Joumal of the American Mosquito Control Association, 12(4):669-675, 1996Copyright O 1996 by the American Mosquito Control Association, Inc.

METAPHASE KARYOTYPES OF ANOPHELES OF THAILANDAND SOUTHEAST ASIA. VI. THE PYRETOPHORUS

AND THE NEOMYZOMYIA SERIES, SUBGENUSCELLIA (DIPTERA: CULICIDAE)

VISUT BAIMAI.I UDOM KIJCHALAO' eIo RAMPA RATTANARITHIKUL,

ABSTRACT, A total of 6 species of the Pyretophorus (4 species) and Neomyzomyia (2 species) series of thesubgenus Cellia of Anopheles were used for metaphase karyotype analysis. Anopheles subpictus and An. vagusexhibit 4 and 2 forms of mitotic karyotypes, respectively, which are attributable to different types of Y chro-mosomes. Such distinctive mitotic chromosomes may reflect interspecies differences within each of these 2 taxa.Two distinct species, An. indefinitus and An. sundaicus, show similar metaphase karyotypes, particularly withregard to the size and shape of the sex chromosomes. Likewise, An. tessellatus and An. kochi, which are distinctspecies of the Neomyzomyia Series, also have metaphase karyotypes that resemble each other. They exhibit atypical feature of telocentric sex chromosomes resembling those of the An. dirus complex and the other speciesof the Leucosphyrus Group. Like the other cases of the Oriental Anopheles, heterochromatin has played asignificant role in chromosome evolution of the 6 species.

INTRODUCTION

The subgenus Cellia of the gents Anophelescomprises 4 series, namely, Neocellia, Myzomyia,Pyretophorus, and Neomyzomyia (Reid 1968).Metaphase karyotypes of many species belongingto the first 2 series have been reported previously(Baimai et al. 1993b, 1994, 1996). Since some ofthe species belonging to the Neomyzomyia Series,particularly the Leucosphyrus Group, which in-cludes the An. dirus and the An. leucosphyrus com-plexes, are primary vectors of human malarial par-asites in Thailand and neighboring countries, theyhave received a priori attention. The results of ourstudies on metaphase karyotypes of these specieswere reported previously (Baimai et al. 1987, 1988;Baimai 1988a for reviews). Our earlier reports haveshown that analysis of mitotic karyotypes is a use-ful cytotaxonomic tool for separating some crypticspecies of the Oriental Anopheles. In this final re-port of the series, we present the results of meta-phase karyotype analysis of the remaining 2 speciesof the Neomyzomyia Series found in Thailand and4 species of the Pyretophorus Series occurring inthe Oriental Region.

MATERIALS AND METHODS

Totals of 4 and 2 species of the Pyretophorus andNeomyzomyia series, respectively, from Thailand,Indonesia, and Bangladesh were examined cytolog-ically. They include An. subpictus Grassi, An. in-definitus Ludlow, An. sundaicus Rodenwaldt, An.vagus Doenitz, An. tessellarus Theobald, and An.kochi Doenitz. Adult females of these species werecollected from bovine or human bait at different

I Department of Biology, Faculty of Science, MahidolUniversity, Rama VI Road, Bangkok IO4OO, Thailand.

2 Department of Medical Entomology, U.S. Army Med-ical Component, Armed Forces Research Institute of Med-ical Sciences (AFRIMS), Bangkok 10400, Thailand.

localities (Table 1). Fully bloodfed, wild-caught fe-males were identified morphologically to species asfar as possible. All females were individually setup to produce F, larval progeny for chromosomestudy. Brain ganglia of 4th-instar larvae from eachisofemale line were used for mitotic chromosomepreparations and analysis employing the techniquesdescribed by Baimai (1977) and Baimai et al.(1993a).

RESULTS

The 6 species (including forms) examined cyto-logically in this study exhibit uniformity in chro-mosome number (2n : 6) except for the existenceof supernumerary (B) chromosomes in some sam-ples of An. indefinitus from Kanchanaburi Province,western Thailand. Thus, the metaphase karyotypeof these species consists of, as a general rule, twopairs of autosomes and a pair of heteromorphic sexchromosomes that may vary in size and shape de-pending on the amount and distribution of consti-tutive heterochromatin. Differences in mitotic chro-mosomes found in this study are briefly describedbelow.

Anopheles subpictus: This morphological spe-cies is widely distributed in Southeast Asia. The 2pairs of autosomes are quite uniform in all speci-mens examined. Differences in mitotic chromo-somes were found in the X and Y chromosomes.Two types of X chromosome and 4 types of Y chro-mosome were observed among the 15 families ex-amined. The X, is submetacentric. The short armof the X, is entirely heterochromatic while the longarm consists of a euchromatic portion and a con-spicuous block of centromeric heterochromatin(Figs. 1-9). The X, appears to be metacentric, dif-fering from the X, in having a major block of het-erochromatin at the distal end of the heterochro-matic arm. This difference between the X, and theX, can be readily seen in a heterozygous female

669

670 JounNll or rue AvpnrcAN Moseurro CoNrr.ol AssocrenoN VoL. 12 , No.4

Table 1. The number of females (isolines) of 4 and 2 species of the Pyretophorus and the Neomyzomyia series,respectively, collected and examined cytologically from different wild populations in Thailand, Indonesia, the

Philippines, and Bangladesh. All localities are listed as districts and provinces.

Species/form Locality

No. ofisolines

examinedDate of

collection

Pyretophorus Series

An. subpictusForm A

Form B

Form CForm D

An. indefnitus

An. sundaicusAn. vagus

Form AForm B

Neomyzomyia Series

An. tessellatusAn. kochi

Flores, IndonesiaLuzon (Montalban), PhilippinesPathiu, ChumphonFlores, IndonesiaLuzon ( Montalban), PhilippinesPathiu, ChumphonSawee, ChumphonSrinakarin Dam, KanchanaburiTaknaf, BangladeshTakuapa, PhangngaNangrong Fall, Nakhon NayokMaetang, ChiangmaiSadao, Songkhla

Tirng Ka Ngok, PhangngaTung Ka Ngok, PhangngaMaetang, Chiangmai

221IJ

I5J

6^J

22

April 1984August 1986March 1986April 1984August 1986March 1986March 1986August 1982November 1984March 1990August 1982August 1982January 1986

June 1982June 1982September 1982

J

24

(Figs. 4, 6, and 9). The Y, is subtelocentric (acro-centric) (Fig. l) while the Y, is metacentric (Fig.3). The Y, and Y, are apparently similar in size.The Y, has a large submetacentric shape (Fig. 5)when compared with the large metacentric Yo chro-mosome (Fig. 7). Thus, these types of Y chromo-some clearly differ from each other in the amountof heterochromatin and the position of the centro-mere. Based on the different types of Y chromo-some, 4 forms of metaphase karyotypes are rec-ognized in An. subpictus, i.e., form A (X,, Y,), formB (X,, Xr, Yr), form C (X,, Xr, Y.) and form D(X,, Xr, Y4). Of these forms, form B appears to bewidely distributed since it was found in Thailand,Indonesia, and the Philippines. Form A appears tobe common in Indonesia and the Philippines. Incontrast, forms C and D were found only in Thai-land, with form C occurring in sympatry with formB at Chumphon Province. Such distinctive forms ofmitotic chromosomes may reflect interspecies dif-ferences within the taxon An. subpictus. Unfortu-nately, we did not have information on polytenechromosomes for comparison with the fixed inver-sion of the 4 species of the An. subpictus complexdescribed by Suguna et al. (1994). This interestingpossibility of cryptic species warrants further in-vestigations on the population genetics of An. sub-

pictus, especially in the areas where this taxon hasplayed a major role in malaria transmission.

Anopheles indefinitus: Samples of this speciesfrom Thailand and Bangladesh show generally sim-ilar metaphase karyotypes. Interestingly, specimensfrom Thailand exhibit 2 supernumerary (B) chro-mosomes as previously reported (Baimai et al.1984). Two types of X chromosome were observed.The X, is telocentric with a very large portion ofcentromeric heterochromatin occupying about onehalf of the chromosome length (Figs. 11 and l2).The X, is a larger telocentric chromosome due tothe presence of extra block(s) of heterochromatin,possibly in the distal area of centromeric hetero-chromatin (Figs. 10 and 12). The Y chromosome isa telocentric chromosome of considerable size com-pared with the X, (Figs. 10 and l l). The autosomesII and III consist of prominent blocks of pericentricheterochromatin (Figs. 10-12).

Anopheles sundaicus; The metaphase karyotypeof this species is, in general, similar to that of An.indefinitus mentioned above, although An. sundai-czs is morphologically and ecologically quite a dis-tinct species. Nevertheless, An. sundaicus exhibitslarge telocentric X and Y chromosomes (Figs. 13and 14) compared with those of An. indefinitus.Two types of telocentric X chromosome were re-

Figs. 1-12. Metaphase karyotypes from larval neuroblast cells. l-9, Anopheles subpictus. l,2.Male and female,respectively, of form A; 3, 4. Male and female, respectively, of form B; 5, 6. Male and female, respectively, of formC;7,8-9. Male and female, respectively, of form D; 10-ll, 12. Male and female, respectively, of An. indefinitus(arrows indicate supernumera"ry chromosomes).

DECEMBER 1996 METAPHASE KARYOTYPES OF ANOPHELES

riltltI U FormD

! : l

lilr!!U

[] Xl X2Y2

ll E Form B

l ! lsundoicus

n

l ! l l l t !LlLlXX2YLlLlI u [ | I I

t! lhULI \X2YI t [

subpicius

ffi lll [[! Irlxt x2Y3Form CI U FormB I r lI t I | t

xr YlForm A

vogus

Xr XeYl

Form A

Fig. 25. Diagrammatic representation and comparison of metaphase karyotypes of 4 species (including forms) ofthe Pyretophorus Series and 2 species of the Neomyzomyia Series. Only one set of autosomes II and III is presented.Variable heterochromatin is indicated in black or shaded. The centromeres are indicated by constrictions of eachchromosome. Chromosome lengths, arm ratios, and heterochromatic portions are shown in proportion.

corded in this study. The X, is clearly larger than C, which were discovered recently from the Indo-the X, primarily due to the acquisition of hetero- nesian populations (Sukowati and Baimai 1996).chromatin in the centromeric region (Fig. 14). This Anopheles vdg&s: This species exhibits conspic-type of metaphase karyotype is referred to as form uous blocks of pericentric heterochromatin in bothA to distinguish it from 2 additional forms, B and pairs of autosomes. There are 2 types of X chro-

Figs. 13-24. Metaphase karyotypes from larval neuroblast cells. 13, 14. Male and female, respectively, of Anoph-

eles sundaicus; t5-19, An. vagus. 15, 16. Male and female, respectively, of form A; 17, 18-19. Male and female,

respectively, of form B; 20, 21. Male and female, respectively, of An. tessellatus; 22, 23-24. Male and female, re-

spectively, of An. kochi.

tessellotus kochi

il[EE,IIHEIuu

XX2Y ll Ll

rX2Y[ u u t r I

674 JounNeL op rgg Avgnlcnx Mosqutro Conrnol AssocrnrroN Vor-. 12, No.4

mosome. The X, is submetacentric. Its long armconsists of a euchromotic portion and a large blockof centromeric heterochromatin, while the shortarm is totally heterochromatic (Figs. 15-19). TheX, is also submetacentric but its heterochromaticarm is slightly longer than that of the X, due to thepresence of an extra block of heterochromatin(Figs. 16-18). The general figure of these types ofX chromosome corresponds with that of An. vagusfrom the Indian subcontinent (Avirachan et al.1969). Two types of Y chromosome were also ob-served. The Y, has a normal submetacentric shape(Fig. 15), while the Y, is a larger submetacentricchromosome due to the presence of an extra blockof heterochromatin in the short arm (Fig. 17). Thus,2 forms of metaphase karyotype are recognized inAn. vagus based on the two types of Y chromo-some, viz., form A (X,, Xr, Y,) and form B (X,, Xr,Yr). Form B seems to be widespread since it wasfound in Chiangmai and Songkhla provinces innorthern and southern Thailand, respectively, whileform A was detected in Nakhon Nayok Province.It is not known at this stage whether these 2 formsof metaphase karyotype represent intra- or inter-species differences.

Anopheles tessellatus: This species shows dis-tinctive X and Y chromosomes which are typicalof the Neomyzomyia Series as demonstrated in theLeucosphyrus Group. The X chromosome is telo-centric, consisting of large blocks of centromericheterochromatin occupying approximately twothirds of the chromosome length. Two distinctivetypes of X chromosome were found among oursamples. The X, is shorter than the Xr, which ob-viously contains a larger amount of centromericheterochromatin (Fig. 21). The Y chromosome hasa short telocentric shape compared with that of theX, (Fig. 20). The autosome pairs show a limitedamount of pericentric heterochromatin.

Anopheles kochi: The metaphase karyotype ofthis species resembles that of An. tessellatus, de-scribed above. It consists of 2 types of X chromo-some. The telocentric X, is slightly longer than theX, chromosome due to the presence of centromericheterochromatin (Figs. 23 and 24). Tl;ie Y chro-mosome is also telocentric and is only slightlyshorter than the X, (Fig. 22); the Y chromosome ofthis species is longer than that of An. tessellatus. Alimited amount of pericentric heterochromatin ispresent in the autosome pairs.

Diagrammatic representations of the metaphasekaryotypes of the 6 species, including forms, foundin this study are shown in Fig. 25.

DISCUSSION

Analysis of metaphase karyotypes of the 15 is-ofemale lines derived from the wild-caught femalesof An. subpict s collected from different localitiesin Thailand, Indonesia, and the Philippines has re-vealed remarkable differences in the Y chromo-

some with respect to the amount of constitutive het-erochromatin. Four forms of mitotic karyotypes arerecognized based on the different types of Y chro-mosome. However, none of these forms corre-sponds with the mitotic karyotype of "An. subpic-trrr" reported by Avirachan et al. (1969). Further-more, Suguna et al. (1994) described a paracentricfixed inversion on the X chromosome of 4 species(A, B, C, and D) in the An. subpictus complex inIndia. We are unable to say what species of the An.subpictus complex occurs in Thailand because wedid not have polytene chromosome data from ourmaterial for comparison with that of Suguna et al.(1994). In our earlier work, we reported somegroups of closely related species which are knownto have different types of the Y chromosome, forexample, the An. dirus complex (Baimai 1988a),the An. macukttus complex (Baimai et al. 1993b),and the An. minimus and the An. culicifacies com-plexes (Baimai et al. 1996). It is possible that someof these forms of metaphase karyotypes of An. sub-pictus might represent distinctive characters at aninterspecific level. Further cytogenetic study of thispossible species complex is required to clarify thisspecies problem since An. subpictus plays an im-portant role in malaria transmission in certain areasof Southeast Asia, including Indonesia (Kirnowar-doyo 1988) and Malaysia (Loong 1988). A similarsituation is found in the 2 forms of An. vagzs whichexhibit distinctive Y chromosomes.

There has always been some taxonomic confu-sion in separating An. subpictus and An. indefinitusbased on morphological criteria alone (Reid 1968).The evidence from metaphase karyotype analysisshown in this study clearly demonstrates that these2 closely related species are quite distinct cytolog-ically, particularly in the size and shape of hetero-morphic sex chromosomes (Fig. 25). On the con-trary, An. indefinitus and An. sundaicus exhibit sim-ilar metaphase chromosomes despite their distinctmorphologies. The metaphase karyotypes of An.tessellatus and An. kochi generally resemble thoseof the other species of the Neomyzomyia Seriesdescribed previously (Baimai 1988b) despite theirdistinctiveness in morphology.

Analysis of metaphase karyotypes has proved tobe useful as a cytotaxonomic tool in separatingsome cryptic species of anopheline mosquitoes, atleast in the Southeast Asian region, as demonstratedin this series of publications (Baimai et al. 1993a,1993b, 1994, 1996). Most, if not all, differences inmitotic chromosomes observed in this study and inthe previous reports are due to the acquisition ofextra blocks of constitutive heterochromatin. Thusthe accumulation of heterochromatin in the sexchromosomes and, to a lesser extent, in the centro-meric region of the autosomes, may play a signif-icant role during the processes of species diver-gence, although the functional aspect of hetero-chromatin remains an unsolved problem (John andMiklos 1979). Further studies of mitotic chromo-

METAPHASE KARYOTYPES OF ANOPHELES 675

some variation attributable to gain of heterochro-matin in the genome of a large number of speciesof Anopheles would contribute to a better under-standing of the processes of speciation and possibly

coevolution between the Plasmodiutn parasites andtheir mosquito vectors (Baimai 1988a).

ACKNOWLEDGMENTS

We thank P. J. Grote for valuable comments onthe manuscript. We also thank K. Vejsanit and PPanthusiri for preparing the illustrations. This workhas been supported in part by UNDP/World BanVWHO Special Program for Research and Tfainingin Tropical Diseases and by the Armed Forces Re-search Institute of Medical Sciences (AFRIMS),

Bangkok.

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