Chapter 2: REVIEW OF LITERATURE

11

The microbial control of mosquitoes is a very promising and rapidly developing

area for the management of mosquito borne diseases. The growing interest in the

microbial control of mosquitoes in the recent times is primarily due to various limitations

posed by chemical insecticides viz., resistance in mosquitoes, prohibitive cost of alternate

insecticides and growing environmental concerns. To cut down dependence on these

chemicals, Bacillus thuringiensis israelensis and Bacillus sphaericus, being biological

control agents of mosquitoes, provide an excellent alternative and cutting edge over

chemical insecticides being environmentally safe and target specific besides being reliable

and efficient tools. Obviously the journey from the first isolation to recent

commercialization and field application has been quite long and indeed challenging.

Winston Churchill once said 'those who ignore history are doomed to repeat it'.

Edward Steinhaus, the father of modern insect pathology, while expressing a similar view,

stressed the importance of understanding the thoughts and philosophies that guided our

predecessors in the research on microbial insecticides as we develop our own programmes

on them. In this chapter, therefore, the available literature has been reviewed on all round

progress made in the research on mosquito-pathogenic bacilli, especially from the earlier

days of their search, isolation, characterization and identification to the more recent

research on bio-efficacy trials, their persistence in the environment, safety profile and large

scale field application in the mosquito control programmes.

2.1 Isolation of mosquito-pathogenic bacilli

2.1.1 The Source: The earliest report on isolation of Bacillus sphaericus comes from

Kellen and Meyers (1964). Kellen, during routine surveillance of rock holes near Big

Creek, Fresno, California, collected several moribund fourth instar larvae of Culiseta

12

incidens. From these larvae, he isolated several bacteria besides B. sphaericus. On the

other hand, Goldberg and Margalit (1977) isolated B. thuringiensis israelensis from larvae

obtained from the stagnant ponds located in a dry desert in Israel. The silt and water

samples containing these larvae were taken to the laboratory and refrigerated, and

processed for isolation and assayed for larvicidal activities. The one which was extremely

toxic to mosquito larvae was designated as ONR -60A from which was derived the now

widely used B. thuringensis israelensis serotype H-14 (de Barjac, 1978).

_ Menon et al. (1982) isolated rapidly larvicidal spores bearing Bacilli (ISPC-5)

from diseased Culex fatigans larvae and identified them as B. sphaericus. Weiser (1984)

isolated mosquito larvicidal Bs strain 2362 from blackfly. Subsequently, Lysenko et al.

(1985) isolated 5 new strains of B. sphaericus, possessing high larvicidal activity from

non-mosquito sources ie. Catterpillars and grasshoppers against which, they were

ineffective.

Manonmani et al. (1987) collected soil, water and larval samples from the

transient pools, coconut gardens, Casuarina plantations, paddy fields, garden lands, river

beds, polluted habitats, irrigation canals, ponds, estuaries and deer sanctuaries. From these

they isolated 101 strains of B. thuringiensis H-14 and 11 non H-14 serotypes showing

activity against Culex quinquefasciatus larvae. They discovered a subgroup of serotype H-

20 and designated it as B. thuringiensis var. pondicherriensis. Martin and Travers (1989)

using acetate selection method examined soil samples for mosquito-pathogenic bacilli from

five continents, Africa, Asia, Europe, North and South America and their associated

islands. They isolated Bt in 785 out of 1115 soil samples and found that insect control

agent Bt was a ubiquitous soil microorganism.

13

In 1991, Gupta et al. isolated a highly toxic strain of B. sphaericus H5a(9001)

from the diseased mosquito larvae and soil samples from Kheda, Gujarat state, India.

Manonmani et al. (1991) also isolated 86 B. sphaericus and 23 B. thuringiensis strain

active against Cx. quinquefasciatus larvae from the root surface of hydrophytes using a

selective medium, nutrient agar containing 0.01% streptomycin. Novel variants of B.

thuringiensis were isolated from the phylloplanes of deciduous and coniferous trees as

well as other plants by Smith and Couch (1991). Hastowa et al. (1992), have isolated B.

thuringiensis from the soils of sericulture areas and natural environments form various

regions of Indonesia. Ohba (1996) examined mulberry leaves and found many B.

thuringiensis strains and a few of these isolates were toxic for silkworm Bombyx mori and

Aedes aegypti. Damgaard et al. (1996 & 1997) have recently isolated strains of Bt from

the food items (pasta, pita, bread and milk) and from the phylloplane of organically grown

cabbage leaves in Slangerup, Denmark.

2.1.2 Methodology for isolation of bacilli: Goldberg and Margalit (1977) and many

other workers isolated mosquito larvicidal bacilli by heat treatment of the samples and

plated them on Nutrient Agar from which the bacterial colonies were picked up. Since

then many media and newer techniques have been devised for efficient and selective

isolation & growth of these bacilli. White and Lotay (1980) discovered that B. sphaericus

NCTC 9602, grew and sporulated well in a simple chemically defined medium with pH 7.2

containing only KH2PO4/ NaHPO4 buffer, Ammonium Sulphate, inorganic salts and

Sodium Acetate as sole source of carbon. They also discussed about minimal nutritional

requirements of 26 other strains of the species.

14

B. sphaericus ISPC-5 isolated by Menon et al. (1982) were found to grow well on

simple media like nutrient broth alone or when supplemented with 0.3% molasses but

showed poor and delayed sporulation when cultured on brain heart infusion broth. Smith

(1982) demonstrated the effect of strain and medium variation on mosquito toxin

production by Bti. He showed that both insect mortality and degree of sporulation were

dependent on the media as well as strain and time of exposure. He further observed that

toxin production was associated with sporulation and the spore count was generally not

- proportionate to toxins produced for those strains and media used. Obeta and Okafor

(1984) formulated five types of media from the seeds of legumes, dried cow blood and

mineral salts, and assessed the growth and production of insecticidal toxins of Bti which

were found effective against Ae. aegypti, Cx. quinquefasciatus and An. gambiae. The

powder containing ground seeds of Voandzeia subterranean was most effective which

also compared well with the standard (IP578). Yousten et al. (1985) defined a selective

medium known as BATS. They reported that this medium containing Streptomycin

Sulphate and Arginine as the sole carbon and nitrogen source allowed the growth of 18

strains of mosquito-pathogenic B. sphaericus but inhibited the growth of 68% of the non-

mosquito pathogenic B. sphaericus strains as well as other bacilli species and aquatic

bacteria.

Travers et al. (1987) devised a selective process for efficient isolation of soil

Bacillus spp. In this process, germination of B. thuringiensis spores was selectively

inhibited by sodium acetate whilst most of the undesired spore-formers germinated. In the

next step, all the non-sporulated microbes were eliminated by heat treatment for 3 min at

15

80°C. The surviving spores were then plated on the rich agar medium and allowed to grow

till sporulation occurred.

Eziofor & Okafor (1988 & 1989) reported the production of Bacillus sphaericus

2362 using fermented cowpea (Vigna unguiculata) medium containing mineral substitutes

from Nigeria. They have shown that entomo-pathogenic Bs can be grown on a medium

compounded entirely from locally available materials and advocated that larvicidal Bt H-14

should also be produced on a similar medium.

Earlier Okafor (1987) had shown that by controlling pH (above 8.7) the growth of

naturally occurring lactic acid bacteria could be enhanced. This in turn actively converted

much of the proteins present in the cowpeas to smaller peptides and amino acids. These

along with minerals that leached out of the cowpeas were actively utilized by Bacillus

sphaericus for growth.

Russel et al. (1989) used NYSM (Nutrient Yeast Sporulating Medium) to study

the carbohydrate metabolism in B. sphaericus. Using BATS and acetate medium,

Carboulec and Priest (1989) isolated 40 strains of B. sphaericus and examined their ability

to use carbon and other energy sources. They tried to establish the correlation between

DNA homology group and nutritional profiles. Gupta et al. (1991) maintained B.

sphaericus 9001 isolated from Gujarat in laboratory at 30 °C in Polymedium in liquid

cultures. Carozzi et al. (1991) have reported the use of modified version of acetate

selection protocol of Travers et al. (1987) for isolating spore forming Bacillus strains

from soils and insect cadavers. Asimeng and Mutinga (1992) developed a baiting

technique for recovering bacteria from the environment. Using the healthy mosquito larvae

16

held in fully permeable plastic bottles, they were able to isolate toxic bacilli from mosquito

breeding habitats.

2. 2 Identification and characterization of mosquito-pathogenic bacilli

The rod shaped bacteria that aerobically form endospores are assigned to the genus

Bacillus. According to Bergey's Manual of Systematic Bacteriology (1989) they are the

endospore forming gram positive rod shaped bacteria which are mostly catalase positive.

Bacillus thuringiensis produces oval spores and its most distinctive characteristic is the

presence of a parasporal body. The parasporal body consists of insecticidal proteins that

are produced during sporulation (Angus, 1965; Heimpel, 1967; Hofte and Whitley 1989).

Since the discovery of Bti (H-14) by Goldberg and Margalit (1977) various authors have

proposed different schemes for their identification.

Heimpel (1966) described some 32 varieties of 2 species of crystalliferous bacteria

and proposed a taxonomic key for differentiating them. According to him the serological

classification coincides with the gross finding of biochemical and bioassay investigations.

They have given several morphological and biochemical tests for the differentiation of

several varieties of Bacillus thuringiensis. deBarjac and Bonnefoi (1968) proposed a new

key for the identification of Bacillus thuringiensis Berliner based on serological and

biochemical characterization, supported by the analysis of 161 isolates. They recognised 9

serotypes of Bt with 12 type strains or varieties. According to them the production of

thermostable toxin does not justify the criteria for naming new varieties.

Ohba and Aizawa (1978) investigated the distribution of Bt and related spore-

forming bacteria in Japan and performed serological identification of these new isolates.

They have described the preparation of H-antisera and H-factor sera and the methodology

17

for H & 0 agglutination tests. Logan & Berkley (1984) have described a system using a

matrix of results from the tests in the API 20 E and API 50 CHB strips and from the

supplementary tests for rapid and accurate identification of Bacillus isolates. According to

them, the API tests are more reproducible than the other methods and the taxonomy based

on them is also in good agreement with that of other methods. According to Berkely et al.

(1984) pyrolysis mass spectroscopy is the most promising method of general application

to the genus. They have also modified a tentative key for identification of the common

Bacillus species, devised earlier by Gordan (1973) which is based on the biochemical

tests.

de Barjac (1990) reported the classification of all Bt strains according to H-

antigens into 27 groups and 7 subgroups. Recently, Lecadet et al. (1999) have devised

and updated H-antigen classification of Bacillus thuringiensis. According to them, 63

serotypes and 13 sub antigenic groups of this species had been identified giving 82

serovars among the 3500 Bt Isolates available at the IEBC collection Centre at Pasteur

Institute, Paris, France.

Unlike Bt, B. sphaericus was recovered as saprophytic organism. The B.

sphaericus vegetative cells are rod-shaped, straight and motile, 0.6-1 pm wide and 1.5-5

pm long. They form nearly spherical heat resistant spores (Holt et al., 1975). The earlier

studies on B. sphaericus failed to demonstrate the presence of a paraspore in some strains

(c.r. from Yousten & Davidson, 1990)). But Yousten (1984) and Payne & Davidson

(1984) opined that the B. sphaericus synthesizes a parasporal inclusion that contained

proteins toxic to the mosquito larvae.

18

According to Bergeys Manual of Systematic Bacteriology (1986), there were some

40 valid and recognized species of B. sphaericus. Microbiological identification of this

species is presently done on the basis of morphological characteristics and rounded spores

located terminally in swollen sporangia. According to Claus and Berkeley (1986), B.

sphaericus can be differentiated by several phenotypic characteristics. Briefly, B.

sphaericus is a strict round spore former and is unable to use sugars as carbon source for

growth (Russel et al., 1989).

Various other schemes are noteworthy for the identification of B. sphaericus.

Yousten et al. (1980) recommended bacteriophage typing, while de Barjac et al. (1985)

proposed H-serotyping for identification and differentiation. Alexander and Priest (1990)

proposed the use of numerical classification and identification of Bs based on DNA

homologies. They used a combination of carbon-source utilization tests and other

physiological and biochemical characteristics in their study for phenotypic classification.

They results obtained were found to be completely compatible with those based on DNA

homologies. This enabled separation of several clusters that contained strains that were

combined in the previous studies under B. sphaericus (Priest et al. 1988).

Besides these schemes of identification, according to Frachon et al. (1991), the

fatty acid patterns revealed by gas chromatography, is another method to recognize

mosquito larvicidal Bs strains.

2.3 Bacilli toxins and their mode of action

Since the discovery of mosquito-pathogenic bacilli, attempts have been made by

various workers to understand the very nature and characteristics of the toxins, their mode

of action and pathogenesis. As stated earlier the most distinctive characteristic of Bti is the

19

parasporal body produced during sporulation that contains insecticidal proteins (Angus,

1965; Heimpel, 1967; Hofte & Whitley, 1989). According to Luthy & Ebersold (1981), in

most sub-species of Bt, the parasporal body is bi-pyramidal crystal containing one or more

similar proteins of about 135 kDa that are toxic to Lepidopterous larvae. When ingested

by the larvae, this toxin containing inclusion dissolves in the gut juices. The midgut

proteases cleave the protoxin, yielding an active peptidoglycan of 60-70 kDa, the delta-

endotoxin. The intoxication occurs as a result of osmotic imbalance across the midgut

epithelial cell membrane leading quickly to hypertrophy and lysis of midgut cells. Lysis is

followed by the disruption of basement membrane and leakage of digestive juices into the

haemocoel which is followed by larval death (Fig.2.1).

The majority of the toxins of Bti have been cloned and sequenced. Attempts have

also been made to express them in other organisms (Aronson, 1993). Kawalek et al.

(1995) by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

have revealed that the crystals of B. thuringiensis subsp. jegathesan were composed of

77, 74, 72, 68, 55, 38, 27 &23 kDa and were found to be distinct from that of Bti.

Schnepf et al. (1998) have discussed in details the ecology, genetics, toxin structure,

mechanism of action and biotechnology of Bt in their review "Bacillus thuringiensis and

its pesticidal crystal proteins."

Thomas & Ellar (1983) and Chilcott et al. (1990) obtained three major proteins

from the Bti crystals by FPLC on a mono Q column. They also determined the relative

toxicity of all the three proteins by estimating their LC50 value (i.e. concentration required

to kill 50% of mosquito larvae) and found that 65 kDa protein component was the most

toxic.

20

Invasion of haemolymph and haemocoel by bactena

Alkaline gut contents in haemolymph & haemocoel

A

Ingestion of biolarvicide with food

Ilir

0

Spore

Bacterial multiplication

Fig. 2.1: Mechanism of action of bacilli toxins in the mosquito larval gut.

21

Various toxic proteins of Bt are however reported to have a synergistic

interaction by several workers (Wu & Chang, 1985; Ibarra & Federici, 1986 and Chilcott

& Ellar, 1988). They concluded that all the major parasporal body proteins are

mosquitocidal but none alone is as toxic as the parasporal body per se. Hoftey and Whitely

(1989) have classified these endotoxins into several classes based on their toxicity Cry I

(Cry 1)- Lepidoptera specific; Cry II (Cry2)- Lepidoptera and Diptera specific; Cry III

(Cry 3,Cry 7 and Cry8)- Coleoptera specific and Cry IV (Cry 4, Cry 10, Cry 11)- Diptera

specific.

According to Hofmann et al. (1988 a & b) and van Rie et al. (1990) the delta-

endotoxins are synthesized as inactive protoxins that are solublized and proteolytically

activated in the insect gut. They suggested that the active toxin binds to specific, high

affinity receptors in the apical microvilli of epithelial cells in the insect midgut. It was

earlier stated by Knowles & Ellar (1987) that this bound toxin inserts irreversibly into the

plasma membrane to form pores or lesions leading to colloid-osmotic lysis. There is a

general accepted view that Bt toxins interact with the midgut membrane causing

physiological changes which lead to larval paralysis, cessation of feeding and eventually

death (Ellar et al. 1986; Knowles & Ellar, 1987; WHO Expert Committee on

Onchocerciasis, 1987; Gill et al. 1987; Hofte & Whitley, 1989, Drobniewsky & Ellar,

1988; 1989 and Chillcott et al. 1990).

Fast (1981) also attributed insecticidal activity of Bt to the production of toxin

protein crystals (the delta-endotoxin) which according to him account for 20-30% of dry

weight of sporulating culture. Jarret (1985) has concluded that the genes coding for the

production of toxins or for their expression may be located on both the plasmids or on the

22

chromosomes. Various theories proposed to explain the mode of action of Bt are summed

up in the Table 2.3. From these theories, it can be assumed that Bt toxins have detergent

like activity as they finally cause neurotoxicity or colloid-osmotic lysis. Neurotoxicity of

Bt has been reported by different workers (Chilcott et al. 1984; Cheung et al. 1985 &

1987; Singh and Gill, 1985 and Singh et al. 1986).

As mentioned earlier the first studies on Bacillus sphaericus failed to demonstrate

the presence of a parasporal body (c.r. from Davidson & Yousten, 1990). However later

studies revealed the presence of a paraspore in some strains, which are produced in the

course of sporulation and contain protein toxic to mosquito-larvae (Payne & Davidson

1984; Kalfon et al., 1984 and Yousten, 1984).

Baumann et al. (1985) and Broadwell et al. (1990) reported extraction of the toxic

proteins of 100kDa, 110kDa & 125kDa from the spores using various isolation

techniques. They discovered that these proteins further split into 63kDa & 43kDa and

51.4kDa & 41.9kDa. It is now accepted that of these, two proteins of molecular weights

51.4 and 41.9kDa are toxic to mosquito-larvae and both are required for the toxicity.

The pathology of B. sphaericus infections in susceptible larvae has been

studied both by light and electron microscopy. Kellen et al. (1965) who discovered

the first mosquitocidal strain of Bs also described its pathology. According to them

the death of larvae required up to 7 days and appeared to result in the progressive

degeneration of the gut cells. Davidson et al. (1975) found that after ingestion of

Bs strain SS11-I toxins, the epithelial cells of the intoxicated larvae show visible

ultra structural changes. These were separated from one another at the base and

also resulted in swelling of the gut. Large cytolysosomes appeared in the posterior

23

Table 2.3: Theories for Mechanism of Action of Bacillus thuringiensis Toxins

(source: Chilcott et al., 1990).

S. No Target of Action Reference

1. Plasma membrane (Ionophore) Angus, 1968 Nickerson and Schnell, 1983

2. Mitochondria Travers et al., 1976

3 Plasma membrane (General breakdown)

Luthy and Ebersold 1981 Nishitsusuji-Uwo, Endo, and Himeno, 1979

4 Goblet cell K+ pump Griego et al. 1979, Harvey and Wolfersberger, 1979

5 Plasma membrane (B. t. i.) (detergent like action )

Thomas and Ellar, 1983b

6 Neuromuscular system Chilcott et al. 1984 Cheung et al. 1985, 1987 Singh et al. 1986

7 Na+ and /or K+ transport Himeno et al. 1985 Gupta et al. 1985 Sacchi et al. 1986

8 Plasma membrane (colloid-osmotic lysis)

Knowies and Ellar, 1987 Haider and Ellar, 1987 Drobniewski and Ellar, 1988a

24

midgut cells and eventually these cells were observed to slough off from the basement

membrane. Invasion of the larval haemocoel by gut bacteria did not occur until after the

death of larvae (Davidson, 1979). The larvae experienced tremors, became sluggish and

eventually died while hanging from the water surface. According to Davidson (1981 &

1984), the mortality in larvae that fed on toxins may begin as early as 4 hrs after injestion

but generally 48 hrs are required for full expression of mortality in bioassays. Davidson &

Titus (1987) reported rapid swelling of the mitochondrial cristae and endoplasmic

reticulum which was noticed within 5 minutes of the treatment of the cultured cells.

Charles (1987) also observed pathological changes in the larvae following

ingestion largely involving the midgut cells. Lakshmi and Gopinathan (1988) reported that

the toxins inhibited the activity of choline acetyl transferase and oxygen uptake by the

mitochondria. Some workers have observed that following the death of the larvae the Bs

cells multiply in the larval cadavers and form new spores, indicating re-infection which

may be crucial in the recycling capability of Bs in the environment (Davidson et al. 1984;

Des Rochers & Garcia, 1984 and Charles & Nicholas, 1986)

2.4 Application of biocides: Formulations and testing of toxic strains

2.4.1 Formulations: For the successful control of vectors, it is necessary that the

biolarvicide is in such a form that is readily available to the larvae in their feeding zone and

is easily ingested by them. Besides, it should be economical to use and safe to the non-

target insects. The activity of the Bti and Bs toxic strains can also be enhanced through

appropriate formulation strategies (Lacey, 1984).

The earlier formulations were liquid concentrates of the bacterial cells and spores

or the primary lyophilized powders. The concentrated liquid cultures are dispersible and

25

simpler in preparation but they are bulkier and hence difficult to transport (Mulla, 1985).

According to Duimage et al. (1970) the primary powders could be obtained by

lyophilization or freeze drying, but this method resulted in the significant loss of spores

and cells. They devised a protocol for obtaining spore crystal complexes in dry form by

Lactose-Acetone Co-precipitation method.

According to Lacey et al. (1984) formulation that provides an adequate level of

toxin for the initial control of older mosquito larvae and continuous subsequent release of

toxin for the control of younger larvae would be optimal. They compared aqueous

suspension of formulated Bacillus thuringiensis (H-14) (Bactimos ® WP) and B.

sphaericus (1593) acetone precipitated spore powder with slow release floating pellet

formulation of each pathogen for the control of Culex quinquefasciatus breeding in urban

settings and found that the pellet formulations gave prolonged activity.

The Bti formulations (Table.2.4.1A) have been commercialized by many firms viz.,

Sandoz (Teknar ®), Abbot (Vectobac ®), Solvay (Bactimos) and used on a large scale in the

bio-control of mosquitoes and blackflies in many countries (de Barjac, 1990). More

recently, WP formulation of Bti named bacticide has been field tested in a multi-centric

trial in India. This is Russian formulation and marketed by Biotech International Limited,

New Delhi in India and in many other countries. The Bs formulations produced for

commercial purpose include Solvay liquid 2362, Abbot granules 2297, HIL-8, HIL-9 &

HIL-10 (M/s Hindustan Insecticides Ltd.), Spherix (Berdesk Plant in Russia marketed by

Biotech International Ltd., New Delhi), etc (Table 2.4.1B).

26

Table 2.4.1A Formulations of B. thuringiensis israelensis (H-14) tested against

various mosquito species.

S. No. Formulation Trade name Mosquito Species

Reference

1. Flowable concentrate

Bactimos® Vectobac®

An. arabiensis Mulla, 1990 Romi et al., 1993

2. Suspension concentrate

Teknar, SAN 402-1 SC

Mansonia spp. Foo & Yap, 1983

3. Corncob Vectobac-G® Culiceta spp., Psorophora spp., Aedes spp.

Charbonneau et al., 1994

4. Granular Floating granules Sinking granules Fine powdery granules

ABG-6108IIG® ABG-61081IG® Bactisand®

Cx. quinquefasciatus -do- -do-

Mulla,1990

5. Powder Bactoculicide/ Bacticide

Bactimos®WP Bactoculicide/ Bacticide Vectobacl2AS

An. stephensi An. subpictus An. culicifacies Ae. albopictus Ae. aegypti Cx. quiquefasciatus Cx. quinquefasciatus

An. stephensi Cx. sitiens Ae. vigila:c

An. stephensi An. culicifacies Ae. aegypti Cx. quinquefasciatus

Dua et al.,1993

Lacey et al., 1984 Kumar et al., 1995

Brown et al., 1998 Brown et al., 1999

Mittal et al., 1993

6. Briquetts Bactimos® Aedes spp. Culex Spp.

Logan & Linthicum, 1992

7. Tablets NM Ae. aegypti Becker et al., 1991

8. Sustained release formulation.

NM Cx. quinquefasciatus Lacey et al., 1984

NM Not menlicrvecl.

27

Table 2.4.1B Bacillus sphaericus formulations tested against various mosquito

species.

S. No.

Strain Formulation Trade Name Mosquito species

Reference

1. 1593 Acetone precipitated spore powder

NM Cx. quinquefasciatus Lacey et al., 1984

2. 1593 Wettable powder

NM An. annulipes, Cx. annulirostis, Cx. quinquefasciatus

Davidson et al., 1981

1593I1 -do- IIIL- 8 An. culicifacies, Cx. quinquefasciatus

Mittal et al., 1985

1593-4 -do- Stauffer WP 4920-34-3

Cx. quinquefasciatus, Cx. tarsalis

Mulligan et al., 1978; 1980

3. 1593 Sustained release pellets

.

Cx. quinquefasciatus, Cx. restuans

Lacey et al., 1984; 1988

2362 Cx. restuans Theobald, Cx. nigripalpus Theobald, Cx quinquefasciatus Say, Cx. salinarus Coquillett

Lord, 1991

Cx. territanus Walker Ps. Columbiae

4. 2362 Briquettes NM Cx. quinquefasciatus Lacey et al., 1988

5. NM Granules NM Cx. peus Mulla et al., 1988

NM Cx. pipiens, Cx. pipiens, Ae. trivittatus Berry et al., 1987 Ps. Columbiae

Abbot granules 2297

Vectolex

An. culicifacies, An. stephensi, An. subpictus, Cx. quinquefsciatus

Ansari et al., 1989

Karch et al., 1992 An. gambiae, Culex spp., Mansonia spp.

6. 2362 Flowable concentrate

NM

NM

An. quadrimaculatus, Ps. Columbiae, Cx. peus

Lacey et al., 1986

Mulla et al., 1988 Vectolex 2.5AS Spherimos

Cx. quinquefasciatus, An. stephensi, An. culicifacies

Ansari et al., 1995

7. 1593M Dust formulation

HIL-9 & HIL- 10 Cx. quinquefasciatus, An. culicifacies

Mittal et al.,1985

8. NM Floating type Spherix I'm Mansonia spp. Rajendran et al., 1991

9. 101 Powder Spherix ® An. stephensi Kumar et al., 1994 Cx. quinquefasciatus Kumar et al., 1996

ISM.- t\k-E 118nca_ 28

2.4.2 Assays of the biocides: The lab evaluation of biolarvicides is necessary before their

field trials and use in the vector control programmes. Assaying microbial insecticides is

very much difficult as compared to chemical insecticides. In the case of latter, the assayer

already knows the quantity and purity of the test insecticide and hence assay is an

accessory to the production process and it is confirmatory in nature. But in the case of Bt

or Bs larvicides, the assay is a measure of all the stages of the production process viz.,

quality of the fermentation; losses of activity occurring during the recovery process; the

quality of the product recovered and the characteristics of the final formulations. All these

must be known accurately and this knowledge could be achieved through bioassays at

different levels.

Essentially, a bioassay measures the interaction between the test insect and the

toxin being assayed. According to Dulmage et al. (1990) the most dramatic response of an

insect to the microbial insecticide and the one which is easiest to observe is the death. The

killing power of a biolarvicide, like any other insecticide, is expressed in terms of LC

(Lethal concentration), LD (Lethal Dose) or LT (Lethal Time), related to the

concentration, dose and the time of lethality respectively. But the most accurate and

accepted expression is the LC 50 and LC90 value i.e. a concentration of the sample that

causes 50% and 90% mortality of the test mosquito species larvae respectively. The major

steps in the bioassay are exposing the larvae to the biolarvicide, incubating them for a set

period of time, determining the percentage mortality at each concentration and then

plotting the Probit regression curve to determine the LC 50 and LC90 values. It is however

very important to perform bioassay following accurate and recommended procedure.

29

Several workers have proposed different methods, of which the two most popular ones

are those of de Barjac (1983) and McLaughlin et al. (1984).

The larvicidal activity of the very first strain of Bt was studied by the Goldberg &

Margalit in 1976. They found this strain to be very effective against 5 species of

mosquitoes viz. Culex pipiens (LC 50 = 6 X 103 spores/ml), C. univittatus (LC 50 = 2 X 104),

Aedes aegypti (LC 50 = 1 X 104), Uranotaenia unguiculata (LC50= 3 X 104) and Anopheles

sergentii (LC50= 5 X 105).

Ramoska et al. (1977) bioassayed B. sphaericus strains 1593, 1404 and SS11 -I for

infectivity against field collected Psorophora columbiae, Culex nigripalpis and Aedes

taeniorhynchus. They found that Aedes larvae were less susceptible to all the three strains

but most susceptible to strain 1593. They also observed that 43% of the larvae that were

to die in the course of experiment, died within 24hrs of incubation with the bacteria.

Tyrell et al. (1979) described the toxicity of Bacillus thuringiensis israelensis

parasporal crystals in larvae of three medically important mosquito vector species. The

number of larvae killed was in relation to crystal dry weight. The LC 5o values obtained

were: Aedes aegypti Linnaeus, 1.9 X 1ettg/m1; Culex pipiens var. quinquefasciatus

(now Culex quinquefasciatus) Say, 3.7 X 10 -4 µg/m1 and Anopheles albimanus

Weidmann, 8.0 X le pg/ml.

Myers et al. (1979) performed bioassay of two strains of B. sphaericus against

second instar larvae of Culex quinquefasciatus. The bacterial cells were removed from

growth medium by centrifugation and re-suspended in the equal volume of sterile de-

chlorinated tap water. They tested three cups each containing 10 larvae in 20m1 de-

chlorinated tap water per 10-fold dilution of bacterial cells and recorded the larval

30

survival. The strain SSII-1 had LC 50 at 2.5 X 108 spores/m1 while LC5 0 of strain 1593 was

2.9 X 102 spores/ml. Smith (1982) performed bioassays with Bti (H-14) against fourth

instar larvae ofAedes aegypti. As differences among log dilutions were detected, they did

not calculate LC 50 values. Instead they devised a rating system based on 10-fold dilution

and on the number of surviving larvae at the lowest effective sample dilution to define the

effectiveness of the samples. Balaraman et al. (1983) cultured Bt H-14 (VCRC B-17) and

using different additives prepared six different formulations. They then determined their

potency against early fourth instar Cx. quinquefasciatus larvae using IPS-78 as the

standard. Many other workers conducted lab bioassays using different commercial

formulations viz., Mittal et al. (1985 & 1993); Rettich (1987); Balaraman & Hoti (1987);

Ansari et al. (1989 & 1995); Klein et al. (1989) and Theoduloz et al. (1997).

Brown et al. (1998) conducted laboratory bioassays based on the standard methods

to determine effectiveness of Vectobac® 12 AS. The larvae were exposed to serial

dilutions of formulations in filtered water habitats. They maintained 5 replicates each of 20

late III/early IV instar larvae in 250m1 glass beakers containing 200m1 of the test

concentration. Initially they conducted a number of range-finding assays with widely

spread exposure concentrations. Based on these, they then conducted the main bioassays

with a narrow effective range of concentrations and calculated LC 50 and LC95 values.

Rodrigues et al. (1998) conducted lab bioassays to test 10 strains of Bs isolated from

Brazilian soils against third instar larvae of Anopheles species. They used lyophilized

bacteria for the bioassays in six or seven concentrations ranging from 0.5ppb to 50ppb.

Twenty larvae from each Anopheline species were used per concentration tested in one

31

single bioassay. They calculated Relative Activity (RA) of the isolates using the following

formula for 48 hrs. results:

LC50 Standard RA =

LC50 Sample

Extensive literature is also available on the successful small and large scale field

trials of biolarvicides in mosquito control from all over the world. Reports are available on

testing of various preparations and formulations of Bs and Bti against different species of

mosquitoes in a variety of habitats. Field trials of Bs as a mosquito larvicide were first

reported by Ramoska et al. (1978), who found it to be a promising biological control

agent of mosquitoes. Mulligan et al. (1980) conducted field trials to test the efficacy and

persistence of Bs strain 1593-4 and Bt (H-14) against mosquitoes. They found that both

were more active in raw and sewage effluent than in tap water. They observed that an

aerial application of Bt (H-14) at 1 kg/ha could bring about C. tarsalis control apparently

without adverse effects on predator populations.

Davidson et al. (1981) conducted comparative field trials to test the efficacy of Bs

strain 1593 and Bti commercial formulations in test ponds and found them to be very

similar in efficacy. Foo & Yap, 1983 conducted field trials on the use of Bt serotype H-14

against Mansonia mosquitoes in Malaysia. They tested a suspension concentrate Teknar ®

SAN 402-1 SC against lab reared as well as naturally occurring Mansonia mosquito larvae

using a Hudson knapsack sprayer in small plots in swampy ditches on Penang Island. They

obtained comparable dosage/response values for natural and introduced populations and

32

suggested the use of caged populations in bioassay methods for Mansonia larvae in the

field tests.

Mulla et al. (1984) reported excellent initial control of Culex tarsalis and Cx. peus

using primary powders of two strains of BS 2362 (IF-97) and 1593 (IF-94) at the rates of

0.1 and 0.2 lb/ha and also found them to be safer towards non-target organisms. Sandoski

et al. (1986) applied Bt (H-14) against Anopheles quadrimaculatus larvae in rice fields

using Beecomist® spray head and affirmed that this method of application is a cost

effective for high volume ground application in large areas. Bhalwar et al. (1993) and

Kumar et al. (1996) carried out field trials with B. sphaericus (Spherix formulation) in

Delhi and Goa, India respectively and at a dose of 1g/sq. m. demonstrated effective

control of Culicine breeding in the polluted water habitats such as drains, septic tanks,

cesspits, cesspools, etc.

Mittal et al. (1993) from their field trials concluded that Spherix formulations of

Bs @ 0.25-2 g/sq. m. produced 90-95% reduction in the larval densities of both An.

stephensi and Cx, quinquefasciatus within 48hrs. in different habitats and the larvicidal

activity persisted for 2-4 weeks in these trials. Dua et al. 1993 from their field trials with

Bactoculicide (Bti) sprayed @0.5g/sq. m. against Culex, Aedes and Anopheles larvae

breeding in industrial scrap observed that mosquito breeding was controlled from 96-100

% up to 5 weeks.

Ansari et al. (1995) evaluated the efficacy of two flowable formulations of B.

sphaericus i.e. Spherix and Vectolex 2.5AS and found that they produced control for

longer duration in deep wells than in shallow ponds and also recommended their use in

polluted habitats. Shulda et al. (1997) observed in Haldwani, District Nainital, India, that

33

B. sphaericus gives better control of Anopheline breeding in cemented tanks and small

ponds than B. thuringiensis.

Kumar et al. (1994 & 1997) demonstrated control of Malaria utilizing B.

thuringiensis israelensis against Anopheles stephensi in major field trials in Goa, India.

Brown et al. (1998) conducted field trials with Vectobac ® 12AS (active ingredient: 1200

ITU/mg Bti) against late third/fourth instar field specimens of Cx. sitiens in floating mesh

cylinders introduced in salt marsh pools near Coomera Marina, Southeast Queensland,

Australia. They achieved 100% mortality of Cx. sitiens larvae at 24hr post treatment at

the rate of 0.5 litre/ha.

Brown et al. (1999) evaluated in the field efficacy of four insecticides including 2

organophosphate compounds (temephos & pirimiphos methyl), a growth regulator (delta-

methoprene) and Bti against Aedes vigilax (Skuse). Based on high selectivity ratios

excellent field efficacy and lack of influence on abiotic water characteristics, they

suggested that delta-methoprene and Bti were ideal for insecticidal control of Ae. vigilax

in Australia.

Many more reports are available on the successful small and large field trials

with biolarvicides from all over the world (Lacey et a/. 1986; Rettich, 1987; Ansari et al.

1989; Rajendran et al. 1990; Logan & Linthicum, 1992; Karch et al. 1992; Romi et al.

1993; Rettich & Ryba, 1993 and All et al. 1994).

2.4.3. Factors affecting biolarvicide activity: The efficacy of the biolarvicide is

influenced by various biotic and abiotic factors including the target species, type of

formulation, feeding behaviour of the larvae and ecological factors like temperature, pH,

34

exposure to UV radiation in the sunlight, stream discharge, presence of micro-organisms,

etc.

2.4.3.1. Feeding behaviour: Ramoska & Pacey (1979) reported that the efficacy of Bs

Neide. strain 1593 was inversely related to the amount of food available to the mosquito

larvae and that the larvae of Culex quinquefasciatus consumed lethal quantities rapidly

after bacterial application whereas those of Aedes aegypti did so less rapidly, resulting in

low mortality even after 90 minutes of incubation at high dosage (7 X 10' cells/m1)

2.4.3.2 Temperature & pH: The effect of temperature and pH at which the mosquito

larvae are exposed to Bs and Bt formulations has been reported by Muligan et al. (1980);

Wraight et al. (1981); Subramonian et al. (1981); Molloy et al. (1981) & Mittal et al.

(1993).

2.4.3.3. Exposure to sunlight: Mulligan et al. (1980) reported that the active ingredient

of B. sphaericus was readily degraded by exposure to direct sunlight, while that of Bt. H-

14 was not even after 5 hours exposure to direct sunlight. Burke et al. (1983) reported

that the UV light from a germicidal lamp reduced the viability of B. sphaericus 1593

spores but insecticidal activity was resistant to inactivation after continuous exposure to

UV light for 4 hour.

2.4.3.4. Pollutants: Lab and field trials with Bs and Bti have suggested the efficacy of

biolarvicide is lowered in waters which are turbid or contain sewage with high chloride

content or other waste effluents (Mulligan et al.1 1980; Ramoska et alb 1982 and Car,

1984).

35

2.4.3.5. Other Factors: A number of other factors known to affect the efficacy of the

biolarvicides include strain of the biocide & media used for growth (Smith, 1982), larval

instars (Ramoska et al. 1977; Wraight et al. 1981), larval breeding habitat (Huang et al.,

1993; Romi et aL, 1993; Ansari et al., 1995), species of the mosquito (Habib, 1983) and

the stream discharge.

2.5. Persistence and recycling of biolarvicides in the environment

One of the most desirable qualities of a biocide is that it should be able to produce

long lasting control with one or few treatments. This is important to compensate for the

high cost of production and applications of these bio-control agents. Therefore it is

desirable to use only those agents that are able to persist or recycle in the treated habitats

for reasonably long period and provide satisfactory control apart from possessing the

other desired qualities.

Soon after the discovery of the two entomo-pathogenic bacteria (Bs and Bti) and

their use as biolarvicide, various researchers made efforts to study their persistence and

recycling potential in the host, soil, mud and water (Hertlein et al., 1979; Chilcott et al.,

1983; Davidson, 1984; Hoti & Balaraman, 1991; Yousten et al., 1991& 1992; Khawaled

et aL, 1992 and Becker et al., 1995).

Saleh et al. (1970) reported that B. thuringiensis spores can remain viable for long

periods of time and that the organisms can compete successfully under conditions

favouring the Bacillus component of soil microbial populations. Ignoffo & Garcia (1978)

however reported that the UV light inactivates the cells and causes their death. West et al.

(1984) studied the persistence of viable and heat killed vegetative cells, parasporal crystals

36

and the spores of B. thuringiensis in the soil monitoring them by immuno-fluorescence and

found that the number of spores remains unaltered throughout 91 days incubation at 25 °C.

Persistence and the residual activity can be enhanced through proper formulation.

Lacey et al. 1984 had reported that some slow release formulations provided persistent

release of toxins and control of larvae.

Menon & Mestral (1985) conducted field and laboratory studies to assess the

survival of Bt var. kurstaki in waters and reported that Btk could survive for relatively

long periods in fresh water and marine environment at 20 °C.

According to Aly et al. (1985), Bti sporulates and produces crystals in cadavers of

mosquito larvae and thus helps in the recycling of the organism in the environment. Petras

& Casida (1985) reported good survival of B. thuringiensis spores in the soils. However

Aronson et al. (1986) opined that the rather sluggish germination of B. thuringiensis

spores may contribute to the lack of persistence of these organisms. According to Mulla

(1990), most of the commercial formulations give high levels of initial control but young

mosquito larvae soon appeared after a few days of the treatment. This according to them

shows that Bti fails to recycle in nature.

Recently Mansherob et al. (1998) reported bio-encapsulation of the spores of

Bti in protozoan, Tetrahyemna pyriformis as the new mode of Bti recycling in nontarget

organisms. They have observed that spores indeed germinated, grew and sporulated in

excreted food vacuole of T. pyriformis forming new active ICPs during this cycle.

The first reports of the recycling potential of Bacillus sphaericus came from

Hertlein et al. (1979). They reported that Bs remained viable and infective even 9 months

after applications as a larvicide agent of mosquitoes in a roadside ditch. Burke et al.

37

(1983) have demonstrated the effect of UV light from a germicidal lamp on Bacillus

sphaericus, 1593. They found that due to this exposure spore viability was considerably

reduced but insecticidal activity was resistant to the inactivation even after continuous

exposure to UV light for four hours. Thus the sprayed habitats which have low light

intensity would probably exhibit longer larval control.

Mulligan, et al. (1980) found that larvae were continuously killed by exposure to

water taken from the bottom of the same basins for up to one month after treatment with

Bs suggesting its settlement and the prolonged activity. On the contrary, Karch & Charles

in 1987 reported that spores that had settled at the bottom of cesspools rapidly lost their

toxicity against Culex pipiens larvae.

There are, on the other hand, several reports suggesting that B. sphaericus cells

multiply in the larval cadaver and formed fresh spores (Silapanuntakul et al., 1983;

Davidson et al., 1984; Des Rochers & Garcia, 1984; Charles & Nicolas, 1986). The

recycling potential of Bs has been attributed to the ingestion of spores by non-target

invertebrates to the role of cadavers (Yousten et al., 1991; Becker et al., 1995 and

Carvalho-Pinto et al., 1995)

2.6 The Safety of Bacillus species as mosquito control agents

With the discovery of a number of larvicidal strains of B. sphaericus and B.

thuringiensis there emerged a need to test these for safety against man, fish, wildlife and

other non-target biota before they could be recommended for the biocontrol of disease

vectors. Since one member of the genus Bacillus i.e B. anthrax is a known mammalian

pathogen, it has become necessary to evaluate the mosquitocidal strains of Bs and Bti for

their safety to the man and environment.

38

There is a large volume of information on the impact of these two

entomopathogens on non-target biota. As early as in 1975, Kingsbury had reported that

the aerial applications of Bti had no adverse effect on aquatic insects. Ever since then,

various authors have shown Bs and Bti to be safer to most of the non-target fauna

(Mulligan et al. 1978 & 1980; Molloy & Jamnback 1981; Walton & Ladle, 1983; Car &

Moor, 1984; Pistrang & Burger, 1984; Mulla et al. 1984; Rettich, 1987; Majori et al.

1987; Molley, 1992; Richardson & Perrin. 1994; Chui et al. 1993; Wipelli & Merrit, 1994

and Roberts, 1995)

A few other reports are briefly discussed here. Moulinier et al. (1981) had

conducted experiment with Bti to show its innocuity against Oyster larvae and hence

suggested its possible use near the areas of Oyster culture. Davidson (1982) reported that

Bti itself has no effect on aquatic organisms such as water mites, shrimps and oysters.

Bslararnan et al. (1983) found it to be safer for fishes, copepods, notonectids and other

aquatic fauna. Lacey and Mulla, 1990 have written a comprehensive review on the safety

of Bti and Bs on the non-target organisms. Charbonneau et al. 1994 conducted field and

laboratory studies to determine the effects of Bti (Vectobac® G) on water fowl macro-

invertebrate food resources at Minnesota valley National Wild life Refuge Bloomington,

Minnesota. They concluded that chironomids (a major water fowl food source) were

adversely affected by Vectobac G under controlled laboratory situation. However,

environmental factors reduce the efficacy of the biolarvicide in the field. Hershey et al.

(1995) studied the effects of methoprene and Bti on non-target insects in USA and did not

observe any negative effects of biolarvicide treatment on density or biomass of

invertebrate groups nor any decrease in richness of benthic invertebrate taxa. Brown et al.

39

(1999) from their study concluded that Bti was an ideal insecticide for the control ofAe.

vigilax in Australian salt marsh pools based on its high selectivity ratio, excellent field

efficacy, and lack of influence on abiotic water characteristics.

Several reports are available on biolarvicide safety to humans as well as other

mammals. Burges (1981) reported the testing of several species of B. sphaericus and B.

thuringiensis israelensis against mammals including human volunteers and concluded

them to be safe for use as insecticides. Shuddack et al. (1980) could recover Bs from

rodents 14-18 days after intra-peritoneal and intra-cerebral injections. Seigel et al. (1987)

reported that B. thuringiensis israelensis could be recovered for as long as 7 weeks after

injection. Seigel & Shadduck (1990) summarized that oral, intra-peritoneal and aerosol

exposure to Bti produced no significant illness or mortality. According to them the

entomo-pathogens disappeared rapidly from the lungs of rats, and there was no evidence

of their multiplication. Subcutaneous injections of Bti produced abscesses but these

abscesses appeared following injection with autoclaved material as well. Abscesses most

likely arose from the presence of heat stable foreign material in the injection site. The

ocular irritation to Bti depended upon the physical state of the preparation; fine powders

produced no irritation and large clumps caused considerable corneal irritation, conjuctival

swelling, congestion and discharge. Intra-cerebral injection of 10 7CFU resulted in

mortality of rats. As cited by these workers, the toxicological studies performed by

experimental exposure of various animals (mice, rats, guinea pigs and rabbits) showed the

absence of acute and prolonged toxicity.

Mammalian safety of Bacillus sphaericus has been discussed in detailed by Siegal

and Shadduck (1990). They concluded that exposure to Bs isolates 1404, 1593 and NCTC

40

11025 did not result in clinical illness or death. Occular irritancy of B. sphaericus was

minimal and although this organism persisted for 8 weeks in the conjuctival cul-de-sac of

rabbits, there was no evidence of infection. B. sphaericus 1404 and 1593 did produce

lesions when injected intra-ocularly and intra-cerebrally. The safety data of strain 2362

however are more variable, although dose greater than 10 7 CFU proved toxic to mice.

Clearance studies conducted by them and those by de Barjac et al. (1987) indicated that B.

sphaericus disappeared from blood within two weeks following parenteral injection. They

concluded that these biolarvicides could be used safely in the environment where exposure

to human beings might occur.

Recently some workers have indicated the risk of gastroenteritis outbreak due to

B. thuringiensis in humans (CarLston et al. 1994; Damgaard, 1995; Damgaard et a1.1996).

A review by Drobniewsky in 1994 entitled 'The safety of Bacillus species as vector control

agents' sums up the safety profile of Bti and Bs stating that these species may cause

disease in man but the reported cases are very less. In comparison to their benefits to the

community the risk to the public health is very small and at present there is no good

evidence to discontinue their use.

2.7 The problem of resistance to biolarvicides

The first reports of the insect resistance to Bacillus thuringiensis came from

McGaughey, 1985 who found that Plodia interpunctella, a major lepidopteran pest can

develop resistance to the insecticide within few generations. According to him the strains

of P. interpunctella collected from the treated grain bins were more resistant than the

strains from the untreated bins indicating that the resistance can develop quickly in the

field. They also found that resistance was inherited as a recessive trait. van Rei et al. 1990

41

studied the mechanism of insect resistance to B. thuringiensis that was due to the

alteration in toxin membrane binding. Resistance to Cry I A(b) toxin was found to be

correlated with reduction in the affinity of Cry I A(b) toxin binding, whereas increased

sensitivity to Cry I(c) toxin is reflected in an apparent increase of the Cry I (c) binding site

correlation. Georghiou et al. (1991) reported that the cross resistance between Bs strains

harbouring different gene sequences has not been found to occur, which suggest that the

toxins may be binding to different receptors.

Adak et al. (1995) for the first time reported the B. sphaericus resistance to Culex

quinquefasciatus. According to them initially B. sphaericus was very effective but within a

year i.e. after 20-25 rounds of application, field populations of Cx. quinquefasciatus

developed resistance up to 150 fold. Through genetic studies they found that resistance

was recessive autosomal chromosome linked and was controlled by more than one gene.

Several authors have tried to explain the mechanism of insect resistance and have

suggested methods to overcome the problem. McGaughey & Whalon (1992) have

mentioned several theories for managing the development of resistance to Bt toxins but

their utility has not been assessed in the field. Among their recommendations is the

rotation or the alteration of Bt toxins with other toxins. They have also suggested three

other ways to manage resistance viz. application of low dose, high dose and ultra high

dose. According to them the low dose tactics include the reduction in the rate of toxin

application and reduced frequency of application and they aim at reducing the pest

populations slightly or slowing down the pest larval development to the point that the

number of generations per year is reduced. Probably this reduces the selection pressure.

High dose kills heterozygotes, which are the most abundant carriers of insect resistance

42

genes, whereas, the 'ultra high dose' is sufficient to kill both the heterozygous and

homozygous resistant insects. Ferre et al. (1995) found that the insect resistance to Bt

insecticidal proteins is inherited as a single recessive or partially recessive major gene.

According to them the binding site modification seems to be the most significant resistance

mechanism under field conditions. Moar et al. (1995) suggested that the use of individual

protoxins or toxins especially expressed in transgenic organisms induce resistance in the

field populations more readily than the formulated materials containing multiple Cry

proteins and spores. Charles et al. (1996) reported that the resistance in field seems to

decline very quickly when the treatment is suspended. They have also suggested methods

to overcome the problem of resistance through the use of Bs strains belonging to different

serotypes that display no cross-resistance. Besides, according to them the best way to

prevent or retard resistance may be to produce bacterial strains that simultaneously

express different toxins binding to different receptors. Thus the diversification and the

combination of Bt and Bs strains could help in the management of resistance in target

mosquito species.

van Frankenhuyzen et al. (1997) discovered that toxin Cry 9Ca I binds to different

receptors than do other ICP's that are currently used in conventional sprays or transgenic

plants. Such toxins may become valuable tools to manage resistance problem. Koskella &

Stotzy (1997) reported that the clay bound toxins become resistant to degradation and

retain insecticidal activity for long, thus the toxins from transgenic plants and microbes

may accumulate in soils and thereby enhance the development of insect resistance. They

have recommended that this mechanism of persistence of toxins in soil should be

incorporated in the future management plans to reduce the development of resistance to

43

biolarvicides. Georghiou & Wirth (1997) studied the influence of single versus multiple

toxins of Bacillus thuringiensis subsp. israelensis on the development of resistance in

Culex quinquefsciatus and concluded that the usage of natural mixture of four Bti toxins

would be advantageous in suppressing or delaying the resistance in Cx. quinquefasciatus.

Liu et al. (1998) opined that the absence of spores in Bt toxin-expressing transgenic plants

and bacteria would accelerate the evolution of pest resistance. Cross-resistance to B.

sphaericus has been reported by Poopathi et al., 1999. They have indicated the judicial

use of Bti for the management of resistance in Bs resistance Cx. quinquefasciatus and

have suggested the use of other alternate mosquito control strategies in the microbial

resistance management. It may therefore be concluded that insect resistance to B.

thuringiensis and B. sphaericus can occur and to overcome this serious problem and for

the judicious deployment of these tools in insect control, the resistance management

strategies recommended by experts should be adopted.

2.8 Genetic manipulation of B. sphaericus and B. thuringiensis

The narrow target range of mosquitocidal bacilli, lack of persistence in the

environment for a long time, high cost of production especially that of B. sphaericus are

the major limitations of naturally occurring strains being used extensively in biocontrol.

The cost of production can be reduced considerably using locally available cheaper raw

materials that support good growth and toxin production (Eziofor &Okafor, 1988 and

1989). The problem of narrow host range can be addressed by genetic engineers and new

strains expressing the new and existing combination of mosquitocidal toxins could be

obtained. The lack of persistence of the biolarvicides in the environment for a long time

has been attributed to the settling of the spore -crystal complexes (Davidson et al. 1984

44

and Ohana et al. 1987). This settling problem can be solved by the improvement of

formulation technology and with the use of genetically engineered organisms in which the

mosquitocidal toxin genes of Bacillus sphaericus and B. thuringiensis, could be expressed

in those micro-organisms that do not settle at the bottom of ponds and lakes and rather

suspend in feeding zones of larvae.

Porter et al. (1993) summed up recent advances on new type of recombinant

microorganisms and new cloning strategies in their review. According to them these

genetically transformed microorganisms have the potential to provide effective control of a

wider range of mosquito species for a longer duration than the naturally occurring bacilli.

A few other important research articles on this topic are briefly reviewed here.

According to Huber (1986), attempts have been made to express these

mosquitocidal toxins in E. coli and Baculoviruses. But these new organisms are of not

much importance because viruses take a long time for activity and E. coli. is a mammalian

pathogen. Angsuthanasombat and Panyim (1989) have reported the introduction of 130

kDa mosquitocidal toxin gene cloned from Bti into the Cynobacterium, Agmenellum

quadruplicatum PR-6 by plasmid transformation and the synthesis of mosquitocidal toxin

by the transformed cells. They demonstrated the use of engineered Cynobacterium for

biological control of mosquitoes. The Cynobacteria are widely distributed in the upper

layer of water and thus the produced toxin would reach the target mosquito larvae more

effectively. Thanabalu et al. (1992), reported the production of recombinant Caulobacter

crescentus by transformation with broad host range plasmids carrying genes encoding

larvicidal toxins from either B. sphaericus or B. thuringiensis subsp. israelensis. They

further demonstrated the mosquito larvicidal activity of the intact recombinant C.

45

crescentus. Bacteria belonging to Caulobacter species are found in almost every aquatic

habitat predominantly near water surface.

Liu et al. (1996) expressed the binary toxin of Bs in the gram-negative bacterium

Asticacaulis excentricus and reported that the plasmid transformed bacterium exhibited

toxicity to Culex and Anopheles larvae. A. excentricus has the potential advantage as a

larvicide compared with the bacilli, especially persistence in the larval feeding zone,

resistance to UV light, lack of toxin degrading proteases and low production cost.

Thiery et al. (1997) isolated a gene, Cyt 1 AbI encoding the 27,490 Da protein

from B. t. var. medellin (H30 serotype) by using an oligonucleotide probe corresponding

to Cyt 1 AbI gene. It was expressed in crystal negative Bt strains. They found that the

large inclusion of cytlAbl protein was twice as haemolytic as that from the wild strain.

Besides, transgenic plants have been obtained using toxin genes from Bt and Bs. (Vaeck et

al. 1987; Hofte & Whitely, 1989 and Peferoen, 1997). These transgenic plants of tomato,

tobacco, cotton, corn and potato are protected from the feeding damage by the insect

pests. Whether the genetically engineered mosquitocidal organisms will possess all the

necessary qualities of biocide is however still a question of concern for many workers.

2.9 Economics of biolarvicide usage in vector management

For the commercialization and wide spread use of biocide in insect control, it is

very important that these should be economical. Many authors have tried to evaluate and

compare their costs and have suggested ways to reduce costs of production or application.

Margalit & Dean (1985) suggested that the newer strain of entomopathogens may be

constructed which grow faster, produce more toxin and grow on cheaper carbon source,

thus reducing the cost of production and formulation (Table 2.9.1).

46

Table 2.9.1: List of potential raw materials for use in formulating fermentation media for local production of B. sphaericus and B. thuringiensis israelensis (Source: B hum iratana, 1990)

Raw Material Comment; Reference

Agricultural Products: These materials are extracted in boiling water and

added to basal medium containing dried cow blood

and mineral salts

Obeta & Okafor, 1983.

Vandekar and Dulmage, 1983

Bambara bean, cowpea,

Groundnut cake, soya bean

Ground corn, Cottonseed meal,

Cassava, Yams

Animal Products: Vandekar and Dulmage 1988.

Hertlein et al' 1981 Blood, beef bones, chicken parts,

Fishmeal, animal dung

Industrial By-products: Dh arm sthiti et al., 1985.

Byproduct from monosodium-

Glutamate factory.

Other Vandekar and Dulmage, 1983

Coconut milk, corn-steep,

Liquor, molasses, whey

Table 2.9.2 Production cost of one batch* of B. thuringiensis H-14 and B. sphaericus strains in laboratory fermenter, of 13 lit. working capacity (Source: Balaraman and Hoti, 1987).

Inputs B. thuringiensis B. sphaericus H5a5b strain H-14 strains

VCRC-B17 VCRC-MB24 VCRC- B42 Us $ Us $ Us $

Raw materials 1.25 1.17 1.25 Equipment 0.42 0.42 0.42 Personnel 6.67 6.67 6.67 Energy 3.17 2.17 4.33 Building 0.25 0.25 0.25 Laboratory Maintenance 0.17 0.17 0.25 Servicing and Maintenance of Equipment 0.17 0.17 0.17 Total 12.10 11.02 13.34

47

For the economical use of a biolarvicides, it is also desirable that the cost of

application of the biocide should be less and affordable. Yates (1984) and Sandoski

et al. (1986) affirm that Beekomist ® spray head as a cheaper alternative to high

volume aerial application of Bti on large areas. According to Sandoski et al.

(1984) if the effective swath width, optimum flight path interval and flow rate are

more precisely determined, the cost of treating large areas of rice for mosquito

control will be further decreased.

Mulla (1990) opined that the lower dosages of entomopathogens should be

sufficient to control rising populations of insects after their first application. Hence

reducing the dose by 50% to 75% after the first application yielded equivalent

reduction in larval populations, while it resulted in significant saving in cost of the

treatment.

Balaraman & Hoti (198'7) not only worked out the comparative cost of

production of one batch of Bti and Bs strains in the laboratory fermenter (Table

2.9.2) but also compared the costs involved in controlling mosquito larvae in an

area of one hectare with the bacilli and chemical larvicides. They concluded from

their study that the cost of larval control with bacilli was more or less equivalent to

that incurred if Fenthion was used but the cost was significantly lesser if malaria

oil, abate or Paris green were used (Table 2.9.3).

48

Table 2.9.3: Comparative cost of larval control (in unpolluted waters) per hectare for four weeks with chemical and microbial larvicides (Source: Balaraman & Hoti, 1987).

Larvicides Unit price US$

Dosage/ Ha

Cost of larvicide (US$)

Cost of labour (US$)

Total cost of larval

control for 4 weeks (US$)

For 1 appli- cation

For 4 applic- ation

For I appli- cation

For 4 appli- cation

Chemical

1. Malaria Oil 0.25 lit 142 lit 35.50 142.00 4.25 17.00 159.00

2. Fenthion 25.00/lit 500 ml 12.50 50.00 4.25 17.00 67.00

3.Paris Green 7.08/ kg 4kg 28.32 113.28 5.42 21.67 134.95

4. Abate (50%)

41.67 lit 400 ml 16.67 66.68 4.28 17.00 83.68

Microbial

1.Bt H-14(B17) 12.10/120 660 brig. 66.55 - 0.83 - 67.38

2.Bt H-14

(MB24)

11.02/120 660 brig. 60.61 - 0.83 - 61.44

3.Bs (B42) 13.34/120 660 brig. 73.37 - 0.83 - 74.20

49

According to Sutherland (1990), the ground application cost per acre treatment

with Bti and Abate® respectively was US$3.2 and US$4.39 and consequently for

economy, Bti was chosen. But for the aerial application cost/acre worked out was US$3.2

and US$2.3 respectively. According to them, suitable formulation development of Bti

through cheaper sources could further reduce this difference.

According to Hougard (1990) despite the high concentration of B. sphaericus

recommended for the treatment of polluted waters (10g/m 2), it was found to be promising.

To substantiate this view, he cited the example of the use of a liquid formulation of the

strain 2362 in Dar-as-salaam (Tanzania) by the sanitary services of the town (Table

2.9.4).

Moffat (1991) reported that the higher insurance premiums were associated with

the use of chemicals than the biocides, due to which the people were switching over to the

latter. According to Priest (1992) the bacterial insecticides will become popular only if

their treatment cost is equal or less than the chemical alternatives. There is surely

possibility of improvement of the strain and fermentation process to reduce the cost of

production of both Bti and Bs. It was also opined that effective products at the realistic

prices could ensure a bright future for the bacterial insecticides.

50

Table 2.9.4: Comparison of the approximate cost (U.S.$) of operational use

for a one-year Period of B. sphaerius (BSP1) at 10g/m 2 and Chlorpyrifos

(Dursban®) at 1 g/m 2 in Dar-as- salaam (Tanzania) for Control of Cx.

quinquesfasciatus (Source: Hougard, 1990).

Parameter B. Sphaericus 2362 (flowable conc.)

Chlorpyrifos (E.C. 480g11)

Dosage 10g/m2 (0.01 lt/m2) 1 g/m2(0.001 lt/m 2)

Frequency of larviciding 9 cycles a year 24 cycles a year

Quantity of formulation 45 liters 25 liters

Staff' $12 x 9 = $108 $12x 24=$288

Equipment b $30 x 9 = $270 $30 x 24 = $720

Insecticide $8 x 45 = $360 $15 x 25 = $ 375

TOTAL $738 $1,383

NOTE : The cost is calculated on the basis of 500 m 2 oftreated sites.

a- About $12 for two technicians for one cycle of larviciding.

b- About $30 for one vehicle for one cycle of larviciding.

51

Becker & Margalit (1993) have stated that the application of Bti can be

cost effective as compared to the conventional insecticides. They have given the

example of the KABS programme of German Mosquito Control Association, by

which it was possible to suppress the mosquitoes from a catchment area of more

than 500km2 including 100km2 of the existing breeding grounds. According to

them a total yearly budget of US $ 1 million protects more than 2 million residents

in the area. They have also compared the relative costs of larvicides in the

Onchocerciasis Control Program and have reported that the cost of Bti treatment is

more or less comparable to the cost of treatment using chemical insecticides such

as temephos or chlorophoxim when the discharge of the streams is low. However

the cost of Bti treatment is slightly higher when the stream discharge is several

hundred m3/sec. Furthermore, they have also stated that the cost of development

and registration of Bti (app. US $ 50,0000) are many times lower than those than

for a conventional chemical insecticides (about US $20 million)

2.10 The test mosquito species

2.10.1 Identification of the mosquitoes: Christopher (1933 & 1934) has given

the key for the identification of the families Culicidae and Anophelini. According to

him the adult anophelines are at once recognised by their spotted wings and from

their characteristic posture of resting on the walls or other objects. He described

43 species and 10 varietal forms collected at that time in India. Barraud (1934) has

given keys to the identification of adults & larvae belonging to family Culicidae

tribes Megharhinini and Culicinae. According to him the characteristics taken into

consideration for the identification of the adults are found chiefly in the pleural

52

chetotaxy and in the venation of the wings. Besides, the other characteristics taken

into consideration for morphological identification were scaling and the length of

palpi. He also used structure of larvae and pupae which often shows more definite

generic characteristics than adults. Puri (1954 & 1960) provided suggested rapid

identification method for the adult and larvae of Anopheline mosquitoes. Rao

(1984) in his book has provided comprehensive information on biology and basic

principles of control of Anopheles mosquitoes. This book also contains a key for

the rapid identification of Indian Anopheles species. Das et al. (1990) proposed a

simplified and easy pictorial key to 54 species of Indian Anopheles mosquitoes and

contains 177 illustrations and 3 tables. Nagpal & Sharma (1994) have also

prepared a key to the identification of all Anopheline mosquitoes reported from

India with illustrations of the various species, their morphological variations,

distribution and zoogeography.

2.10.2 Field sampling: For the bioassay studies a table top insectary facility for

the regular supply of healthy mosquito larvae of particular instar, age and species is

a must. Initially, the immature were collected from the field directly by sampling,

brought to the insectary, maintained under suitable light, temperature and rearing

conditions by providing larval food.

A manual of WHO (1975) entitled 'Practical Entomology in Malaria'

provides detailed information on the construction of exit traps, larval collection

methods, maintenance of culture in the lab, etc. Service (1976) has also described

procedures of larval sampling, types of traps, and methods used to collect

53

mosquitoes and methods for the analysis of the results that are of great use for the

field workers.

2.10.3 Mosquito rearing procedure: Ansari et al. (1978) described a method for

the mass production of Anopheles stephensi Liston. For mass production, standard

wooden cages of 70 x 60 x 60cm sizes were used. Adults were held in cages for

oviposition (egg production) using cyclic and non-cyclic population methods.

Mosquitoes were fed on water soaked raisins and offered blood meals at regular

interval. Eggs were collected in ovipositional jars (6 x 8 x 10cm) containing 250m1

water and lined with filter paper and they were held in these jars for 72 hrs. for

hatching. Newly hatched larvae were transferred to plastic trays (63 X 60 X 9 cm).

Larvae were fed on powdered food that consisted of 60% dog biscuit and 40%

brewer's yeast. They have described the establishment of the adult colonies, larval

rearing and pupal collection. They concluded that the maximum of 15000 larvae of

An. stephensi can be reared in a plastic tray (63 x 60 x 9 cm) as against 30,000 of

Cx. p. fatigans or Ae. aegypti. Ramoska & Pacey(1979) maintained mosquito

larvae in the lab for bioassays and fed them on yeast and rabbit chow.

2.10.4 Bioassays using mosquito larvae: This topic has been already reviewed

under applications. Literature on some aspects such as calculation of lethal doses

has been reviewed again here briefly. Ramoska et al. (1977) bio-assayed three

strains of Bs against field collected Psorophora columbiae, Culex nigripalpus and

Aedes taeniorhynchus larvae. They found that nearly 75% of the mortality that

occurred in the course of Bs exposure occurs within 48 hrs post-incubation with

the bacteria and also that susceptibility of P. columbiae larvae decreased to

54

Bacillus with increase in larval age (instar). They calculated Median Lethal Dose

(LDso) by plotting dose vs. mortality for a series of doses on log probit paper.

They then recorded LD so in terms of the reciprocal of the log dilution of the

original culture medium. Mulligan et al. (1978) used lab reared second instar of the

southern house mosquitoes, Culex quinquefasciatus Say. for bioassaying Bs strains

1593-4 and SSII-1 in the lab and in the field. They found strain 1593-4 to be more

virulent. Tyrell et al. (1979) used lab reared II instar larvae of Ae. aegypti , Cx.

pipiens quinquefasciatus and An. albimanus to test the toxicity of the parasporal

crystals of Bti. They calculated LCso in terms of the dry weight of the crystals/ml

and found the relative sensitivities as follows: Ae. Aegypti (LCs0=1.9 X 10-4

14,/m1) > Cx. pipiens var. quinquefasciatus (LCso= 3.7 X 10-4 µg/m1) > An.

albanimus (LCso= 8.0 X le RAW).

Wraight et al. (1981) used field collected I, II & III instar Aedes stimulans

larvae to study the effect of temp. and instar on the efficacy of Bs strain 1593.

They recorded larval mortality after 12hr interval and considered the larvae dead if

they were unable to return to the water surface after being forced to the tray

bottom.

Smith (1982) studied the effect of strain & medium variation on mosquito

toxin production by Bti and performed the bioassays against IV instar Ae. aegypti

(Linneaus). After the treatment the containers with larvae were incubated at 30 °C

and surviving larvae were counted. As he detected differences among log dilutions,

he did not determine LC so instead proposed a rating system based upon the 10-fold

dilutions and on number of surviving larvae at the lowest effective sample dilution.

55

Foo & Yap (1983) obtained comparable dosage response values for the

caged and introduced populations in the field trials ofBti serotype 11-14 against

Mansonia mosquitoes and suggested that lab reared populations can be used in

bioassay methods in the field.

Mulla et al. (1984) tested the larvicidal and field activity of Bs strains

against II and III instar larvae of the 5 species of mosquitoes. They calculated the

percentage reduction as per following formula (Mulla et al., 1971):

Ci X T2 %Reduction (R) = 100 X 100

C2x -ri Where

C 1 = No. of larvae in control pre-treatment

C2= No. of larvae in control post-treatment

T1 = No. of larvae in treated pre-treatment

T2= No. of larvae in treated post-treatment

Obeta & Okafor (1984) used lab reared third instar larvae ofAe. aegypti

and Cx. quinquefasciatus and field collected third instar larvae of An. gambiae to

test the powder formulation of Bti produced from 5 local media. For this they

suspended 100 mg of the powder in 1000m1 distilled water containing 1% (vol/vol)

Tween 80.Then they added 20 mosquito larvae to each 250 ml capacity plastic cup

containing 150m1 distilled water. They kept three replicates and maintained

simultaneously controls for the comparison. The Aedes and Culex larvae were fed

with small portions of ground oat flakes mixed with dried yeast, while the

Anopheles larvae were fed with baby cereal. The observations were made for

56

paralysis and knockdown effect and the mortality counts were made after 24 & 48

hrs post-exposure. The larvae that did not move on touching were presumed dead

and moribund larvae were counted among the survivors. They used Abbot's

formula for correction of the mortality and rejected the assays if mortality in the

control exceeded 20% in any of the experiments.

Ansari et al (1989) lab evaluated Bs formulations viz. Solvay liquid and

Abbott granules (ABG6185) against colonized larvae of An. culicifaciens, An.

stephensi, An. subpictus and Cx. quiquefasciatus. They recorded larval mortality

after 24 & 48 hrs and corrected it by Abbot's formula given below:

% test mortality - % control mortality Corrected % mortality = X 100

100 - % control mortality

Mittal et al. (1993) used lab reared larvae of An. stephensi, Cx.

quinquefasciatus and Ae aegypti to test two biolarvicides Spherix (B. sphaericus)

and Bactoculicide (B. thuringiensis) and observed the effect of temperature on

their toxicity. They exposed the II instar larvae to serially diluted concentration of

biolarvicide suspension in water and kept three replicates of each concentration.

They used Abbot's formula for the % mortality correction and calculated LC so and

LC90 values.

Brown et al. (1998) conducted lab and field trials to evaluate the

efficacy of Vectobac ® 12AS (Bti) against Cx. sitiens larvae. Initially they

conducted a large number of range-finding bioassays with widely spread exposure

concentrations. Based on these they obtained a narrow range of concentrations in

57

which lay the effective concentration. They conducted bioassays at 25 ° C under

12hr light and dark cycles and used probit analysis to calculate LC 50 & LC95 values.

The field trials were conducted by suspending field collected mosquito larvae in

salt marsh pools treated with biocide.

Rodrigues et al. (1998) recently carried out studies on the Bs larvicidal

activity against Malaria vector species in Amazonia. They conducted bioassays

with the 20 III instar larvae per concentration for each Anopheline species tested.

They calculated LC soin each period of observation (24 & 48hrs) using POLO-PC

programme. They also calculated relative activity (RA) using B. sphaericus 2362

as standard, according to the following formula:

LC50 standard RA=

LC50 sample The values of RA were calculated for 48 hrs results.

2.11 Conclusion

From all the literature reviewed in this chapter, it is evident that it has

taken decades of research from the point of initial isolation of mosquito-pathogenic

B. sphaericus and B. thuringiensis israelensis to their current status of commercial

availability for the vector control programmes. Their efficacy and safety aspects

have been studied and they provide a viable operational alternative to chemical

larvicides. Depending upon their suitability to the local ecological conditions and

vector breeding behaviour, these biolarvicides can be judiciously deployed either

individually or with other vector control agents in an integrated manner. Their

large-scale and prolonged use may however, pose problems like development of

58

resistance in the target vectors in the long run. Therefore suitable resistance

management strategies are being proposed and adopted. Various studies to

enhance their recycling potential are also being carried out. At the same time, there

is felt need for the isolation of potent indigenous strains for the better compatibility

between the local vector and the pathogenic bacilli which when commercialised

and indigenously mass produced is expected to be cost effective. Efforts are also

being made to express toxin genes of virulent strains in other organisms like algae

and cynobacteria in order to overcome their limitations like settling to ensure ready

availability in the feeding zones of larvae. Continued research on mosquito-

pathogenic bacilli therefore is a rewarding enterprise.

59