;

shorterthan hairs

6&9

Shorter thansub apical dark

band

Back waters(0.5574-5.3554)

C a+b 27(25-29)

Semi-transpa-

rent

3-branched(rarely 2)

Branched -2; shorter,but longerthan in sp.

B

D +ab 22(21-24)

Semi-transpa-

rent

3-branched Branched -3; shorter

Equal to subapical dark

band

Same as A


in inland regions, while in coastal areasresistance to permethrin was observed in thisspecies (Kelley-Hope et al., 2005).Abhayawardana, Wijesuriya and Dilrukshi(1996) reported species A’s predominance ininland regions and that of species B in coastalareas. Following this, Kelley-Hope et al.(2005) suggested that different resistantmechanisms may prevail in the two siblingspecies, with species A being more resistantto organophosphates and species B topyrethroids, with little evidence ofintrogression between the two sibling species.

Now that four sibling species areidentified, the distribution of the siblingspecies should be mapped in all the areaswherever An. subpictus is prevalent and isconsidered a vector, as in Indonesia and SriLanka, and the biological characters of thesibling species should be studied.

3.13 The Sundaicus ComplexAnopheles sundaicus belongs to the subgenusCellia and the Ludlowae Group in thePyretophorus Series (Harbach, 2004). An.sundaicus is widely distributed in the Orientalregion. The distribution extends from India,and eastward to China through Bangladesh,Myanmar, Viet Nam, Cambodia, Thailand,Malaysia, Singapore, Philippines andIndonesia (Rao, 1984). In India it has nowdisappeared from the mainland (Rao, 1984;Dash et al., 2000), except for a small focus inthe Kutch area of Gujarat state (Singh, Nagpaland Sharma, 1985) and is now foundabundantly and widely in the Andaman andNicobar islands (Rao, 1984). This species isconsidered an important vector throughoutthe areas of its distribution.

An. sundaicus is generally a salt-waterbreeder. The major breeding places areswamps and pits along bunds (embankments)containing brackish water (Sundararaman,Soeroto and Siran, 1957). Rao (1984) reportsthat the most suitable breeding places are

those in which sea water and fresh watermingle, but it was also found adapted to freshwater in some areas. In Vishakapatnam,Andhra Pradesh state, India, it was foundexclusively in fresh water-breeding placessuch as tanks, ponds and borrow pits. In VietNam and Indonesia, shrimp/fish ponds foundalong the coast or those irrigated by inlandsea water were reported as favourablebreeding sites for this species (Collins et al.,1979).

Its populations were found both indoorsand outdoors in Malaysia (Reid, 1968), andin Car Nicobar, Andaman and NicobarIslands, India (Kumari and Sharma, 1994). InIndonesia, in brackish water-breeding areas,this species is exophilic except in KampundLaut, Cilacap, where it is endophilic(Kirnowardoyo and Gambiro, 1987). Thepreference to feed on man varies in thisspecies. In Vishakapatnam area, India, theanthropophilic index was found to be 5.6 percent (Rao, 1984). In Car Nicobar Island(Andaman and Nicobar islands), India, thisspecies was found to be predominantlyzoophagic, with an anthropophilic index of2.5 per cent, but in human dwellings it was18 per cent (Kumari et al., 1993). However,in Indonesia, it is primarily anthropophagicexcept in some places like Purworejo (CentralJava) and Kulon Progo (Yogyakarta), wherethe human blood index was only 1.3 per cent(Kirnowardoyo, 1988). Because of suchdistinct differences in the populations of An.sundaicus, this species has long beensuspected to be a species complex (Kalra,1978; Rao, 1984).


Species A, B and C (Sukowati et al., 1999)Sukowati and Baimai (1996), for the first time,reported three cytological forms designatedas A, B and C differing in their polytenechromosome complement and withheterochromatic variation in their Y-chromosomes. The three cytological forms


were identified from the examination ofnatural populations from different areas inThailand and in Java and Sumatra, Indonesia.The differences observed between the threeforms are summarized in Table 11. A standardphotomap of the ovarian polytenechromosomes with a detailed description ofeach chromosome is given by Sukowati andBaimai (1996).

In both Trat and Phangnga, two areassurveyed in southern Thailand, only the Aform was found. In Asahan, in northernSumatra, all three forms were found while insouth Tapanali, also in northern Sumatra, onlythe A and B forms were found in the twosurveys conducted. In Lampung, situated inthe most southern part of Sumatra, and insouth Banten, Panganadaran and Cilacap inJava, only the A form was observed. In asingle survey conducted in Purworejo in Java,both A and B were found with apredominance of the A form (90 per cent).

In spite of the fact that the cytologicalforms were found to be sympatric, the authorscould not give them the species statusbecause the forms were identified on the basisof Y-chromosome variation, and differencesin the polytene chromosomes were not asclear- cut as when there are inversions, forwhich any heterozygotes are readily available.

Sukowati et al. (1999) collected An.sundaicus from bovine and human baits fromsix localities and the cytological identificationrevealed A, B and C. Other Indonesian siteswith the forms found were as follows:Purowerojo (forms A and B) and Cilacap (formA) in Central Java; Panganduari (form A) inwest Java; Lampung (form A) in south Sumatraand Asahan (forms A, B and C), and southTapanuli (form B) in north Sumatra. An.sundaicus breeds in fresh water in southTapanuli while it breeds in brackish water inthe other five localities. F1 progenycytologically identified for forms A, B and Cwere analysed for 11 enzyme systems. Datawere analysed by BIOSYS-1 (release 1.7University of Illinois programme). Genetic

variability in terms of mean heterozygosity,which ranged between 0.101 and 0.179, andpolymorphism between 0.267 and 0.533,were compared with those observed for otherspecies complexes. No unique electromorphswere found fixed in any of the threecytological forms studied in thesepopulations, but frequency differences andWright’s F statistics for the Mpi enzymedistinguished the three cytological forms witha high degree of probability. The data fromthe Mpi locus strongly indicated a lack ofrandom mating. Therefore, the threecytological forms were considered as threeisomorphic species and the Sundaicus as aspecies complex.

A new cytotype (Nanda et al., 2004)Nanda et al. (2004) reported a new cytotype,D, from the Andaman and Nicobar Islands,India. In the polytene chromosomescomplement, the X-chromosome resembledthat of species A (Xa type), while thechromosome 2 had a prominent looselydiffuse area in Zone 19 proximal to thecentromeric regions (2RB type) as in speciesC (Sukowati and Baimai, 1996). Thus, thesespecimens did not completely resembleeither species A or species C. In the mitotickaryotype, both the X and Y chromosomeswere telocentric and the Y-chromosome hadtwo heterochromatic blocks as in species Aand C (Table 12). The new cytotype couldnot be given a new species status, as it wasnot found sympatric with any of the otherforms.

Evidence for another sibling species, An.sundaicus s. s. (Dusfour et al., 2004)An. sundaicus specimens collected from twosites, Lundu and Miri in Sarawak, Malaysia,two in Thailand and two in southern Viet Namwere examined for differences, if any, in thesequences of partial regions of cytochrome band cytochrome oxidase I of themitochondrial DNA (Dusfour et al., 2004).The specimens from Thailand and Vietnam


Table 11: Cytological descriptions of forms A, B, C and D in An. sundaicus

Source: Sukowati and Baimai, 1996; Nanda et al., 2004.* Differences between the Xa and Xb type of chromosomes are slight. The Xb type differs from the standard arrangement

at the free end of the X-chromosome. Xa at the tip has four diffuse bands while Xb has two prominent dark bandsat the tip.

** The 2 refers to the new arm designation for chromosome 2R. In this paper the authors gave both types ofdesignations to the arms. 2Rb differs from 2Ra in having a loosely diffuse area ni zone 19 (this zone covers thecentromeric region).

# It should be noted that the designations "a" and "b" used here are not inversion designations, but have been used toindicate different types.Species C exhibits prominent pericentric heterochromatin for all chromosome arms compared with those of formsA and B. An additional block of heterochromatin is observed near the centromeric region of chromosome 2.Sukowati and Baimai(1996) consider that the diffuse heterochromatin observed in zone 19 of polytene chromosome2R in this form may be because of this extra block of heterochromatin.

were found distinctly different from the onescollected from Malaysia, and the geneticdivergence and cladistic analysis relationshipestablished that they represent two siblingspecies: An. sundaicus s. s. from Malaysia andAn. sundaicus species A from Viet Nam andThailand.

Formal designation of An. sundaicus fromMalaysia and of species ADusfour et al. (2004) designated specimensfrom the Sarawak region as An. sundaicus s.s., because Linton et al. (2001) reported An.sundaicus from Lundu province as neotypeof this species (as no type specimens of thisspecies exist). Linton et al. (2005) designatedspecies A as An. epiroticus Linton & Harbachbased on ITS2, cytochrome b andcytochrome oxidase I sequences. Linton etal. (2005) also reported that nomorphological characters were observedwhich could be used as diagnostic charactersat adult, larval and pupal stages between An.sundaicus s. s. and An. epiroticus.


Mitotic karyotypesThe three cytological forms have now beengiven species status, and cytologicaltechniques can be used to identify these threespecies. Species B can be differentiated fromspecies A and C by examining the Y-chromosome in mitotic karyotypes. Asspecies A and C have similar Y-chromosomes,and if it is clearly known that only A and B oronly B and C exist in a given area, observationof the Y-chromosome can give valididentifications.

Polytene chromosomesAll three species and new cytotype D can bedifferentiated by examining the X andchromosome 2 in the polytene chromosomecomplements (Sukowati and Baimai, 1996).The differences observed in the Y-chromosomein the mitotic karyotype and the X-chromosomeand 2 (2R) are given in Table 11.

Polytene chromosomesCytological forms/Species X* 2R/2**

MitoticY-chromosome

Species A Xa#

(standardarrangement)

2Ra#

(standardarrangement)

Y1

Telocentric with 2heterochromatin blocks

Species B Xb# 2Ra# Y2

Telocentric but longer thanY1 and has three hetero-

chromatic blocks

Species C� Xb# 2Rb# Y1

Cytotype D Xa# 2Rb# Y1


Electrophoretic variationsVariations at the Mpi locus could be used todistinguish the three species (Sukowati et al.,1999)

Multiplex PCR assayDusfour et al. (2004) developed a multiplexPCR assay consisting of four secies specificSCAR (sequence characterized amplifiedregion) primers derived from randomamplified polymorphic region. This assaydifferentiated An. sundaicus s. s. fromMalaysia and species A from Thailand andViet Nam. Species A specimens used in theassay were neither cytogically norelectrophoretically identified, but were fromthe areas where, in earlier studies, onlyspecies A was found. The assay needs to bevalidated with other sibling species in thecomplex.

Distribution and biological charactersIn southern Thailand only species A wasfound, and in Sumatra, Indonesia, all threespecies, A, B and C, were found in thenorthern part, but in the southern part therewere only species A and B. Cytotype D wasfound in laboratory colonies established fromlarvae collected from brackish and freshwater-breeding sites found in Andaman andNicobar Islands (Nanda et al., 2004). Salinityin the breeding sites in these islands was ashigh as 14 gm/l (Das et al., 1998 and Sharmaet al., 1999). Recently, Alam et al. (2005),based on D3 and ITS2 sequnce analysis, alsoconcluded that An. sundaicus breeding infresh water and brackish water sites isidentical. The three cytological forms reportedby Sukowati and Baimai (1996) were alsofound in both brackish and fresh water-breeding sites. Similarly, Dusfour et al. (2004)found An. sundaicus s. s. in fresh water andsaline water breeding sites in Malaysia. Thus,breeding in both fresh water and saline waterdoes not appear to be a characterstic of anyone sibling species in the complex. In CarNicobar (Andaman and Nicobar Islands,India) where only cytotype D was found, An.

sundaicus was predominantly zoophagic(Kumari et al., 1993). Cytotype D was foundin indoor collections from human dwellingsand cattle sheds and also in outdoorcollections. It may be noted that An.sundaicus is the sole malaria vector in theAndaman and Nicobar islands. Though onlycytotype D was found in this study (Nanda etal., 2004), the authors did not rule out thepossibility of another species existing in theseislands. Alam et al. (2005), however, foundno differences in the D3 and ITS2 sequencesof the specimens collected from four isolatedislands of the Andaman and Nicobar groupof islands. Dusfour, Harbach and Manguin(2004) presented a consolidated account ofthe bionomics of An. sundaicus s. l. and ofthe sibling species identified across the rangeof their distribution in the oriental region. An.epiroticus was collected resting inside humanhabitations in Viet Nam (Linton et al., 2005).

This is an important species complexresponsible for the transmission of malaria inSouth-East Asian countries, hence, thebionomics of all the members of the complexneeds to be studied in detail to plan effectivecontrol strategies. Now that there are differenttechniques, mitotic and polytenechromosomal differences, electrophoreticvariation, and PCR assay for the identificationof sibling species, these techniques should bevalidated for their specificity and sensitivitythroughout the range of An. sundaicusdistribution. This is necessary to establishsibling species distribution and their hostfeeding preferences, vectorial potential andresponses to different control measures indifferent geographical regions.

3.14 The Anophelesstephensi variants

Anopheles stephensi belongs to the subgenusCellia and the Neocellia Series (Harbach,2004). This species is found in all mainlandzones of India but is rarely found at highaltitudes in the Himalayas (Rao, 1984). The


distribution of An. stephensi extends beyondthe west of India to Pakistan, Afghanistan,Iran, Iraq, Bahrain, Oman and Saudi Arabia,and in the east to Bangladesh, south Chinaand Myanmar. It is not reported in the northof India in Nepal nor in the south in Sri Lanka.

This species is considered an importantvector of malaria in Iran, Pakistan and India.Extensive studies have been carried out onthis species, but so far there is no evidencewhich indicates that this is a species complex.Sweet and Rao (1937) identified twopopulations, type form and variety mysorensisdiffering in egg morphometrics, whichinhabited urban and rural areas respectively.These populations were designated as racesby Sweet, Rao and Subba Rao (1938), and assubspecies by Puri (1949). Stone, Knight andStarke (1959) accepted them as subspeciesand included them in the catalogue ofmosquitoes. Rutledge, Ward and Bickley(1970) found the two forms sympatric andconsidered them as variants and notsubspecies. Subbarao et al. (1987) reportedan additional type, intermediate, andconsidered all three as “ecological variants”.In addition to egg morphometrics, studies oninversion polymorphism (Mahmood andSakai, 1984; Subbarao, 1996) have alsoshown rural and urban populations to bedistinct..

Evidence for considering/notconsidering urban and ruralpopulations as distinct species

Egg morphometricsSweet and Rao (1937) reported two races,An. stephensi type form and An. stephensi

variety mysorensis, on the basis of differencesin the egg width and length and number ofridges on the egg float.

Subbarao et al. (1987) studied severallaboratory colonies established from urbanand rural populations of An. stephensi forvariation in ridge numbers on egg float. Inaddition to typical An. stephensi and var.mysorensis strains, some strains were foundto have egg float ridge numbers fallingbetween type form and var. mysorensisvalues. The three categories of egg ridgenumbers were : 14 - 22, 12 - 17 and 9 -15.The mode number of ridges among the eggslaid by individual females in these stocks were16 - 19, 13 - 16 and 10 - 14 respectively.The categories with the highest ridge numbercorresponded with the type form, and thelowest with var. mysorensis of Rao, Sweet andSubba Rao (1938), and the new category withintermediate numbers was designated as“intermediate”. The authors reported thatareas where only mysorensis and“intermediate” were found were the typicalrural villages and peri-urban localities. In theheart of Delhi, in two rural areas surroundedby high-income urban residential colonies,all three forms were observed and the typeform was in the majority. The authors referredto these areas as semi-urban localities.Subbarao et al. (1987) referred to the threeforms as ‘ecological variants’.

Spiracular indicesMosquitoes have two pairs of spiracles on thethorax and six pairs on the abdomen. Thesize of the spiracle is a key factor in theadaptation of mosquitoes to different climatic

Egg Characters Type form Var. mysorensis

Length in microns + SD 555+24 476+24

Width in microns + SD 204+26 126+12

Length of float in microns + SD 294+23 218+20

No. ridges on float + SD 18+1.5 13+1.2

Table 12: Egg morphometrics in An. stephensi ecological races

Source : Sweet and Rao (1937) , SD- standard deviation


stresses. Vinogradskaya (1969) demonstratedthat the anterior thoracic spiracular index canbe used as a morphological indicator todistinguish xerophilic, mesophilic andhygrophilic mosquitoes. Nagpal et al. (2003)examined the spiracular index of type formand var. mysorensis collected from Jodhpurdistrict, Rajasthan state, India. In type form,the average spiracle length was 0.11 - 0.12mm and the average spiracle index was 8.09– 9.23, whereas in mysorensis, thecorresponding figures were 0.09 - 0.1 mmand 6.82 - 7.60. These parameters showedconsistent differences between thepopulations of mosquitoes which emergedduring the monsoon and those in the summerseasons. The spiracular indices correlatedsignificantly with the number of ridges on theegg float. Thus, using this index the twoecological races can be identified at the adultstage.

Crossing experimentsSweet, Rao and Subba Rao (1938) reportedfailure of egg-laying by a majority of thefemales in reciprocal crosses between typeform and var. mysorensis, and of the eggs laid,a majority failed to hatch.

Rutledge, Ward and Bickley (1970)observed varied levels of fertility in reciprocalcrosses between three strains established fromIran, Iraq and India. It is not known whetherthese colonies were established from rural orurban populations. The characters exhibitedby these colonies with reference to eggmorphology were of mixed type. Theyresembled mysorensis with respect to egglength but resembled the type form in eggwidth and egg ridge number.

Subbarao et al. (1987) made reciprocalcrosses and back crosses between type formand mysorensis colonies established,respectively, from urban and rural areas inIndia. Only the ridge numbers on the eggfloat were studied in these crosses. Thecrosses were fertile and the F1 hybrid progenywere also fully fertile, indicating no post-

copulatory barriers between the populations.A detailed genetic analysis of the ‘ridgenumber’ character was carried out followingthe likelihood method of Curtis, Curtis andBarton (1985). The results indicated that thevariation in ridge numbers is controlled bymore than one genetic factor. The authorsexplained that intermediate-type strainsprobably have an intermediate number ofpolygenes, and the strains maintainintermediate-type ridge numbers in thelaboratory strains because of the absence ofany selection pressure.

Polytene chromosomes and inversionpolymorphismA photomap of polytene chromosomes of An.stephensi is presented by Mahmood andSakai (1985). In this species, six autosomalinversions were reported by Coluzzi, Di Decoand Cancrini (1973a) and 16 polymorphicinversions (12 were new inversions) werereported in Pakistan populations byMahmood and Sakai (1984). They observed13 inversions in urban populations and, incontrast, only three in rural populations; thethree inversions observed in rural areas werenot found in urban localities. Furthermore,the number of females observed withinversions was very low in rural areas (4/225in Kasur district and 4/168 in Lahore district).In Karachi city, 28 females out of 40 examinedwere found with inversions. Similarly in India,10 inversions were observed in urbanpopulations of An. stephensi, while in ruralpopulations only the b inversion onchromosome arm 2 in a low frequency andanother inversion h1 were observed in theheterozygous form in one specimen(Subbarao, 1996). The h1 inversion was notobserved in urban populations. Break-pointsof inversions reported by Coluzzi, Di Decoand Cancrini (1973a) and Mahmood andSakai (1984), and of those observed in Delhi,India, are shown on photomaps of polytenechromosomes by Subbarao (1996).

Though rural and urban populationswere found to be distinct with reference to


inversion polymorphism, no pre-copulatorybarriers have been observed so far. Onecannot, however, rule out the possibility ofthe existence of homosequential specieswithin this taxon.

Y-chromosome variationRishikesh (1959) and Aslamkhan (1973)reported the mitotic karyotype of An.stephensi. The two autosomal pairs and theX-chromosome are metacentric, and the Y-chromosome is submetacentric. Suguna(1992) reported two types of Y-chromosomes,one submetacentric (as reported by earlierworkers) and a new metacentric type fromtwo urban localities in Tamil Nadu, India. InCuddalore, 91.4 per cent of single femalecultures examined had submetacentric (Y2)

Y-chromosomes and the remainder were ofthe metacentric type (Y1). However, inPondicherry, Y1 was found in 31.9 per centand Y2 in 68.1 per cent of the An. stephensimale population.

Distribution and biological charactersIn Pakistan, both the forms were reported,and in India, three forms were seen, includingthe new “intermediate” form (Subbarao etal., 1987). Senior-White (1940) reportedboth type form and mysorensis from Kolkata,India. Subbarao et al. (1987) did not findtype form in rural localities but in semi-urbanlocalities they found all three forms, with amajority of type form. These authors did notsurvey typical urban populations.

f1

c

d

a.b

d

c

a

g1

h1

g1

h1

b

a

g .d1

i1

d1

b1

m1

h1

b

a

i1

c1

f1

e1.g

1

c.c1

c.b1

d h1. 1

b

q1

o1

m .n1 1 1

.r

q1

n1

r1

o1

q1

p1

c

c

q

q

f1

e1.f

1

d

p1

X 2 3 4 5

Figure 14: Photomap of An. stephensi polytene chromosomes showing the break-points of the inversions. Inversionsincluded are from Coluzzi et al. (1973a), Mahmood and Sakai (1984) and inversions observed in Delhi populations

(Subbarao, 1996). Re-naming of inversions compared with the names used by Mahmood and Sakai and Coluzzi et al., are as follows:inversions on chromosome 2 (2R)- e as f1, and f as g1; inversions on chromosome arm 5 (2L), d as q1; inversions on chromosome arm 4(3R)- a as m1, b as n1 and c as o1, and inversions on chromosome arm 3(3L), d as b1, e as c1, f as d1, g as e1, h as f1, i as g1 and j as h1

(Source : Subbarao, 1996). Subbarao (1996) renamed the inversions identified by the earlier workers to have an unified inversiondesignations for species in the Neocellia Series as proposed by Green (1982) (For details see Chapter 2 in this book).


Coluzzi, Di Deco and Cancrini (1973b)reported that 2Rb inversion homozygotes had“mysorensis type” of eggs. Suguna (1981)and Subbarao et al. (1987) observed no suchcorrelation in laboratory and fieldpopulations.

In rural areas where mysorensis isprevalent, a wide range of breeding sites, suchas streams and channels, tanks and ponds,seepages, irrigation wells, etc., were found,and in urban areas the breeding sites werewells, overhead tanks, cisterns, fountains andwater collections at construction sites (Rao,1984).

Sweet and Rao (1937) and Rao, Sweetand Subba Rao (1938) reported that An.stephensi type form is an efficient vector,while var. mysorensis is a poor vector.Recently, Nagpal et al. (2003) studied thebioecology of type form and var. mysorensisin an arid zone (Jodhpur, Rajasthan state) inIndia. The study was carried out during thepre-monsoon, monsoon and post-monsoonseasons. The type form was found breedingindoors in domestic and peri-domesticcontainers and it was found inside, especiallyon hanging objects, throughout the year. Var.mysorensis was found breeding in the samesites and resting indoors during the summer,but it moved outside with the onset of rains.The authors do not consider var. mysorensis

to be involved in the transmission of malariadue to its predominantly animal feeding andlow parity during the transmission season.However, in Iran, var. mysorensis isconsidered an important vector. Suguna(1992) reports that in Cuddalore (Tamil Nadu,India) where the submetacentric Y-chromosome was found in about 91 per centof the male population, An. stephensi wasimplicated in malaria transmission (Suguna,1981), but not in Pondicherry where thesubmetacentric type was about 68 per cent.In villages around Delhi, as well as in Delhicity, An. stephensi was incriminated (Sharmaet al., 1993). Recently, Rowland et al. (2002)incriminated An. stephensi along with sevenother anophelines in rural areas of easternAfghanistan. Positive mosquitoes were foundwith P. vivax (CSP210 and 247) and P.falciparum CSP antigens, and the sporozoiterate was 0.35%. Laboratory-feedingexperiments revealed that type form takes ashorter period for the development ofsporozoites than var. mysorensis (Vasanthi,1996).

There are 16 microsatellite markersdeveloped for this species (Verardi et al.,2002). Now, the taxonomic status of the twoecological races in this species could againbe addressed using these markers.


The importance of recognizing speciescomplexes and identifying their membersbecame obvious as early as the 1930s withthe discovery of An. maculipennis as a speciescomplex in Europe. During the Second WorldWar, in Italy, G. Davidson (personalcommunication from C. F. Curtis) had the jobof locating the dangerous anthropophagicspecies An. labranchiae and the lessdangerous and generally zoophagic An.maculipennis s.s. so as to site military campsin safe locations. But soon after the war,malaria was eradicated from Europe. Malariacontinues to be endemic in more than 100countries and is a serious health problem inall the South and South- East Asian countries.With the escalating prices of insecticides anddrugs, and beset with problems of resistanceof vectors to insecticides and of parasites todrugs, almost all countries in this region areexperiencing challenges to contain andcontrol malaria.

It is now increasingly being accepted bymany control programmes that a singlestrategy for an entire country, and even for asingle province/district, is not applicable.Situation-specific, and at times even species-specific, strategies need to be employed.Now that almost all important malaria vectorsin South and South-East Asia have beenidentified as species complexes, it is importantto map the distribution of the sibling speciesof the complexes and to establish the role ofeach sibling species in malaria transmissionand monitor their response to controlmeasures. This requires the study of thebiological characters of each sibling species.

The Culicifacies and Fluviatilis in Indiaand Dirus and Maculatus Complexes inThailand are the most thoroughly investigatedones in this region. The data generated sofar have clearly established the importanceof identifying sibling species in malaria controlprogrammes. However, the informationgenerated on these complexes so far isinsufficient for planning and implementingeffective control strategies suitable fordifferent situations. Since each species has adifferent gene pool and hence may havedifferent behaviour and genetic potential,data on the following aspects of all membersof each complex would be of interest:

(i) Breeding habitats

(ii) Resting behaviour (indoors/outdoors)

(iii) Feeding preference with reference to siteand host (human/animal; indoors/outdoors)

(iv) Biting rhythms (peak biting time)

(v) Susceptibility to plasmodial infections inthe laboratory to establish whether thelow vectorial capacity is due to genetic/physiological factors or because of poorlongevity, preference to feed on hostsother than humans, etc.

(vi) Response to commonly-used insecticideswhich could indicate the difference inthe rate of development of resistance

(vii) Molecular mechanisms responsible forresistance (if possible).

In addition to these, studies should beencouraged on more subtle, but equally

4. Prospects for the future


relevant and not so easily observed,differences such as (a) density regulation ofsympatric species in breeding places; (b)factors responsible for distinct distributionpattern; (c) physical and chemical propertiesof water in different breeding places todetermine the factors influencing speciespreferences; (d) population structure, smallestbreeding unit, population bottlenecks, geneflow over the entire range of each speciesdistribution; etc.

Serious consideration is now being givento genetic strategies as alternative vectorcontrol tools. Efforts are under way todevelop genetically manipulated/engineeredmosquitoes for use in vector control. Manyresearch groups are working on differentaspects to contribute to the development ofanopheline strain(s) which will not besusceptible to parasite development, andconsequently if these could be made toreplace the wild vector population tointerrupt malaria transmission. For this finalachievement, other necessary aspectsessential for the release of such a desirablestrain and spread it in nature are beingaddressed with great vigour and care. Withthe completion of the sequencing of An.gambiae genome (Holt et al., 2002), it isexpected that more novel targets will beidentified for anopheline vector control.Volume 219 of Science (2002), in which thissequencing data was published, alsocontained viewpoints of several distinguishedscientists on the implications of genomesequencing in developing vector controlstrategies and for better understanding of thebiology of vectors. For these and for otheralready existing vector control strategies, it isimportant to recognize all the species present(whether they are morphologicallydistinguishable or indistinguishable) and thebiological features of each species (asmentioned above).

If identification of sibling species is to bedone routinely in malaria control activities,

techniques which are simple, accurate andaffordable need to be developed.

By definition, sibling species aremorphologically very similar, but members ofa complex should nevertheless be examinedfor any morphological variation. Membersof the Subpictus Complex can easily bedistinguished by morphological characters,while for those of the Maculatus Complex,more skill and effort are needed. There areother complexes, for which there are noreports of morphological variations.Examination of polytene chromosomes forspecies-specific diagnostic inversiongenotypes is the cheapest technique nowavailable, but it requires specialized skill andaptitude for microscopic examination. In spiteof certain limitations, this technique wasefficiently used for studying the Culicifaciesand Fluviatilis Complexes in India andMaculatus in Thailand. In the 1980s, therewas more interest in the development ofallozymes as diagnostic tools than there isnow. Advancements and advantages of DNA-based techniques have shifted the interest ofresearchers towards molecular methods.Initial establishment costs are high and alsoexpertise is required to develop DNA-basedmethods. However, once these techniquesare standardized, the procedures are simpleand can routinely be used to identify thespecies and a large number of samples canbe screened on a single day.

An informal consultancy meeting onMalaria vector species complexes and intra-specific variations: Relevance for malariacontrol and orientation for further researchwas held in Bangkok, Thailand, from 29October to 3 November 1984. During thismeeting several recommendations weremade. More than two decades have sincepassed. The author considers the re-commendations of the meeting are still validand require attention. Therefore, therecommendations are reproduced toemphasize the importance of identifyingspecies complexes and for further researchon these complexes.


“In order to simplify epidemiologicalmapping and stratification at the countrylevel, as part of the programme planningprocess, attempts should be made to linkgenetically-determined sibling species andassociated variants with topographical andvegetational indicators.”

“Species distribution does not recognizepolitical boundaries, and it is stronglyrecommended that collaboration betweencountries and regions be encouraged ininvestigations involving species complexes ofmalaria vectors.”

Thus, it becomes imperative thatresearch findings on species complexesshould find a place in the planning of nationalvector control strategies and should not beconsidered merely as academic achievementsor pursuits. In order for this to beaccomplished, both researchers andprogramme planners/managers have to makean effort to:

(i) Establish close linkages betweenorganizations/universities where researchon species complexes is carried out andauthorities/directorates who plan controlstrategies;

(ii) Create a database on species complexesto which national control programme canhave an access to (with the advancementin communication techniques, it wouldnot be difficult to create such linkages);

(iii) Organize intra-country courses to imparttraining to personnel in the nationalprogrammes to learn methods to identifysibling species and to utilize knowledge onspecies complexes in malaria entomology.

As a follow-up to this meeting, twoworkshops, one for Indian participantssponsored by the Indian Council of MedicalResearch in 1994 and the other an inter-country workshop sponsored by the WHORegional Office for South-East Asia in 1997were held in New Delhi, India. The MalariaResearch Centre (now renamed as NationalInstitute of Malaria Research), Delhi

organized both these workshops. In theintercountry workshop, experts fromAfghanistan, Bangladesh, India, Indonesia,Myanmar, Sri Lanka and Thailandparticipated. Both these workshops providedhands-on-experience to the participants onthe techniques used in the identification ofsibling species. This is an indication of thecommitment of the organizing agencies andparticipating countries to study speciescomplexes. There is need for organizing moresuch workshops, especially in view of thetechniques (molecular methods) that havebeen developed recently.

Another important requirement for anycontrol programme, especially for geneticcontrol strategies, is information on populationstructure, gene flow and geographical barriers,if any. Workshops covering these aspects andpractical training on the tools that can be usedfor these studies are urgently required in thisregion. Training on genotyping of populationsusing microsatellite markers should beorganized as markers are available for fourimportant vectors, An. culicifacies, An. dirus,An. maculatus, and An. stephensi. Studentsfrom developing countries will greatly benefitstudying for their masters and doctoral degreesat universities in developed countries in thespecialized aspects of field biology related tomalaria vectors.

Lastly, it should be noted that siblingspecies are not a special kind of species andought to be treated as any other species. Thisis an appeal to researchers that they shouldformally designate sibling species with formalbinomial nomenclature at the earliestopportunity and drop the provisionaldesignations given to them. It can be assumedthat entomologists and programme planners/managers will not hesitate to accept siblingspecies with formal designations. For manythe concept of biological species/siblingspecies is still alien and they continue toconsider them as races or variant taxa, whichhave been conclusively proved to be separatebiological species that do not generallyexchange genes by mating in the field.


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2. Techniques used in therecognition of SpeciesComplexes

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Zheng, L.B., Cornel, A.J., Wang, R., Erfile, H., Voss, H., Ansorge,W., Kafatos, F.C. and Collins, F.H. (1997). Quantitativetrait loci for refractoriness of Anopheles gambiae toPlasmodium cynomolgi B. Science 276: 425-8.

3.1 The Annularis ComplexAlam, M. T., Das, M. K., Dev, V., Ansari, M. A. and Sharma, Y. D.

(2006). Identification of two cryptic species in theAnopheles (Cellia) annularis complex using ribosomal DNAPCR-RFLP. Parasitol. Res. (in press) (Epub Dec. 6, 2006).

Atrie, B. (1994). Cytogenetic studies of different field populationsof Anopheles annularis Van Der Wulp and Anophelessubpictus Grassi. Ph.D. thesis, Dept. of Zoology, Universityof Delhi, Delhi, India.


Atrie, B., Subbarao, Sarala K., Pillai, M.K.K., Rao, S.R.V. andSharma, V.P. (1999). Population cytogenetic evidence forsibling species in Anopheles annularis (Diptera: Culicidae).Ann. Entomol. Soc. Amer. 92 (2): 243-9.

Baker, E.Z., Beier, J.C., Meek, S.R. and Wirtz, R.A. (1987).Detection and quantification of Plasmodium falciparumand P. vivax infections in Thai-Kampuchea Anopheles(Diptera: Culicidae) by enzyme-linked immunosorbentassay. J. Med. Entomol. 24: 536-41.

Bang, Y.H. (1985). Implications in the control of malaria vectorswith insecticides in tropical countries of South- East Asia.Part I — Insecticide Resistance. J. Commun. Dis. 17: 199-218.

Dash, A.P., Bendley, M.S., Das, A.K., Das, M. and Dwivedi, S.R.(1982). Role of An. annularis as a vector of malaria ininland of Orissa. J. Commun. Dis., 14: 224.


Gunasekaran, K., Sahu, S.S., Parida, S.K., Sadanandane, C.,Jambulingam, P. and Das, P.K. (1989). Anopheline faunaof Koraput district, Orissa state, with particular referenceto transmission of malaria. Indian J. Med. Res. 89: 340-3.


Harrison, B.A. (1988). The role of population genetics in thestudy and control of Anopheles malaria vectors in SoutheastAsia. Paper presented at IMR, Kaula Lumpur, June, 28-30, 1988.

Kondrashin, A.V. and Rashid, K.M. (1987). Epidemiologicalconsiderations for planning malaria control in South-EastAsia Region. WHO Regional Publications, Series No. 17.WHO/SEARO, New Delhi.

Ramasamy, R., DeAlwis, R., Wijesundere, A. and Ramasamy,M.S. (1992). Malaria transmission at a new irrigationproject in Sri Lanka: The emergence of Anopheles annularisas a major vector. Am. J. Trop. Med. Hyg. 47: 547-53.

Rao, T.R. (1984). The anophelines of India. Malaria ResearchCentre (ICMR), Delhi. p 518

Rowland, M., Mohammed, N., Rehman, H., Howitt, S., Mendis,C., Ahmed Kamal, M. and Wirtz, R. (2002). Anophelinevectors and malaria transmission in eastern Afghanistan.Trans. Roy. Soc. Trop. Med. Hyg., 96(6): 620-6.

World Health Organization (WHO), (1983). Report of thesteering Committee of the scientific working group onmalaria (1980-83).TDR/MAL/SC-SWG980-83)/83.3.

3.2 The BarbirostrisComplex

Baimai, V., Rattanarithikul, R and Kijchalao, U. (1995).Metaphase karyotypes of Anopheles of Thailand andsoutheast Asia : IV The Barbirostris and umbrosus speciesgroups, subgenus Anopheles (Diptera : Culicidae). J. Am.Mosq. Contr. Assoc. 11 (3): 323–8.

Chowdiah, B.N., Avicharan, T.T. and Seetharaman, P.l. (1970).Chromosome studies of Oriental anophelines. Expolientia26: 315-7.

Choochote, W., Sucharit, Supat and Abeyewickreme, A. (1983).Experiments in crossing two strains of Anopheles

barbirostris Van Der Walp 1884 (Diptera : Culicidae) inThailand. J. Am. Mosq. Contr. Assoc. 14(2): 204–9.


Rao, T.R. (1984). The anophelines of India. Malaria ResearchCentre (ICMR), Delhi. p 518.

Reid, J.A. (1968). Anopheline mosquitoes of Malaya and Borneo.Stud. Inst. Med. Res., Malaya 31: 1-520.

Sukowati, S. Andris, H. and Sondakh, J. (2003). Cytogeneticstudy of the malaria vector Anopheles barbirostris inIndonesia (Abstract).

3.3 The Culicifacies ComplexAbhayawardena, T.A., Dilrukshi, R.K.C. and Wijesuriya, S.R.E.

(1996). Cytotaxonomical examination of sibling speciesin the taxon Anopheles culicifacies Giles in Sri Lanka.Indian J. Malariol. 33: 74-80.

Adak, T., Subbarao, S.K., Sharma, V.P. and Rao, S.R.V. (1994).Lactate dehydrogenase allozyme differentiation of speciesin the Anopheles culicifacies complex. Med. Vet. Entomol.8: 137-40.

Adak, T., Kaur, S., Wattal, S., Nanda, N. and Sharma, V.P. (1997).Y-chromosome polymorphism in species B and C ofAnopheles culicifacies complex. J. Amer. Mosq. Contr.Assoc. 13(4): 379-83.

Adak,T., Singh, O.P., Nanda, Nutan, Sharma, V.P. and Subbarao,Sarala K. (2006). Isolation of a Plasmodium vivax refractoryAnopheles Culicifacies strain from India. Trop. Med. Int.Hlth., 11(2): 1-7.

Adak, T., Kaur, S. and Singh, O.P. (1999). Comparativesusceptibility of different members of the Anophelesculicifacies complex to Plasmodium vivax. Trans. Roy. Soc.Trop. Med. Hyg., 93: 573-7.

Akoh, J.I., Beidas, M.F. and White, G.B. (1984). Cytotaxonomicevidence for the malaria vector species A of the Anophelesculicifacies complex being endemic in Arabia. Trans. Roy.Soc. Trop. Med. Hyg. 78: 698.

Baimai, V., Kijchalao, U. and Rattanarithikul, R. (1996).Metaphase karyotypes of Anopheles of Thailand andSoutheast Asia : The Myzomyia series sub-genus Cellia(Diptera: Culicidae). J. Am. Mosq. Contr. Assoc. 12: 97-105.

Cornel, A.J., Subbarao, S.K., Chandra, D., Raghavendra, K.,Nanda, N., Sharma, V.P., Porter, C.H. and Collins, F.H.(2005). Separation of Anopheles culicifacies species A andD from species B,C and E (Diptera: Culicidae) using PCRprimers selected from within the D2 domain of theribosomal DNA 28S unit (unpublished).

Curtis, C.F. and Townson, H. (1998). Existing methods of vectorcotrol and molecular entomology. British Med. Bull. 54(2):311-25.

de Silva, B.G., Gunasekera, M.B., Abeyewickreme, W.,Abhayawardana, T.A. and Karunanayake, E.H. (1998).Screening of Anopheles culicifacies population of Sri Lankafor sibling species A. Indian J. Malariol. 35(1): 1-7.

Ghosh, S.K., Tiwari, S.N., Satyanarayana, T.S., Sampath, T.R.R.,Sharma, V.P., Nanda, N., Joshi, H., Adak, T. and Subbarao,S.K. (2005). Larvivorous fish in wells target malaria vectorsibling species of Anopheles culicifacies complex inKarnataka, India. Trans. Roy. Soc. Trop. Med. Hyg. 99: 101-5.


Goswami, G., Nanda, N., Raghavendra, K., Gakhar, S.K. andSubbarao, S.K. (2005). PCR-RFLP of mitochondrialcytochrome oxidase subunit II and ITS-2 of ribosomalDNA: markers for the identification of members of theAnopheles culicifacies complex (Diptera : Culicidae). ACTATropica 95: 92-9

Goswami, G., Raghavendra, K., Nanda, N., Singh, O.P., Gakhar,S.K. and Subbarao, S.K. (2006). PCR diagnostic assays forthe identification of all members of the Anophelesculicifacies complex. Am. J. Trop. Med. Hyg. 75(30): 454-460.

Green, C.A. and Miles, S.J. (1980). Chromosomal evidence forsibling species of the malaria vector Anopheles (Cellia)culicifacies Giles. J. Trop. Med. Hyg. 83: 75-8.

Gunasekera, M.B., de Silva, B.G.D.N.K., Abeyewickreme, W.,Subbarao, S.K., Nandadasa, H.G. and Karunayayake, E.H.(1995). Development of DNA probes for the identificationof sibling species A of the Anopheles culicifacies (Diptera:Culicidae) complex. Bull. Entomol. Res. 85: 345-53.


Jambulingam, P., Sebason, S., Vijayan, V.A., Krishnamoorthy, K.,Gunasekaran, K., Ranendran, G., Chandrahas, R.K. andRajagopalan, P.K. (1984). Density and biting behaviour ofAnopheles culicifacies Giles in Rameshwaram Island (TamilNadu). Indian J. Med. Res. 80: 47-50.

Joshi, H., Vasantha, K., Subbarao, S.K. and Sharma, V.P. (1988).Host feeding patterns of Anopheles culicifacies species Aand B. J. Am. Mosq. Contr. Assoc. 4: 248-51.

Kar, I., Subbarao, S.K., Eapen, A., Ravindran, J., Satyanarayana,T.S., Raghavendra, K., Nanda, E. and Sharma, V.P. (1999).Evidence for a new malaria vector species, species E, withinthe Anopheles culicifacies complex (Diptera: Culicidae).J. Med. Entomol. 36(5): 595-600.

Kaur, S., Singh, O.P. and Adak, T. (2000). Susceptibility of speciesA,B,C of Anopheles culicifacies complex to Plasmodiumyoelii yoelii and Plasmodium vinckei petteri infections. J.Parasitol. 86: 1345-8.

Mahmood, F., Sakai, R.K. and Akhtar, K. (1984). Vectorincrimination studies and observations on species A andB of the taxon Anopheles culicifacies in Pakistan. Trans. R.Soc. Trop. Med. Hyg. 78: 607-16.

Miles, S.J. (1981). Unidirectional hybrid male sterility from crossesbetween species A and species B of the taxon Anopheles(Cellia) culicifacies Giles. J. Trop. Med. Hyg. 84: 13-6.

Milligan, P.J.M., Phillips, A., Molyneux, D.H., Subbarao, S.K. andWhite, G.B. (1986). Differentiation of Anophelesculicifacies Giles (Diptera: Culicidae) sibling species byanalysis of cuticular components. Bull. Entomol. Res. 76:529-37.

Mittal, P.K., T. Adak, O.P. Singh, K. Raghavendra and S.K.Subbarao (2002). Reduced susceptibility to deltamethrinin Anopheles culicifacies s.l. in district Ramanathapuramin Tamil Nadu: selection of pyrethroid resistant strain. Curr.Sci. 82(2): 185-8.

Nanda, Nutan, R.S. Yadav, Sarala K. Subbarao, Hema Joshi andV.P. Sharma (2000). Studies on Anopheles fluviatilis andAnopheles culicifacies in relation with malaria in forest anddeforested riverine ecosystems in northern Orissa, India.J. Amer. Mosq. Contr. Assoc. 16(3): 199-205.

Raghavendra, K., Vasantha, K., Subbarao, S.K., Pillai, M.K.K. andSharma, V.P. (1991). Resistance in Anopheles culicifaciessibling species B and C to malathion in Andhra Pradeshand Gujarat states, India. J. Am. Mosq. Contr. Assoc. 7:255-9.

Raghavendra, K., Sarala K. Subbarao, K. Vasantha, M.K.K. Pillaiand V.P. Sharma (1992). Differential selection of malathionresistance in Anopheles culicifacies A and B (Diptera:Culicidae) in Haryana state, India. J. Med. Entomol. 29:183-7.


Rowland, M., Mohammed, N., Rehman, H., Howitt, S., Mendis,C., Ahmed Kamal, M. and Wirtz, R. (2002). Anophelinevectors and malaria transmission in eastern Aghanistan.Trans. Roy. Soc. Trop. Med. Hyg., 96(6): 620-6.

Saifuddin, U.T., Baker, R.H. and Sakai, R.K. (1978). Thechromosomes of Anopheles culicifacies. Mosq. News38: 233-9.

Satyanarayan, T.S. (1996). Field and laboratory studies onselected ecological and behavioural aspects of siblingspecies of the An. culicifacies complex. Ph.D Thesis Dept.of Zoology, Delhi University, Delhi.

Sebesan, S., Jambulingam, P., Krishnamurthy, K., Vijayan, V.A.,Gunasekaran, K., Rajendran, G., Chandrahas, R.K. andRajagopalan, P.K. (1984). Natural infection and vectorialcapacity of Anopheles culicifacies Giles in Rameshwaramisland (Tamil Nadu). Indian J. Med. Res. 80: 43-6.

Senior-White, R. (1947). On the anthropophilic indices of someAnopheles found in East Central India. Indian J. Malariol.1: 111-22.

Singh, O.P., Raghavendra, K., Nanda, N., Mittal, P.K. andSubbarao, S.K. (2002). Pyrethroid resistance in Anophelesculicifacies in Surat district, Gujarat, West India. Curr. Sci.82(5): 547-50.

Singh, O.P., Goswamy, G., Nanda, N., Raghavendra, K., Chandra,D. and Subbarao, S.K. (2004). An allele-specificpolymerase chain reaction assay for the identification ofmembers of Anopheles culicifacies complex. J. Biosciences29: 275-80.

Subbarao, S.K. (1984). Biological species in malaria vectors ofIndia. In Indo-UK Workshop on Malaria. Proceedings ofthe workshop held at the Indian Council of MedicalResearch, New Delhi, November 14-19, 1983. (ed. V.P.Sharma) pp. 77-83. Malaria Research Centre, Delhi.

———. (1988). The Anopheles culicifacies complex and controlof malaria. Parasitol. Today. 4: 72-5.

———. (1991). Anopheles culicifacies sibling species andmalaria transmission. ICMR Bull. 21: 61-5.

Subbarao, S.K. and Sharma, V.P. (1997). Anopheline speciescomplexes and malaria control. Indian J. Med. Res. 106:164-73.

Subbarao, S.K., Adak, T. and Sharma, V.P. (1980). Anophelesculicifacies sibling species distribution and vectorincrimination studies. J. Commun. Dis. 12: 102-4.

Subbarao, S.K., Nanda, N. and Raghavendra, K. (1999).Malariogenic stratification of India using Anophelesculicifacies sibling species prevalence. ICMR Bull. 29(7):75-80.

Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988a).Cytotaxonomy of certain malaria vectors of India, In :Biosystematics of haematophagous insects. (ed. M.W.Service) pp. 25-37. Clarendon Press, Oxford.


———. (1988b). Studies on the crosses between the siblingspecies of the Anopheles culicifacies complex. J. Hered.79: 300-3.

———. (1988c). Response of Anopheles culicifacies siblingspecies A and B to DDT and HCH in India: implicationsin malaria control. Med. Vet. Entomol. 2: 219-23.

Subbarao, S.K., Vasantha, K., Adak, T. and Sharma, V.P. (1983).Anopheles culicifacies complex: evidence for a new siblingspecies, species C. Ann. Entomol. Soc. Am. 76: 985-8.

Subbarao, S.K., Vasantha, V., Adak, T. and Sharma, V.P. (1987).Seasonal prevalence of sibling species A and B of the taxonAnopheles culicifacies in villages around Delhi. Indian J.Malariol. 24: 9-15.

Subbarao, S.K., Nanda, N., Chandrahas, R.K. and Sharma, V.P.(1993). Anopheles culicifacies complex : Cytogeneticcharacterization of Rameshwaram island populations. J.Am. Mosq. Contr. Assoc. 9: 27-31.

Subbarao, S.K., Vasantha, K., Raghavendra, K., Sharma, V.P. andSharma, G.K. (1988a). Anopheles culicifacies : siblingspecies composition and its relationship to malariaincidence. J. Am. Mosq. Contr. Assoc. 4: 29-33.

Subbarao, S.K., Adak, T., Vasantha, V., Joshi, H., Raghavendra,K., Cochrane, A.H., Nussenzweig, R.S. and Sharma, V.P.(1988b). Susceptibility of Anopheles culicifacies speciesA and B to Plasmodium vivax and Plasmodium falciparumas determined by immunoradiometric assay. Trans. Roy.Soc. Trop. Med. Hyg. 82: 394-7.

Subbarao, S.K., K. Vasantha, H. Joshi, K. Raghavendra, C. UshaDevi, T.S. Satyanarayan, A.H. Cochrane, R.S. Nussenzweigand V.P. Sharma (1992). Role of Anopheles culicifaciessibling species in malaria transmission in Madhya Pradesh,India. Trans. Roy. Soc. Trop. Med. Hyg. 86: 613-4.

Suguna, S.G., Tewari, S.C., Mani, T.R., Hiryan, J. and Reuben, R.(1983). Anopheles culicifacies species complex inThenpennaiyar riverine tract, Tamil Nadu. Indian J. Med.Res. 77: 455-9.

Suguna, S.G., Tewari, S.C., Mani, T.R., Hiriyan, J. and Reuben,R. (1989). A cytogenetic description of a new species ofthe Anopheles culicifacies complex. Genetica 78: 225-30.

Sunil, Sujatha, Raghavendra, K., Singh, O.P., Malhotra, P., Huang,Y., Zheng, Liangbiao and Subbarao, Sarala K. (2004).Isolation and characterization of microsatellite markersfrom malaria vector, Anopheles culicifacies. MolecularEcology notes 4: 440-2.

Surendran, S.N., Abhayawardana, T.A., De Silva, B.G.,Ramasamy, R. and Ramasamy, M.S. (2000). Anophelesculicifacies Y-chromosome dimorphism indicates siblingspecies (B and E) with different malaria vector potential inSri Lanka. Med. Vet. Entomol. 14(4): 437-40.

_________ (2002a). Limnological characterization of larvalbreeding sites of sibling species (B and E) in the Anophelesculicifacies (Diptera: Culicidae) species complex in SriLanka. Proceedings of the Institute of Biology. P. 56.

_________ (2002b). Differential susceptibility to malathion bytwo members (B and E) of the Anopheles culicifacies(Diptera: Culicidae) species complex in Sri Lanka.Proceedings of the Sri Lanka Association for theAdvancement of Science 58: 153.

_________ (2003). Establishment of species E, not B as the majorvector of malaria in the Anopheles culicifacies complex inthe country. Proceedings of the Sri Lanka association forthe Advancement of Science 59: 180.

Van Bortel, W., Sochanta, T., Harbach, R.E., Socheat, B., Roelants,P., Backeljau, T. and Coosemans, M. (2002). Presence ofAnopheles culicifacies B in Cambodia established by thePCR-RFLP assay developed for the identification ofAnopheles minimus species A and C and four relatedspecies. Med. Vet. Entomol., 16: 329-334.

Vasantha, K., Subbarao, S.K. and Sharma, V.P. (1991). Anophelesculicifacies complex: Population cytogenetic evidence forspecies D (Diptera: Culicidae). Ann. Entomol. Soc. Am.84: 531-6.

Vasantha, K., Subbarao, S.K., Adak, T. and Sharma, V.P. (1982).Karyotypic variations in Anopheles culicifacies complex.Indian J. Malariol. 19: 27-32.

Vasantha, K., Subbarao, S.K., Adak, T. and Sharma, V.P. (1983).Anopheles culicifacies : mitotic karyotype of species C.Indian J. Malariol. 20: 161-2.

Zaim, M., Subbarao, S.K., Manouchehri, A.V. and Cochrane,A.H. (1993). Role of Anopheles culicifacies s.l. and An.pulcherrimus in malaria transmission in Ghassreghand(Baluchistan), Iran. J. Am. Mosq. Contr. Assoc. 9: 23-6.

Zavala, F., Gwadz, R.W., Collins, F.H., Nussenzweig, R.S. andNussenzweig, V. (1982). Monoclonal antibodies tocircumsporozoite proteins identify the species of malariaparasite in infected mosquitoes. Nature 299: 737-8.

3.4 The Dirus ComplexAudtho, M., Tassanakajon, A, Boonsaeng, V, Tpiankijagum, S

and Panyim, S. (1995). Simple Nonradioactive DNAHybridization Method for Identification of Sibling speciesof Anopheles dirus (Diptera: Culicidae) Complex. J. Med.Entomol. 32: 107-11.

Baimai, V. (1988). Population cytogenetics of the malaria vectorAnopheles leucosphyrus group. Southeast Asian J. Trop.Med. Publ. Hlth. 19: 667-80.

Baimai, V. (1989). Speciation and species complexes of theanopheles malaria vectors in Thailand. In: Proceedings ofthe 3rd Conference on Malaria Research, Thailand. 18 -20th October 1989. pp. 146-62.

Baimai, V. and Green, C.A. (1988). Cytogenetics of naturalpopulations of the malaria vectors Anopheles dirus andAn. maculatus complexes in Thailand. In: Proceedings ofMahidol University Seminar on Malaria VaccineDevelopment, Bangkok (eds. A. Sabchareon, S. Supavej,P. Attanath and M. Ho). pp. 45-51.

Baimai, V. and Traipakvasin, A. (1987). Intraspecific variation insex heterochromatin of species B of the Anopheles diruscomplex in Thailand. Genome 29: 401-4.

Baimai, V., Andre, R.G., Harrison, B.A., Kijchalao, U. andPanthusiri, L. (1987). Crossing and chromosomal evidencefor two additional sibling species within the taxonAnopheles dirus Peyton and Harrison (Diptera: Culicidae)in Thailand. Proc. Entomol. Soc. Wash. 89: 157-66.

Baimai, V., Green, C.A., Andre, R.G., Harrison, B.A. and Peyton,E.L. (1984). Cytogenetic studies of some speciescomplexes of Anopheles in Thailand and Southeast Asia.Southeast Asian J. Trop. Med. Pub. Hlth. 15: 536-46.

Baimai, V., Harbach, R.E. and Kijchalao, U. (1988). Cytogeneticevidence for a fifth species within the taxon Anophelesdirus in Thailand. J. Am. Mosq. Contr. Assoc. 4: 333-8.

Baimai, V., Harrison, B.A. and Somchit, L. (1981). Karyotypedifferentiation of three anopheline taxa in the Balabacensiscomplex of Southeast Asia (Diptera: Culicidae). Genetica.57: 81-6.


Baimai, V., Kijchalao, U, Sawadwongporn, P. and Green, C.A.(1988). Geographic distribution and biting behaviour offour species of the An. dirus complex (Diptera: Culicidae)in Thailand. Southeast Asian J. Trop. Med. Publ. Hlth 19:151-61.

Baimai, V., Poopittayasataporn, A. and Kijchalao, U. (1988).Cytological differences and chromosomal rearrangementsin four members of the Anopheles dirus complex (Diptera:Culicidae). Genome 30: 372-9.

Bhat, H.R. (1988). A note on Anopheles dirus Peyton andHarrison (1979). [An. balabacensis (Sensu lato) Baisas,1936] in India. Indian J. Malariol. 25: 103-5.

Black, W.C. and Krafsur, E.S. (1985). A fortran program foranalysis of genotypic frequencies and description of thebreeding structure of populations. Theoret. Appl. Genet.70: 484-90.

Choochote, W., Maleewong, W., Sucharit, S. and Tesana, S.(1987). Scanning electron microscopic study of pupal seta9-111-V of Anopheles balabacensis (Perlis form) andAnopheles dirus (Bangkok strain). Southeast Asian J. Trop.Med. Pub. Hlth. 18: 571-3.

Colless, D.H. (1956). The Anopheles leucosphyrus group. Trans.Roy. Entomol. Soc. Lond. 108: 37-116.

Colless, D.H. (1957). Further notes on the systematics of theAn. leucosphyrus group (Diptera: Culicidae). Proc. R.Entomol. Soc. Lond. B. 26: 131-9.

Damrongphol, P. and Baimai, V. (1989). Scanning electronmicroscopic observations and differentiation of eggs of theAnopheles dirus complex. J. Am. Mosq. Contr. Assoc. 5:563-8.

Dutta, P. Bhattacharya, D. R., Khan, S. A. , Sharma, C. K.,andMahanta, J. (1996). Feeding pattern of An. dirus , the majorvector of forest malaria in North-east India. Southeast AsianJ. Trop. Med. Publ. Hlth 27: 378-381.

Green, C.A., Munstermann, L.E., Tan. S.G., Panyim, S. andBaimai, V. (1992). Population genetic evidence for speciesA,B,C and D of the Anopheles dirus complex in Thailandand enzyme electromorphs for their identification. Med.Vet. Entomol. 6: 29-6.

Green, C.A., Rattanarithikul, R., Pongparit, S., Sawadwongporn,P. and Baimai, V. (1991). A newly-recognized vector ofhuman malarial parasites in oriental region, Anopheles(Cellia) pseudowillmori (Theobald, 1910). Trans. Roy. Soc.Trop. Med. Hyg. 85: 35-6.


Hii, J.L.K. (1985). Genetic investigations of laboratory stocks ofthe complex of Anopheles balabacensis Baisas (Diptera:Culicidae). Bull. Entomol. Res. 75: 185-97.

Huong, N.T., Southayanon, P., Ketterman, A.J. and Panyim, S.(2001). A rapid polymerase chain reaction based methodfor identifion of the Anopheles dirus sibling species.Southeast Asian J. Trop. Med. Publ. Hlth 32(3): 615-20.

Manguin, S., Mouchet, J. and Coosemans, M. (2001). Molecularidentification of sibling Anopheles species : example theAnopheles minimus and Anopheles dirus complexes, majormalarial vectors in Southeast Asia. Med. Trop. (Article inFrench). 61(6): 463-9.

Oo, T.T., Storch, V. and Becker, N. (2002). Studies on thebionomics of Anopheles dirus (Culicidae: Diptera) in

Mudon, Mon state, Myanmar. J. Vector Ecol. 27(1): 44-54.

Oo, T.T., Storch, V. and Becker, N. (2003). Anpheles dirus and itsrole in malaria transmission in Myanmar. J. Vect. Ecol. 28(2):175-83.

Panyim, S., Yasothornsrikul, S. and Baimai, V. (1988). Speciesspecific DNA sequences from the Anopheles dirus complex- a potential for efficient identification of isomorphicspecies. In Biosystematics of haematophagous insects (ed.M.W. Service) Clarendon Press, Oxford. pp. 193-202.

Peyton, E.L. (1989). A new classification for the LeucosphyrusGroup of Anopheles (Cellia). Mosq. syst. 21: 197-05.

Peyton, E.L. and Harrison, B.A. (1979). Anopheles (Cellia) dirus,a new species of the leucosphyrus Group from Thailand.Mozq. Syst. 11: 40-52.

Peyton, E.L. and Harrison, B.A. (1980). Anopheles (Cellia)takasagoensis Morishita 1946, an additional species in theBalabacensis Complex of Southeast Asia (Diptera:Culicidae). Mosq. Syst. 12: 335-47.

Peyton, E.L. and Ramalingam, S. (1988). Anopheles (Cellia)nemophilous, a new species of the Leucosphyrus Groupfrom Peninsular Malaysia and Thailand (Diptera:Culicidae). Mosq. syst. 20: 272-99.

Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta,J. (2001). Estimation of vectorial capacity of Anophelesdirus (Diptera: Culicidae) in a forest-fringed village of Assam(India). Vector Borne Zoonostic Dis. 1(3): 231-7.

Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta,J. (2002). Physico-chemical characteristics of breedinghabitats of Anopheles dirus (Diptera: Culicidae) in Assam,India. J. Environ. Biol. 23(1): 95-100.

Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta,J. (2005). Malaria transmission risk by the mosquitoAnopheles baimaii (formerly known as An. dirus speciesD) at different hours of the night in North-east India. Med.Vet. Entomol. 19: 423-427.

Prakash, A., Walton C., Bhattacharyya, D. R., O’Loughlin,Samantha, Mohapatra, P. K. and Mahanta, J. (2006).Molecular characterization and species identification ofthe Anopheles dirus and An. minimus complexes innortheast India using r-DNA ITS2. Acta Trop. 100: 151-61.


Rosenberg, R. and Maheswary, N.P. (1982). Forest malaria inBangladesh II. Transmission by Anopheles dirus. Am. J.Trop. Med. Hyg. 31: 183-91.

Rosenberg, R., Andre, R.G. and Somchit, L. (1990). Highlyefficient dry season transmission of malaria in Thailand.Trans. R. Soc. Trop. Med. Hyg. 84: 22-28.

Sallum, M.A.M., Peyton, E.L. and Wilkerson, R.C. (2005). Sixnew species of the Anopheles leucosphyrus group,reinterpretation of An. olegans and vector implication.Med. Vet. Entomol. 19: 158-199.

Sawadipanich, Y., Baimai, V. and Harrison, B.A. (1990).Anopheles dirus species E: Chromosomal and crossingevidence for another member of the dirus complex. J.Am. Mosq. Contr. Assoc. 6: 477-81.

Samboon, P., Prapanthadara, L. and Suwonket, L. (1999).Selection of Anopheles dirus for refractoriness andsusceptibility to Plasmodium yoelii nigeriensis. Med. Vet.Entomol. 13(4): 355-61.


Tiwari, S.C., Hiriyan, J. and Reuben, R. (1987). Survey of theanopheline fauna of the western Ghats in Tamil Nadu,India. Indian J. Malariol. 24: 21-8.

Trung, H. D., Van Bortel, W., Sochantha, T., Keokanchanh, K.Bret, O. J. T. and Coosemans, M. (2005). Behaviouralheterogeneity of Anopheles species in ecologically differentlocalities in Southeast Asia : a challenge for vector control.Trop. Med. Int. Hlth. 10 (3): 251-62.

Van Bortel, W., Harbach, R.E., Trung, H.D., Roelants, P.,Backeljau, T. and Coosemans, M. (2001). Confirmationof Anopheles varuna in Vietnam, previously misidentifiedand mistargeted as the malaria vector An. minimus. Am.J. Trop. Med. Hyg., 65: 729-32.

Walton, C., Chang, M.S., Handley, J.M., Harbach, R.E., Collins,F.H., Baimai, V. and Butlin, R.K. (2000a). The isolationand characterization of microsatellites from Anophelesdirus mosquitoes. Mol. Ecol. 9(10): 1665-7.

Walton, C., Handley, J.M., Collins, F.H., Baimai, V., Harbach,R.E., Deesin, V. and Butlin, R.K. (2001). Geneticpopulation structure and introgression in Anophelesmosquitoes in South-east Asia. Mol. Ecol. 10(3): 569-80.

Walton, C., Handley, J.M., Kuvangkadilok, C., Collins, F.H.,Harbach, R.E. , Baimai, V. and Butlin, R.K. (1999).Identification of five species of the Anopheles diruscomplex in Thailand, using allele-specific polymerasechain reaction. Med. Vet. Entomol. 13(1): 24-32.

Walton, C., Handley, J.M., Tun-Lin, W., Collins, F.H., Harbach,R.E., Baimi, V. and Butlin, R.K. (2000b). Populationstructure and population history of Anopheles dirusmosquitoes in Southeast Asia. Mol. Biol. Evol. 17(6): 962-74.

Wibow, S., Baimai, V. and Andre, R.G. (1984). Differentiationof four taxa of the Anopheles balabacensis complex usingH-banding in sex chromosome (Diptera: Culicidae). Can.J. Genet. Cytol. 26: 425-9.

Xu, X., Xu, J. and Qu, F. (1998). A diagnostic polymerase chainreaction assay for species A and D of the Anopheles dirus(Diptera: Culicidae) species complex based on ribosomalDNA second internal transcribed spacer sequence. J. Am.Mosq. Contr. Assoc. 14(4): 385-9.

Yasothornsrikul, S., Panyim, S. and Rosenberg, R. (1988).Diagnostic restriction fragment patterns of DNA from thefour isomorphic species of Anopheles dirus. SoutheastAsian J. Trop. Med. Publ. Hlth. 19: 703-8.

3.5 The Fluviatilis ComplexAdak, T., Singh, O. P., Das, M. K. Wattal, S. and Nanda, N. (2005).

Comparative susceptibility of three important malariavectors, Anopheles stephensi, Anopheles fluviatilis andAnopheles sundaicus to Plasmodium vivax. J. Parasitol. 91:79-82.

Bhombore, S.R., Sitaraman, N.L. and Achuthan, C. (1956).Studies on the bionomics of An. fluviatilis in Mysore state,part II. Indian J. Malariol. 10: 23-32.

Brooke Worth, C. and Sitaraman, N.L. (1952). Studies on thebionomics of Anopheles fluviatilis James, 1902 in Mysorestate, India. Review of the literature on bionomics of An.fluviatilis. Ind. J. Malariol. 6: 481-91.

Chen, B., Butlin, R. K., Pedro, P. M.., Wang, X. Z. and harbach,R. E. (2006). Molecular variation, systematics anddistribution of the Anopheles fluviatilis complex in southernAsia. Med. Vet. Entomol. 16: 253-265.

Garros, C., Harbach, R. E. and Manguin, S. (2005). Morphologicalassessment and molecular phylogenetics of the Funestusand Minimus Groups of Anopheles (Cellia). J. Med.Entomol. 42: 522-36.

Gunasekaran, K., Sahu, S.S., Parida, S.K., Sadanandane, C.,Jambulingam, P. and Das, P.K. (1989). Anopheline faunaof Koraput district, Orissa state, with particular referenceto transmission of malaria. Indian J. Med. Res. 89: 340-3


Manonmani, A., Townson, H., Adeniran, T., Jambulinga,, P., Sahu,S. and Vijayakumar, T. (2001). RDNA-ITS-2 polymerasechain reaction assay for the sibling species of An. fluviatilis.Acta Tropica. 1: 7-11.

Manonmani, A., Nanda, N., Jambulingam, P., Sahu, S., Vijakumar,T., Ramya vani, J. and Subbarao, S.K. (2003). Comparisonof PCR assay and cytotaxonomy for identification ofAnopheles fluviatilis sibling species. Bull. Ent. Res. 93: 169-71.

MRC (1995, unpublished). Malaria Research Centre AnnualReport.

Nanda, N., Joshi, H., Subbarao, S.K., Yadav, R.S., Shukla, R.P.,Dua, V.K. and Sharma, V.P. (1996). Anopheles fluviatiliscomplex : Host feeding patterns of species S,T and U. J.Am. Mosq. Contr. Assoc. 12: 147-9.

Nanda, Nutan, R.S. Yadav, Sarala K. Subbarao, Hema Joshi andV.P. Sharma (2000). Studies on Anopheles fluviatilis andAnopheles culicifacies in relation with malaria in forest anddeforested riverine ecosystems in northern Orissa, India.J. Amer. Mosq. Contr. Assoc. 16(3): 199-205.

Rao, T.R. (1984). The anophelines of India. Malaria ResearchCentre (ICMR), Delhi.


Sahu, S.S., Parida, S.K., Sadanandane, C., Gunasekaran, K.,Jambulingam, P. and Das, P.K. (1990). Breeding habitatsof malaria vectors: An. fluviatilis, An. annularis and An.culicifacies, in Koraput district, Orissa. Indian J. Malariol.27: 209-16.

Senior-White, R. (1946). On the outdoor resting of some speciesof oriental Anopheles. J. Mal. Inst. India. 6: 425-35.

Sharma, S.K., Nanda, N., Dua, V.K., Joshi, H., Subbarao, S.K.and Sharma, V.P. (1995). Studies on the bionomics ofAnopheles fluviatilis sensu lato and the sibling speciescomposition in the foothills of Shiwalik range (UttarPradesh), India. Southeast Asian J. Trop. Med. Publ. Hlth.26: 566-72.

Sharma, S. K., Tyagi, P.K., Padhan, K., Adak, T., Subbarao, S. K.(2004a). Malarial morbidity in tribal communities living inthe forest and plain ecotypes of Orissa, India. Ann. Trop.Med. Parasitol. 98: 459-68.

Sharma, S. K., Upadhyay, A. K., Haque, M. A., Singh, O. P., Adak,T., and Subbarao, S. K. (2004b). Insecticide susceptibilitystatus of malaria vectors in some hyperendemic tribaldistricts of Orissa. Curr. Sci. 87: 1722-6.

Sharma, S.K., Tyagi, P.K., Pradhan, K., Upadhyay, A.K., Haque,M.A., Joshi, H., Biswas, S., Nanda, N., Anitha, M., Adak,T., Chitnis, C.E., Chauhan, V.S. and Subbarao, S.K. (2006).Epidemiology of malaria in the forest and plain ecotypesof Sundergarh district, Orissa. Tran. Roy. Soc. Trop. Med.Hyg. 100: 917-25.


Shukla, R.P., Nanda, N., Pandey, A.C., Kohli, V.K., Joshi, H.,Subbarao, S.K. (1998). Studies on bionomics of An.fluviatilis and its sibling species in Nainital district (UttarPradesh), India. Indian J. Malariol. 35: 41-7.

Singh OP, Chandra D, Nanda N, Raghavendra K, Sunil S, SharmaSK, Dua VK, and Subbarao SK. (2004). Differentiation ofmembers of the Anopheles fluviatilis species complex byan allele-specific polymerase chain reaction based on 28Sribosomal DNA sequences. Am. J. Trop. Med. Hyg., 70(1): 27-32.

Singh, O. P., D. Chandra, N. Nanda , S. K. , Sharma, Pe ThanHtun, T. Adak, S. k. Subbarao and A. P. Dash (2006). Onthe Conspecificity of Anopheles fluviatilis species S withAnopheles minimus species C. J. Biosciences 31 (5): 671-677.

Subbarao, S.K., Nanda, N., Vasantha, K., Dua, V.K., Malhotra,M.S., Yadav, R.S. and Sharma, V.P. (1994). Cytogeneticevidence for three sibling species in Anopheles fluviatilis(Diptera: Culicidae). Ann. Entomol. Soc. Am. 87: 116-21.

Vatandoost, H., Shahi, M., Oshaghi, M., A., Naddaf, S., R.,Hanafi-Bojd., A., A. and Abaie, M., R.( 2005). Ecology ofAnpheles fluviatilis James in a malarious area, BandarAbbas, Hormozgan Province, Southern Iran (Abstract).Presented at the International conference on 125 Years ofMalaria Research : Laveran to Genomic, New Delhi.November 4-6, 2005.

Viswanathan, D.K. (1950). Malaria and its control in Bombaystate, Chitrashala, Poona, India.

3.6 The LeucosphyrusComplex

Baimai, V. (1988). Population cytogenetics of the malaria vectorAnopheles leucosphyrus group. Southeast Asian J. Trop.Med. Publ. Hlth. 19: 667-80.

Baimai, V., Harbach, R.E. and Kijchalao, U. (1988). Cytogeneticevidence for a fifth species within the taxon Anophelesdirus in Thailand. J. Am. Mosq. Contr. Assoc. 4: 333-8.

Baimai, V., Harbach, R.E. and Sukowati, S. (1988). Cytogeneticevidence for two species within the current concept ofthe malaria vector Anopheles leucosphyrus in SoutheastAsia. J. Am. Mosq. Contr. Assoc. 4: 44-50.

Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Maricioto, H.,Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria inthe Monoreh Hills of Central Java. Am. J. Trop. Med. Hyg.66(3): 287-92.

Chang, M. S., Doraisingam, P., Hardin, S. and Nagum,, N. (19950.Malaria and filariasis transmission in a village/forest settingin Baram district, Sarawak, Malaysia. J. Trop. Med. andHyg. 98: 192-8.

Harbach, R.E., Baimai, V. and Sukowati, S. (1987). Someobservations on sympatric populations of the malariavectors Anopheles leucosphyrus and Anophelesbalabacensis in a village-forest setting in south Kalimantan.Southeast Asian J. Trop. Med. Publ. Hlth. 18: 241-7.

Hii, J.L.K. (1985). Genetic investigations of laboratory stocks ofthe complex of Anopheles balabacensis Baisas (Diptera:Culicidae). Bull. Entomol. Res. 75: 185-97.

Hii, J.L., Kan, S., Vun, Y.S., Chin, K.F., Tambakau S, Chan, M.K.,Lye, M.S., Mak, J.W.. amd Cochrane, A.H. (1988).Transmission dynamics and estimates of malaria vectorialcapacity for Anopheles balabacensis and An. flavirostris

(Diptra: Culicidae) on Banggi island, Sabah, Malaysia. Ann.Trop. Med. Parasitol. 82(1):91-101.

Peyton, E.L. (1989). A new classification for the leucosphyrusgroup of Anopheles (Cellia). Mosq. syst. 21: 197-205.

Peyton, E.L. and Harrison, B.A. (1979). Anopheles (Cellia) dirus,a new species of the leucosphyrus group from Thailand(Diptera: Culicidae). Mosq. syst. 11: 40-52.

Sallum, M.A.M., Peyton, E.L. and Wilkerson, R.C. (2005). Sixnew species of the Anopheles leucosphyrus group,reinterpretation of An. olegans and vector implication.Med. Vet. Entomol. 19: 158-99.

3.7 The Maculatus ComplexBaimai, V. (1989). Speciation and species complexes of the

anopheles malaria vectors in Thailand. In: Proceedings ofthe 3rd Conference on Malaria Research, Thailand. 18-20th Oct. 1989. pp. 146-62.

Baimai, V. and Green, C.A. (1988). Cytogenetics of naturalpopulations of the malaria vectors Anopheles dirus andAn. maculatus complexes in Thailand. In: Proceedings ofMahidol University Seminar on Malaria VaccineDevelopment. 17 - 18 May, 1988 : (eds. A. Sabachavrn, S.Supavej, P. Attanath and M. Ho). pp. 45-51.

Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Maricioto, H.,Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria inthe Monoreh Hills of Central Java. Am. J. Trop. Med. Hyg.66(3): 287-92.

Christophers, S.R. (1931). Studies on the anopheline fauna ofIndia (Parts I-IV). Rec. Malar. Surv. India. 2: 305-32.

Christophers, S.R. (1933). The fauna of British India, includingCeylon and Burma. Diptera. Vol. IV. Family Culicidae.Tribe Anophelini. Taylor and Francis, London. 371pp.

Crawford, R. (1938). Some anopheline pupae of Malaya with anote on pupal structure. Government Printer, Singapore.110pp.

Ejercito, A. (1934). Anopheles maculatus Theobald, anothermalaria vector in the Philippines. J. Philipp. Is. Med. Assoc.14: 342-6.

Green, C.A. (1982). Polytene-chromosome relationships of theAnopheles stephensi species group from the afrotropicaland oriental regions (Culicidae, Anopheles (Cellia), seriesNeocellia. pp. 49-61. In: Recent Developments in theGenetics of Insect Disease vectors (eds. W.W.M. Steiner,W.J. Tabachnick, K.S. Rai and S. Narang) Stipes publishingCo. Champaign, Illinois.

Green, C.A. and Baimai, V. (1984). Polytene chromosomes andtheir use in species studies of malaria vectors as exemplifiedby Anopheles maculatus complex. In: Genetics: NewFrontiers. Proceedings XV International Congress ofGenetics (eds. V.L. Chopra, B.C. Joshi, R.P. Sharma andH.C. Bansal), Vol. 3, pp. 89-97. Oxford and IBH Publ.Comp., New Delhi.

Green, C.A., Rattanarithikul, R. and Charoensub, A. (1992).Population genetic confirmation of species status of themalaria vectors Anopheles willmori and An. pseudowillmoriin Thailand and chromosome phylogeny of the maculatusgroup of mosquitoes. Med. Vet. Entomol. 6: 335-41.

Green, C.A., Baimai, V., Harrison, B.A. and Andre, R.G. (1985).Cytogenetic evidence for a complex of species within thetaxon Anopheles maculatus (Ditera: Culicidae). Biol. J.Linn. Soc. 4: 321-8.


Green, C.A., Rattanarithikul, R., Pongparit, S., Sawadwongporn,P. and Baimai, V. (1991). A newly-recognized vector ofhuman malarial parasites in the oriental region, Anopheles(Cellia) pseudowillmori (Theobald, 1910). Trans. Roy. Soc.Trop. Med. Hyg. 85: 35-6.


Kittayapong, P., Clark, J.M., Edman, J.D., Potter, T.L., Lavine, B.K.,Marion, J.R. and Brooks, M. (1990). Cuticular lipiddifferences between the malaria vector and non-vectorforms of the Anopheles maculatus complex. Med. Vet.Entomol. 4: 405-13.

Kittayapong, P., Clark, J.M., Edman, J.D., Lavine, B.K., Marion,J.R., Brooks, M. (1993). Survey of the Anopheles maculatuscomplex (Diptera: Culicidae) in Peninsular Malaysia byanalysis of cuticular lipids. J. Med. Ent. 30: 969-74.

Ma, Y., Li, S. and Xu, J. (2006). Molecular identification andphlogeny of the Maulatus group of Anopheles mosquitoes(Diptera: Culicidae) based on nuclear and mitochondriaDNA sequences. Acta Trop. 99: 272-80.

Pradhan, J.N., Shrestha, S.L. and Vaidya, R.G. (1970). Malariatransmission in high mountain valleys of west Nepalincluding first record of Anopheles willmori (James) as athird vector of malaria. J. Nepal Med. Assoc. 8: 89-97.

Puri, I.M. (1931). Larvae of anopheline mosquitoes, with fulldescription of those of the Indian species. Indian Med.Res. Mem. 21: 1-227.

Rao, T.R. (1984). The Anophelines of India. Malaria ResearchCentre (ICMR), Delhi. p 518

Rattanarithikul, R. and Green, C.A. (1986). Formal recognitionof the species of the Anopheles maculatus group (Diptera:Culicidae) occurring in Thailand, including the descriptionsof two new species and a preliminary key to females.Mosq. Syst. 18: 246-78.

Rattanarithikul, R. and Harbach, R.E. (1990). Anophelesmaculatus (Diptera: Culicidae) from the type locality ofHong Kong and two new species of the maculatus complexfrom the Philippines. Mosq. syst. 22: 160-83.

Reid, J.A. (1968). Anopheline mosquitoes of Malaya and Borneo.Stud. Inst. Med. Res. Malaya 31.

Reid, J.A., Wattal, B.L. and Peters, W. (1966). Notes on Anophelesmaculatus and related species. Bull. Indian Soc. Malariol.Commun. Dis. 3: 185-97.

Rongnoparut, P., Yaicharoen, S., Sirichotpakorn, N.,Rattanarithikul, R., Lanzaro, G.C. and Linthicum, K.J.(1996). Microsatellite polymorphism in Anophelesmaculatus, a malaria vector in Thailand. Amer. J. Trop.Med. Hyg. 55(6): 589-94.

Rongnoparut, P., Sirichotpakorn, N., Rattanarithikul, R.,Yaicharoen, S. and Linthicum, K.J. (1999). Estimates ofgene flow among Anopheles macularus populations inThailand using microsatellite analysis. Am. J. Trop. Med.Hyg. 60(3): 508-15.


Torres, E.P., Foley, D.H. and Saul, A. (2000). Ribosomal DNAsequence markers differentiate two species of Anophelesmaculates (Diptera: Culicidae) complex in the Philippines.J. Med. Entomol. 37(6): 933-7.

Upatham, E.S., Prasittisuk, C., Ratanatham, S., Green, C.A.,Rojanasunan, W., Setakana, P., Theeraslip, N., Tremongkol,A., Viyanant, V., Pantuwatana, S. and Andre, R.G. (1988).Bionomics of Anopheles maculatus complex and their rolein malaria transmission in Thailand. Southeast Asian J.Trop. Med. Pub. Hlth. 19: 259-6.

Walton, C., Somboon, P., O’ Loughlin, S.M., Zhang, S., Harbach,R.E., Linton, Y.M., Chen, B.K., Duong, S. Nolan, K., Fong,Y.M., Vythilingumi, I., Mohammed, Z.D., Trung, H.D. andButlin, R.K. (2007). Genetic diversity and molecularidentification of mosquito species in the Anophelesmaculatus group using the ITS2 region of rDNA Infec.Genet. Evol. 7(1): 98-102 (Epub 2006 Jun. 19).

3.8 The Minimus ComplexBaimai, V. (1989). Speciation and species complexes of the

Anopheles malaria vectors in Thailand. In : Proceedingsof the 3rd Conference on Malaria Research, Thailand. 18 -20 October 1989. pp. 146-62.

Baimai, V. and Green, C.A. (1988). Cytogenetics of naturalpopulations of the malaria vectors Anopheles dirus andAn. maculatus complexes in Thailand. In: Proceedings ofMahidol University Seminar on Malaria VaccineDevelopment 17 - 18 May, 1988 (eds. A. Sabachavrn, S.Supavej, P. Attanath and M. Ho). pp. 45 - 51.

Baimai, V., Kijchalao, U. and Rattanarithikul, R. (1996).Metaphase karyotypes of Anopheles of Thailand andSoutheast Asia : The Myzomyia series sub-genus Cellia(Diptera: Culicidae). J. Am. Mosq. Contr. Assoc. 12: 97-105.

Chen, B., Harbach, R.E. and Butlin, R.K. (2002). Molecular andmorphological studies on the Anopheles minimus groupof mosquitoes in southern China: taxonomic review,distribution and malaria vector status. Med. Vet. Entomol.16: 253-65.

Garros,C., Koekemer, L.L., Coetzee, M. Coosemans, M. andManguin, S. (2004a). A single multiplex assay to identifymajor malaria vectors within the African Anophelesfunestus and Oriental An. minimus groups. Am. J. Trop.Med. Hyg. 70: 583-90.

Garros,C., Koekemer, L.L., Kamau, L. Awola, T. S., Van Bortel,W., Coetzee, M., Coosemans, M. and Manguin, S.(2004b)Restriction fragment lengh polymorphism methodfor the identification of major African and Asian malariavectors within the Anopheles funestus and An. minimusgroups. Am. J. Trop. Med. Hyg. 70: 260-65 .

Garros,C., Ron, M. P., Nguyen, T., Nguyen, H. S., and Manguin,S. (2005). First record of Anopheles minimus C andsignificant decrease of An. minimus A in central Vietnam.J. Am. Mosq. Contr. Assoc. 2: 139-43

Green, C.A., Gass, R.F., Munstermann, L.E. and Baimai, V. (1990).Population-genetic evidence for two species in Anophelesminimus in Thailand. Med. Vet. Entomol. 4: 25-34.

Green, C.A., Rattanarithikul, R., Pongparit, S., Sawadwongporn,P. and Baimai, V. (1991). A newly-recognized vector ofhuman malarial parasites in the oriental region, Anopheles(Cellia) pseudowillmori (Theobald, 1910). Trans. Roy. Soc.Trop. Med. Hyg. 85: 35-6.

Harrison, B.A. (1980). Medical entomology studies —XIII. TheMyzomyia Series of Anopheles (Cellia) in Thailand, withemphasis on intra-interspecific variations (Diptera:Culicidae). Contrib. Am. Entomol. Inst. (Ann Arbor) 17:1-195.



Janakara, B., Wajihullah, W.A., Dev, V., Curtis, C.F. and Sharma,V.P. (1995). Deltamethrinimpregnated bednets againstAnopheles minimus transmitted malaria in Assam, India.J. Trop. Med. Hyg. 98: 73-83.

Jambulingam, P., Sahu, S. S. and Manonmani, A. (2005).Reappearance of Anopheles minimus in Singhbhum hillsof East-Central India. Acta Trop. 9: 31-35.

Kanda, T., Ogawa, K., Sucharit, S., Pratchayanusorn, N., Lian,C.G. and Harinasuta, C. (1984). Cytogenetic andhybridization studies among 3 strains morphologicallyvarieted and belonging to Anopheles minimus Theobaldfrom Japan and Thailand. Cytologia 49: 865-81.

Kengue, P., Trung, H.D., Baimai, V., Coosemans, M. and Manguin,S. (2001). A multiplex PCR-based method derived fromrandom amplified polymorphic DNA (RAPD) markers forthe identification of species of the Anopheles minimusgroup in Southeast Asia. Insect Mol.. Biol. 10: 427-35.

Suthas, N., Sawasdiwongphorn, P., Chitprarop, U. and Cullen,J.R. (1986b). The behaviour of Anopheles minimusTheobald (Diptera: Culicidae) subjected to differing levelsof DDT selection pressure in northern Thailand. Bull.Entomol. Res. 76: 303-12.

Suthas, N., Sawasdiwongphorn, P., Chitprarop, U., Cullen, J.R.,Gass, R.F. and Green, C.A. (1986a). A mark releaserecapture demonstration of host-preference heterogeneityin Anopheles minimus Theobald (Diptera: Culicidae) in aThai village. Bull. Entomol. Res. 76: 313-20.

Phuc, H.K., Ball, A.J., Son, L. Hanh, N.V., Tu, N.D., Lion, N.G.,Veradi, A. and Townson, H. (2003). Multiplex PCR assayfor malaria vector Anopheles minimus and four relatedspecies in the myzomyia series from southeast Asia. Med.Vet. Entomol. 17(4): 423-28.

Prakash, A., Walton C., Bhattacharyya, D. R., O’Loughlin,Samantha, Mohapatra, P. K. and Mahanta, J. (2006).Molecular characterization and species identification ofthe Anopheles dirus and An. minimus complexes innortheast India using r-DNA ITS2. Acta Trop. 100: 156-61.

Rao, T.R. (1984). The Anophelines of India. Malaria ResearchCentre (ICMR), Delhi. p 518.

Sharpe, R.G., Hims, M.M., Harbach, R.E. and Butlin, R.K. (1999).PCR-based methods for identification of species of theAnopheles minimus group: allele-specific amplifcation andsingle-strand conformation polymorphism. Med. Vet.Entomol. 13: 265-73.

Sharpe, R.G., Harbach, R.E. and Butlin, R.K. (2000). Molecularvariation and phylogeny of members of the Minimus groupof Anopheles subgenus Cellia (Diptera: Culicidae).Systematic Entomology 25: 263-72.

Somboon, P., Walton, C., Sharpe, R.G., Higa, Y., Tuno, N., Tsuda,Y. and Takagi, M. (2001). Evidence for a new sibling speciesof Anopheles minimus from the Ryukyu Archipelago, Japan.J. Am. Mosq. Contr. Assoc., 17: 98-113.

Somboon, P., Thongwat, D.,Choochote, W., Walton, C. andTakagi, M. (2005). Crossing experiments of Anophelesminimus species C and putative species E. J. Am. Mosq.Control Assoc., 21(1): 5-9.

Sucharit, S. and Komalamisra, N. (1997). Differentiation ofAnopheles minimus species complex by RAPD-PCRtechnique. J. Med. Assoc. Thai. 80(9): 598-602.

Sucharit, S., Komalamisra, N., Leemingsawat, S., Apiwathnasorn,C. and Thongrungkiat, S. (1988). Population geneticstudies on the Anopheles minimus complex in Thailand.Southeast Asian J. Trop. Med. Pub. Hlth. 19: 717-23.

Thanaphum, S., Green, C.A., Baimai, V., Gass, R.F. and Gingrich,J.B. (1990). Genetic linkage relationships of eight enzyme/electromorph loci in Anopheles minimus. Genetica 82:63 - 72.

Trung, H.D., Van Bortel, W., Sochanta, T., Keokenchanh, K,. Briet,O. J. and and Coosemans, M. (2005). Behaviouralheterogeneity of Anopheles species in ecologically differentlocalities in Southeast Asia: a challenge for vector control.Trop. Med. Int. Hlth. 10(3): 251-62.

Van Bortel and Coosemans (2003). Suggesting new species?Comments on “Evidence for a new species of Anophelesminimus from the Ryukyu Archipelago Japan. J. Am. Mosq.Contr. Assoc. 19 (3): 261-4.

Van Bortel, W., Harbach, R.E., Trung, H.D., Roelants, P.,Backeljau, T. and Coosemans, M. (2001). Confirmationof Anopheles varuna in Vietnam, previously misidentifiedand mistargeted as the malaria vector An. minimus. Am.J. Trop. Med. Hyg., 65: 729-32.

Van Bortel, W., Trung, H.D., Manh, N.D., Roelants, P., Verle, P.and Coosemans, M. (1999). Identification of two specieswithin the Anopheles minimus complex in northernVietnam and their behavioural divergences. Trop. Med.Internal. Hlth. 4: 257-65.

Van Bortel, W., Trung, H.D., Roelants, P., Harbach, R.E.,Backeljau, T. and Coosemans, M. (2000). Moecularidentification of An. minimus s.l. beyond distinguishingthe members of the species complex. Insect Mol. Biol. 9:335-40.

Van Bortel, W., Trung, H.D., Roelants, P., Backetjall, T. andCoosemans, M. (2003). Population genetic structure ofthe malaria vector Anopheles minimus A in Vietnam.Heredity 91(5): 487-93.

Van Bortel, W., Trung, H.D., Sochanta, T., Keokenchanh, K.,Roelants, P., Backetjall, T. and Coosemans, M. (2004). Eco-ethological heterogeneity of the members of the MinimusComplex (Diptera:Culicidae) in Southeast Asia and itsconsequences for vector control. J. Med. Entomol. 41(3):366-74.

Wajihullah, Jana, B. and Sharma, V.P. (1992). Anopheles minimusin Assam. Curr. Sci. 63: 7-9.

Yuan, Y. (1987). Studies on the two forms of Anopheles (Cellia)minimus Theobald, 1901 in China (Diptera: Culicidae).Mosq. Syst. 19: 143-5.

Zhou, S.S., Tang, L.H., Gu, Z.C. and Wang, Y. (2002). Sequencedifference of ribosomal DNA second internal trans spacerin Anopheles minimus in different localities. Zhongguo JiSheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. (Articlein Chinese). 20(1): 29-31.

3.9 The Philippinensis-NivipesComplex

Alam, M. T., Das, M. K., Dev, V., Ansari, M. A. and Sharma, Y. D.(2007). PCR-RFLP method for the identification of fourmembers of the Anopheles annularis group of mosquitoes(Diptera: Culicidae). Trans. R. Soc. Trop. Med. Hyg.101(3):239-44 (Epub 2006 Jun.).

Baimai, V., Green, C.A., Andre, R.G., Harrison, B.A. and Peyton,E.L. (1984). Cytogenetic studies of some speciescomplexes of Anopheles in Thailand and Southeast Asia.Southeast Asian J. Trop. Med. Pub. Hlth. 15: 536-46.


Elias, M., Rahman, M., Ali, M., Begam, J. and Chowdary, A.R.(1987). The ecology of malaria carrying mosquitoeAnopheles philippinensis Ludlow and its relation to malariain Bangladesh. BMRC Bull. 13(1): 192-201


Harbach, R. E., (2004). The classification of the genus Anopheles(Diptera : Culicidae): a working hypothesis of phylogeneticrelationships. Bull. Entomol. Res., 94: 537-53.

Klein, T.A., Harrison, B.A., Inlao, I. and Boonyakanist, P. (1982).Colonization of Thailand strains of Anopheles nivipes andAnopheles philippinensis. Mosq. News 42: 374-80.

Klein, T.A., Harrison, B.A., Baimai, V. and Phunkitchar, V. (1984).Hybridization evidence supporting separate species statusfor Anopheles nivipes and Anopheles philippinensis. Mosq.News 44: 466-70.

Krishnan, K.S. (1961). Vectors of malaria - An. philippinensisLudlow, 1902. In: Vectors of malaria in India. NationalSociety of India for Malaria and Mosquito Diseases, Delhi:27-37.

Nagpal, B.N. and Sharma, V.P. (1987). Survey of mosquito faunaof northeastern region of India. Indian J. Malariol. 24:143-9.

Prakash, Anil., Bhattacharya, D.R., Mohopatra, P.K. and Mahanta,J. (2000). Mosquito fauna and malaria vectors in Jirampur,district Changlong, Arunachal Pradesh. Indian J. Malariol.37: 74-81.

Praksash, Anil, Bhattacharya, D. R. Mohapatra,, P. K. andMohanta, J. (2004). Taxonomical observations onAnopheles philippinensis/nivipes group of mosquitoes innorth –east India. J. Commun. Dis. 36: 264-270.

Praksash, Anil, Bhattacharya, D. R. Mohapatra,, P. K. andMohanta, J. (2005). Potential of Anopheles philippinensis-nivipes complex mosquitoes as malaria vector in north –east India. J. Environ. Biol. 26: 719-724.

Prakash, Anil, Walton, C. Bhattacharyya, D. R., Mohapatra, P.K., Mahanta, J. (2006). Characterization of ITS2 rDNA ofAnopheles Philippinensis and An. nivipes(Diptera:Culicidae) from north-east India. South-East AsianJ. Trop. Med. Hyg. 37: 1134-1138.

Rajgopal, R. (1976). Studies on persistant transmission of malariain Burnihat, Meghalaya. J. Commun. Dis. 8: 235-45.


Reid, J.A. (1967). Two forms of Anopheles philippinensis inMalaya. J. Med. Entomol. 4: 175-9.

———. (1968). Anopheline mosquitoes of Malaya and Borneo.Stud. Inst. Med. Res. Malaya 31.

Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988).Cytotaxonomy of certain malaria vectors of India. In :Biosystematics of haematophagous insects. (ed. M.W.Service). pp. 25-37. Clarendon Press, Oxford.

Subbarao, S.K., Kumar, Vasantha, K., Nanda, Nutan, Nagpal,B.N., Dev Vas, and Sharma, V.P. (2000). Cytotaxonomicevidence for the presence of Anopheles nivipes in India.J. Mosq. Am. Contr. Assoc. 16(2): 71-4.

Walton, C., Somboon, P. Harbach, R. E., Zhang, S., Weerasinghe,I., O’ Loughlin, S. M. Phornpida, S. Sochantha, T., Tun-Lin, W., Chen, B. and Butlin, R. k. (2007). Molecularidentification of mosquito species in the Anopheles

annularis group in southern Asia. Med. Vet. Entomol. 21:30-35

3.10 The Punctulatus ComplexBeebe, N.W., Foley, H.D., Saul, A., Cooper, L., Bryan, J.H. and

Burkot, T.R. (1994). DNA probes for identifying themembers of the Anopheles punctulatus complex in PapuaNew Guinea. Am. J. Trop. Med. Hyg. 508: 229-34.

Beebe, N.W. and Saul, A. (1995). Discrimination of all membersof the An. punctulatus complex by Polymerase chainreaction-restriction fragment length polymorphismanalysis. Am. J. Trop. Med. Hyg. 53 (5): 478-81.

Beebe, N.W., Foley, H.D., Cooper, R.D., Bryan, J.H. and Saul, A.(1996). DNA probes for the An. punctulatus complex.Am. J. Trop. Med. Hyg. 54: 395-8.

Beebe, N.W., Ellis, J.T., Cooper, R.D. and Saul, A. (1999). DNAsequence analysis of the ribosomal DNA ITS-2 region forthe Anopheles punctulatus group of mosquitoes. Insect.Mol. Biol. 82: 381-90.

Booth, D.R., Mahon, R.J. and Sriprakash, K.S. (1991). DNAprobes to identify members of the Anopheles farauticomplex. Med. Vet. Entomol. 5: 447-54.

Bryan, J.H. (1970). A new species of Anopheles punctulatuscomplex. Trans. Roy. Soc. Trop. Med. Hyg. 64: 28.

Bryan, J.H. (1973a). Studies on the Anopheles punctulatuscomplex. I. Identification by proboscis morphologicalcriteria and by cross-mating experiments. Trans. Roy. Soc.Trop. Med. Hyg. 67: 64-9.

Bryan, J.H. (1973b). Studies on the Anopheles punctulatuscomplex. II. Hybridization of the member species. Trans.Roy. Soc. Trop. Med. Hyg. 67: 70-84.

Bryan, J.H. and Coluzzi, M. (1971). Cytogenetic observationson Anopheles farauti Laveran. Bull. Wld Hlth Org. 45:266-7.

Bryan, J.H., Reardon, T. and Spark, R. (1990). How many speciesare in the Anopheles punctulatus group? Ann. Trop. Med.Parasitol. 84: 295-7.

Cooper, L., Cooper, R.D. and Burkot, T.R. (1991). Anophelespunctulatus complex: DNA probes for identifying theAustralian species using isotopic, chromogenic, andchemiluminescence detection systems. Rimental Parastiol.73: 27-35.

Cooper, R.D., Waterson, D.G., Bangs, M.J. Beebe, N.W. (2000).Rediscovery of Anopheles (Cellia) clowi (Diptera:Culicidae) is rarely recorded members of the Anophelespunctulatus group. J. Med. Entomol. 37: 840-48.

__________, Frances, S.P., Beebe, N.W., Sweeney, A.W. (2002).Speciation and distributions of the members of theAnopheles punctulatus (Diptera: Culicidae) group in PupaeGuinea. J. Med. Entomol. 39: 16-27.

Foley, D.H. and Bryan, J.H. (1993). Electrophoretic keys toidentify members of the Anopheles punctulatus complexof vector mosquitoes in Papua New Guinea. Med. Vet.Entomol. 7: 49-53.

Foley, D.H. and Bryan, J.H. (2000). Showed salinity toleranceinvalities a test for the malaria vector Anopheles faruatis.s. on Guadal canal, Sukmore Island. Med. Veter. Entomol.14: 450-2.

Foley, D.H., Cooper, R.D. and Bryan, J.H. (1995). Allozymeanalysis reveals a new species within the Anophelespunctulatus complex in Western Province, Papua NewGuinea. J. Am. Mosq. Contrl. Assoc. 11: 122-7.


Foley, D.H., Meek, S.R. and Bryan, J.H. (1994). The Anophelespunctulatus group of mosquitoes in the Solomon islandsand Vanuatu surveyed by allozyme electrophoresis. Med.Vet. Entomol. 8: 340-50.

Foley, D.H., Paru, R., Dagoro, H. and Bryan, J.H. (1993).Allozyme analysis reveals six species within the Anophelespunctulatus complex of mosquitoes in Papua New Guinea.Med. Vet. Entomol. 7: 37-48.

Hartas, J., Whelan, P., Sriprakash, K.S. and Booth, D. (1992).Oligonucleotide probes to identify three sibling species ofthe Anopheles farauti Laveran complex (Diptera:Culicidae). Trans. Roy. Soc. Trop. Med. Hyg. 86: 210-2.

Kondrashin, A.V. and Rashid, K.M. (1987). Epidemiologicalconsiderations for planning malaria control in South-EastAsia Region. World Health Organization, Regional Officefor South-East Asia, New Delhi, p. 411.

Mahon, R.J. and Miethke, P.M. (1982). Anopheles farauti No. 3,a hitherto unrecognized biological species of mosquitowithin the taxon An. farauti Laveran (Diptera: Culicidae).Trans. Roy. Soc. Trop. Med. Hyg. 76: 8-12.

Rozeboom and Knight (1946). The punctulatus complex ofAnopheles (Diptera: Culicidae). J. Parasitol. 32: 95-131.

Schmidt, E.E., Foley, D.H., Hartel, G.F., Williams, G.M. and Bryan,J.H. (2001). Descriptions of the Anopheles (Cellia) farauticomplex of sibling species (Diptera : Culicidae) in Australia.Bull. Entomol. Res. 91: 389-410.

Sweeney, A.W. (1987). Larval salinity tolerances of the siblingspecies of Anopheles farauti. J. Am. Mosq. Contr. Assoc.3: 589-92.

3.11 The Sinensis ComplexBaimai, V., Rattanarithikul, R. and Kijchalao, U. (1993).

Metaphase karyotypes of Anopheles of Thailand andsoutheast Asia : 1. The Hyreanus group. J. Am. Mosq.Contr. Assoc. 9(1): 59-67.

Chow, Y. (1970). Biomics of malaria vectors in the western pacificregion. Southeast Asian. J. Trop. Med. Publ. Helt., 1: 40-57.

Choochote, W., Jitpakdi, A., Rongsriyam, Y., Komalamisra, N.,Pitasawat, B. and Palakul, K. (1998). Isoenzyme studyand Hybridization of two strains of Anopheles sinensis(Diptera: Culicidae) in northern Thailand. Southeast AsianJ. Trop. Med. Publ. Hlth. 29(4): 841-8.

Coleman, R.E., Kiattibut, C., Sattabongkot, J., Ryan, J., Burbett,D.A., Kim. H.C., Lee, W.J. and Klein, T.A. (2002).Evaluation of anopheline mosquitoes (Diptera: Culicidae)from the Republic of Korea for Plasmodium vivaxCircumsporozoite protein. J. Med. Entomol., 39(1): 244-7.

Gao, Q., Beebe, N.W., and Cooper, R.D. (2004). Molecularidentification of the malaria vectors Anopheleslanthropophagus and Anopheles sinensis (Diptera:Culicidae) in central China using polymerase chain reactionand appraisal of their position within the Hyranus group.J. Med. Entomol. 41(1): 5-10


Kanda, T. and Oguma, Y. (1977). Hybridization betweenAnopheles sinensis and Anopheles sineroides. Mosq. News.37(1): 115-7.

Kanda, T. and Oguma, Y. (1978). Anopheles engarensis, a newspecies related to sinensis from Hokkido Island, Japan.Mosq. Syst. 10: 45-52.

Kanda, Tozo, Takai, K., Oguma, Y., Chiang, G.L., Cheong, W.H.,Sucharit, Joesoef, A.M. and Imajo, S. (1981). Evolutionarygenetics of the Anopheles hyrencus group, theLeucosphyrus group and the Pyretophorus group in EastAsia and the Pacific area. In : Cytogenetics and geneticsof vectors (eds. R. Pal, J.B. Kitzmiller and T. Kanda). KoanstaLtd., Tokyo.

Li, B.W., H.L. Lu, K.T. Yao, and D. Liu. 1991. Restriction fragmentlength differences of genomic repetitive DNA from fivesibling species of the Anopheles hyrcanus group. ChineseJ.Parasitol. Para. Dis. 9: 8-11.

Lee, H.I., Lee, J.S., Shin, E.H., Lee, W.J., Kim, Y.Y. and Lee, K.R.(2001). Malaria transmission potential by Anophelessinensis in the Reublic of Korea. Korean J. Paraistol. 39(2): 185-92 (from abstract).

Ma, S.F. 1981. Studies on the Anopheles (A.) sinensis group ofmosquitoes in China, including four new sibling species,[in Chinese] Sinozoologia 1: 59-74.

Ma, Y. J., Qu, F. Y., and Xu, J. J. (1998). Sequence differences ofrDNA-ITS-2 and species diagnostic PCR assay of Anophelessinensis and Anopheles anthropophagus from China. J.Med. College PLA 13: 123-8.

Ma, Y. J., Qu, F. Y., Xu, J. J. and Song, G. H. (2000). Differencesin sequences of ribosomal DNA second internaltranscribed spacer among three members of Anopheleshyrcanus complex from the Republic of Korea. Entomol.Sinica. 7: 36-40.

Ma, Y. Xu, J. (2005). The Hyrcanus Group of Anopheles(Anopheles) in China (Dipter: Culidae): SpeciesDiscrimination and Phylogenetic Relationships Inferred byRibosomal Internal Transcribed Spacer 2 Sequences. J.Med. Entomol. 42 (4): 610-9.

Min, G.S., Choochote, W., Jitpakdi, A., Kim, S.J., Kim, W., Jung,J. and Junkum, A. (2002). Intraspecific hybridization ofAnopheles sinensis (Diptera: Culicidae) strains fromThailand and Korea. Mol. Cells. 14(2): 198-204.

O’Connor, C.T. (1980). The Anopheles lyrcanus group inIndonesia. Mosq. Syst. 12: 293-305.

Oguma, Y. (1978). Crossing studies among six strains ofAnopheles sinensis. Mosq. News. 38(3): 357-66.

Oguma, Y. and kanda, T. (1970). The salivary gland chromosomesof Anopheles koreicus. Jap. J. Sanit. Zool. 21: 114.

Oguma, Y. and kanda, T. (1976). Laboratory colonization ofAnopheles sinensis Weidman 1828. Jap. J. Sanit. Zool. 27:319-24.

Ree, H.I., Hwang, U.W., Lee, I.Y. and Kim, T.E. (2001). Dailysurvival and human blood index of Anopheles sinenesisthe vector species of malaria in Korea. J. Am Mosq. Contr.Assoc. 1791): 67-72.

Rongsriyam, Y., Jitpakdi, A., Choochote, W., Samboon, R.,Tookyang, B. and Suwonkerd, W. (1998). Comparativesusceptibility of two forms of Anopheles sinensisWiedmann 1828 (Diptera: Culicidae) to infection withPlasmodium falciparum, P. vivax, P. yoelii and determinationof misleading factor for sporozoite identification. SoutheastAsian J. Trop. Med. Publ. Hlth. 29: 159-67.

Sleigh, A. C., X.L. Liu, S. Jackson, P. Li, and L.Y. Shang. 1998.Resurgence of vivax malaria in Henan Province, China.Bull. W.H.O. 76: 265-70.

Stricman, D., Miller, M.E., Kim, H.C. and Lee, K.W. (2000).Mosquito surveillance in the demilitarized zone, Republicof Korea during an outbreak of P. vivax of malaria. J. Am.Mosq. Contr. Assoc. 16: 100-13.


Wilkerson, R., C., Li, C., Rueda, L. M., kim, H., Klien, T. A.,Song, G. and Stricman, D. (2003). Molecular confirmationof Anopheles (Anopheles) lesteri from the Republic of SouthKorea and its genetic identity with An. (Ano.)anthropophagus from China (Diptera: Cculicidae). Zootaxa378: 1-14.

3.12 The Subpictus ComplexAbhayawardana, T.A., Wijesuriya, S.R.E. and Dilrukshi, R.K.C.

(1996). Anopheles subpictus complex: Distribution ofsibling species in Sri Lanka. Indian J. Malariol. 33: 53-60.

Amerasinghe, F.P., Amerasinghe, P.H., Peiris, J.S.M. and Wirtz,R.A. (1991). Anopheline ecology and malaria infectionduring the irrigation development of an area of theMahaweli project, Sri Lanka. Am. J. Trop. Med. Hyg. 45:226-35.

Amerasinghe, P.H., Amerasinghe, F.P., Wirtz, R.A., Indrajith, N.G.,Somapala, W., Pereira, L.R. and Rathnayake, A.M.S.(1992). Malaria transmission by Anopheles subpictus(Diptera: Culicidae) in a new irrigation project in Sri Lanka.J. Med. Entomol. 29: 577-81.

Atrie, B. (1994). Cytogenetic studies of different field populationsof Anopheles annularis Van Der Wulp and Anophelessubpictus Grassi. Ph.D. thesis, Dept. of Zoology Dept.University of Delhi, Delhi, India.


Kelley-Hope, L. A., Yapabandara, A. M. G. M., Wickramasinghe,M. B., Perera, Karunaratne, S. H. P. P., Fernando, W. P.,Abeyasinghe, R. R., Herath, P. R. j., Galappaththy, G. N. L.and Hemingway, J. (2005). Spatiotemporal distribution ofinsecticide resistance in Anopheles culicifacies andAnopheles subpictus in Sri Lanka. Trans. Roy. Soc. Trop.Med. Hyg. 99: 751-61.

Kulkarni, S.M. (1983). Detection of sporozoites in Anophelessubpictus in Bastar District, Madhya Pradesh. Indian J.Malariol. 20: 159-60.

Nanda, N., Das, C.N.S., Subbarao, S.K., Adak, T. and Sharma,V.P. (1987). Studies on the development of Plasmodiumvivax in Anopheles subpictus. Indian J. Malariol, 24: 135-42.

Panicker, K.N., Geetha Bai, M., Bheema Rao, U.S., Viswam K.and Suryanarayanamurthy, U. (1981). Anopheles subpictusvector of malaria in coastal villages of South-East India.Curr. Sci. 50: 694-5.


Reid, J.A. (1966). A note on Anopheles subpictus Grassi and An.indefinitus Ludlow (Diptera: Culicidae). J. Med. Entomol.3: 327-31.

Reuben, R. and Suguna, S.G. (1983). Morphological differencesbetween sibling species of the taxon Anopheles subpictusGrassi in India, with notes on relationships with knownforms. Mosq. syst. 15: 117-26.

Singh, S.P., Raghavendra, K., Nanda, N. and Subbarao, S.K.(2004). Morphotaxonomic studies to identify the membersof the Anopheles subpictus Grassi (Diptera: Culicidae)species complex in riverine villages of district Sonepat,Haryana state, India. J. Commun. Dis. 36: 35-40.

Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988).Cytotaxonomy of certain malaria vectors of India, pp. 25-37. In : Biosystematics of haematophagous insects M.W.Service (ed.)., Clarendon Press, Oxford.

Suguna, S.G. (1982). Cytological and morphological evidencefor sibling species in Anopheles subpictus Grassi. J.Commun. Dis. 14: 1-8.

Suguna, S.G., Gopala Rathinam, K., Rajavel, A.R. and Dhanda,V. (1994). Morphological and chromosomal descriptionsof new species in the Anopheles subpictus complex. Med.Vet. Entomol. 8: 88-94.

Sundararaman, S., Soeroto, R.M. and Siran, M. (1957). Vectorsof malaria in mid-Java. Indian J. Malariol. 11: 321-38.

Van Hell, J.C. (1952). The Anopheline fauna and malaria vectorsin South Celebes. Documenta Med. Geogr. Trop. 4: 45-6.

3.13 The Sundaicus ComplexAlam MT, Das MK, Ansari MA, Sharma YD (2005). Molecular

identification of new sibling species of human malariavector Anopheles sundaicus from Andaman and Nicobarislands of India. Acta Trop 1738: 1–9.

Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Marwoto, H.,Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria inthe Menoreh hills of Central Java. Am. J. Trop. Med. Hyg.,66(3): 287-92.

Collins, R. T., Jung, R. K., Anoez, H., Sutrisno, R. H. and Putut,D. (1979). A study of the coastal malaria vectors, Anophelessundaicus (Rodenwaldt) and Anopheles subpictus Grassiin south Sulawesi, Indonesia. Geneva: World HealthOrganization. WHO/Mal. 79.

Das, M.K., Nagpal, B.N. and Sharma, V.P. (1998). Mosquito faunaand breeding habitats of anophelines in Car Nicobarislands. Ind. J. Malariol. 35: 197-205.

Dash, A. P., Hazra, R. K., Mahapatra, N., Tripathy, H. K. (2000).Disappearance of malaria vector Anopheles sundaicus fromChilka lake area of Orissa state in India. Med. Vet. Entomol.14: 445-9.

Dusfour, I., Harbach, R. E. and Manguin, S. (2004). Bionomicsand systematics of the Oriental Anopheles sundaicuscomplex in relation to malaria transmission and vectorcontrol. Am. J. Trop. Med. Hyg. 71(4): 518-24.

Dusfour, I., Linton, Y. M., Cohuet, A., Harbach, R. E., Baimai, V.Trung, H. D., Seng, C. M., Matusop, A. and Manguin, S.(2004). Molecular Evidence of Speciation Between Islandand Continental Populations of Anopheles (Cellia)sundaicus (Dipter: Culicidae), a Principal Malaria VectorTaxon in Southeast Asia. J. Med. Entomol. 41(3): 287-95.Harbach, R. E., (2004). The classification of the genusAnopheles (Diptera : Culicidae) : a working hypothesis ofphylogenetic relationships. Bull. Entomol. Res., 94: 537-53.


Kalra, N. L. (1978). National Malaria Eradication Programme,India – its problems, management and research needs. J.commun. Dis. 10: 1-20.

Kirnowardoyo, S. (1988). Anopheles malaria vector and controlmeasures applied in Indonesia. Southeast Asian J. Trop.Med. Pub. Hlth. 19: 713-6.

Kirnowardoyo, S. and Gambiro, P.Y. (1987). Entomologicalinvestigations of an outbreak of malaria in Cilacap on southcoast of Central Java, Indonesia during 1985. J. Commun.Dis. 19: 121-7.

Kumari, R. and Sharma, V.P. (1994). Resting and biting habits ofAnopheles sundaicus in Car Nicobar island. Indian J.Malariol. 31: 103-14.


Kumari, R., Joshi, Hema, Giri, A. and Sharma, V.P. (1993). Feedingpreferences of Anopheles sundaicus in Car Nicobar Island.Indian J. Malariol. 30: 201-6.

Linton, Y. M., Harbach, R.E., Chang, M. S., Anthony, T. G. andMatusop, A. (2001). Morphological and molecular identityof Anopheles (Cellia) sundaicus (Diptera: Culicidae), thenominotypical member of a malaria vector speciescomplex in Southeast Asia. Syst. Entomol. 26: 357-66.

Linton,Y. M., Dusfour, I., Howard, T. M., Ruiz, F., Minh, N. D.,Trung, H. D., Sochanta, T., Coosemans, M. and Harbach,R. E. (2005). Anopheles (Cellia) epiroticus (Diptera:Culicidae), a new malaria vector species in the SundaicusComplex. Bull. Entomol. Res. 95: 329-39

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