Characterisation of three mosquitocidal Bacillus strains isolated from mangrove forest

Characterisation of three mosquitocidal Bacillus strains isolated from mangrove forest

Biological Control 42 (2007) 34–40 www.elsevier.com/locate/ybcon Characterisation of three mosquitocidal Bacillus strains isolated from mangrove fore...

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Biological Control 42 (2007) 34–40 www.elsevier.com/locate/ybcon

Characterisation of three mosquitocidal Bacillus strains isolated from mangrove forest I. Geetha *, G. Prabakaran, K.P. Paily, A.M. Manonmani, K. Balaraman Unit of Microbiology & Immunology, Vector Control Research Centre, Indian Council of Medical Research (ICMR), Pondicherry 605 006, India Received 28 November 2006; accepted 3 April 2007 Available online 12 April 2007

Abstract In an attempt to isolate mosquitocidal bacteria, 460 samples of soil, leaf and water were collected from mangrove habitats of Andaman–Nicobar Islands, India. Out of a total number of 857 bacteria screened, three Bacillus strains showed mosquitocidal activity at promising levels. Two of them were identified, morphologically and biochemically, as Bacillus subtilis, whereas one was Bacillus thuringiensis. These strains were code named as B469, B471 and B474, respectively. Molecular characterisation using 16S rRNA confirmed the identity of B469 and B471 as B. subtilis strains and flagellar serotyping confirmed B474 as B. thuringiensis subsp. israelensis/tochigiensis (H14/H19). Amplification of cry and cyt genes of B474 showed the presence of cry 4A, cry 4B, cry 11, cry 10, cyt 1 and cyt 2. Bioassay of the culture supernatants (CS) of B. subtilis strains showed mosquitocidal activity against larvae and pupae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The mosquitocidal activity was more pronounced with the CS of B471 than that of the B469. Interestingly, CS of both these strains was more active on pupae, a non-feeding stage, than larvae of the mosquitoes tested. The B. thuringiensis strain was able to kill the larvae of all the above mentioned mosquitoes. This is the first study demonstrating mosquito pupicidal activity of B. subtilis strain. Moreover, this is the first report of isolation of a highly active B. thuringiensis subsp israelensis/tochigiensis from India. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Bacillus strains; Culture supernatant; Mosquito pupicidal; Mangrove forest; Characterisation

1. Introduction Mosquito borne diseases such as malaria, filariasis, dengue and viral encephalitis contribute to a larger proportion of health problems of developing countries. In recent years, as a result of changes in public health policy, social factors and development of resistance in mosquitoes as well as the pathogens they transmit, there has been resurgence in the incidence of mosquito borne diseases. Although chemical insecticides provide effective control of mosquitoes, development of resistance to them has been widely reported (WHO, 1992; Rodriguez et al., 2001). An alternative is the microbial pesticides and is advantageous as they are

*

Corresponding author. Fax: +91 0413 2272041. E-mail address: [email protected] (I. Geetha).

1049-9644/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.04.003

eco-friendly and specific to the target organisms. Among various microbial pesticides, Bacillus thuringiensis and Bacillus sphaericus are being used widely as larvicidal bacteria for mosquito control (Balaraman, 1995; Lee and Zairi, 2005; Medeiros et al., 2005; Armengol et al., 2006). However, recent reports on development of resistance to B. sphaericus by vector mosquitoes (Nielsen-Leroux et al., 1995; Poopathi et al., 1999; Su and Mulla, 2004), prompted us to search for new mosquitocidal bacteria. There are reports of isolation of Clostridum bifermentans, an anaerobic spore former, and B. thuringiensis subsp. israelensis/ tochigiensis, an aerobic spore former, from mangrove swamps and mangrove sediments of Malaysia and Japan (de Barjac et al., 1990; Maeda et al., 2001). In the light of this, we explored mangrove forests of Andaman–Nicobar Islands of India for mosquitocidal bacteria, and it has resulted in the isolation of two Bacillus subtilis and

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one B. thuringiensis strains. These strains were identified and characterised through morphological, biochemical and molecular biological methods. Their mosquitocidal effects were assessed against larval and pupal stages of three vector species. 2. Materials and methods 2.1. Sample collection Samples for isolation of mosquitocidal bacteria were collected from mangrove habitats of Andaman–Nicobar Islands, India. Soil samples each measuring 10 g was collected using sterile spatula and stored in sterile screw capped vials. Water samples of 10 ml each were collected from marshy swamps and pits using sterile pasteur pipette. To get samples with minimal effect of ultraviolet (UV) light, soil and water samples were collected from about 2 to 3 cm below the surface of the habitat. Leaf samples were collected using sterile scalpel from mangroves. To get the maximum UV protected phylloplane microbial population, they were obtained from 2.0 to 2.5 m above the ground and 0.3 m inside the outer leaf canopy of each tree or shrub. 2.2. Isolation of bacteria Samples were brought to the laboratory, 1 g of soil was weighed, transferred to a vial containing 10 ml of sterile water, and kept on a rotary shaker (New Brunswick Scientific Co. Inc., NJ, USA) at 100 rpm for 30 min, to dislodge bacterial cells from the soil particles. The supernatant was diluted 10-fold and 0.1 ml was spread on pre-solidified nutrient yeast salt mineral agar (NYSM) containing 5 g glucose (bacteriological), 5 g peptone, 5 g NaCl, 3 g beef extract, 5 g yeast extract, 203 mg MgCl2, 10 mg MnCl2 and 103 mg CaCl2 (Hi-Media, India) per liter of distilled water. Similarly, water samples were diluted 10-fold with sterile water and plated as above. Leaf samples were transferred to 10 ml sterile water, kept on a rotary shaker for 30 min, diluted 10-fold and plated as above. The bacterial suspensions were not subjected to pasteurisation before plating, expecting both gram-positive and gram-negative bacteria with mosquitocidal activity. The plates were incubated at 30 °C for 48 h and bacterial colonies which formed were purified on NYSM agar. Each of the purified colonies was then sub-cultured on NYSM agar slants, allowed to grow for 72 h and stored at 4 °C. These bacterial isolates were screened for mosquito larvicidal and pupicidal activity. 2.3. Screening for mosquito larvicidal and pupicidal activity A loopful of bacteria from the NYSM slant was inoculated to 10 ml of NYSM broth and incubated (30 °C) on a rotary shaker (200 rpm) for 72 h. After incubation, sample of 1 ml each from the whole culture was used to screen for mosquito larvicidal and pupicidal activity through bioas-

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say. Bioassays were conducted in wax coated paper cups containing 25 third instar larvae/pupae of Culex quinquefasciatus/Anopheles stephensi in 125 ml chlorine-free tap water. Appropriate controls without the addition of the bacterial culture, but containing 1 ml of un-inoculated NYSM broth, were maintained. After 24 h of exposure, mortality was scored by counting the number of live larvae/pupae present in the respective cups. A bacterial isolate was considered potent if it caused 100% mortality of the test larvae or pupae. The potential bacterial cultures were further screened to find out whether the bacterial cells or their metabolites exhibited mosquitocidal activity. For this, the cells and culture supernatant (CS) were separated by centrifuging the active cultures at 8000 rpm for 20 min. The cell mass and CS were bioassayed independently, as mentioned above. The dose of CS used was 1 ml/125 ml water and that of cell mass was 1 mg per 125 ml of water. Based on the mosquitocidal activity three strains were selected, code named as B469, B471 and B474 and characterised. 2.4. Characterisation of the potential bacterial strains Morphological, biochemical and physiological characteristics of the potential mosquitocidal isolates were studied according to Bergey’s Manual of Systematic Bacteriology (Sneath, 1986). Biochemical tests namely, fermentation of glucose, arabinose, mannitol and xylose, utilization of citrate, malonate, hydrolysis of starch, decarboxylation of lysine, ornithine, deamination of phenylalanine, arginine dihydrolase, degradation of tyrosine, decomposition of urea, nitrate reduction, production of indole and acetyl methyl carbinol were performed using Rapid Biochemical Identification test kit (Hi-Media Laboratories, India). The ability of the bacterial isolates to grow at temperatures of 5 °C, 30 °C, 50 °C and 60 °C, and their growth in 2%, 5%, 7% and 10% of NaCl was also studied. Flagellar serotyping was done by Dr. Michio Ohba, Kyushu University, Japan for the identification of the B. thuringiensis isolate. 2.5. Molecular characterisation of the mosquitocidal bacterial strains 2.5.1. Polymerase chain reaction (PCR) of 16S rRNA genes of B. subtilis (B469 and B471) The 16S rRNA gene, corresponding to an internal portion of the B. subtilis group, was PCR-amplified using the primers, Bsub 5F (5 0 -AAG TCG AGC GGA CAG ATG G-3 0 ) and Bsub 3R (5 0 -CCA GTT CCA ATG ACC CTC CCC-3 0 ), reported by Wattiau et al. (2001). Genomic DNA was extracted from B469 and B471 using Gen Elute Bacterial Genomic DNA kit (Sigma, St. Louis, USA). The PCR mixture comprised 3.5 mM MgCl2, 200 lM of each dNTP’s (Promega, Madison, USA), 0.4 lM of each primer (Metabion, Bangalore, India), 2.5 ll of Taq buffer and 1.25 units of Taq polymerase (AmpliTaq Gold, Applied Biosystems, NJ, USA). To this mixture, 1 ll of the DNA

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template was added. The control tube was added with 1 ll deionised water in place of DNA sample and the reaction mixture in the tubes were made up to 25 ll volume using deionised water. The reaction was amplified in a Thermal Cycler (Bio-Rad, USA). The PCR conditions were: denaturation at 94 °C for 12 min., 30 cycles of denaturation at 95 °C for 0.5 min, annealing at 65 °C for 2 min, extension at 72 °C for 2 min, and a final extension step at 72 °C for 7 min. Each of the amplification products was checked by electrophoresis of 8 ll on 1.5% agarose (Sigma, St. Louis, USA) gel containing ethidium bromide. To verify the size and purity of the product, a 100 bp ladder (Promega, Madison, USA) was run along with the sample. After confirmation of the size of the amplicon, the amplified PCR products was purified using Qiagen QIA quick gel elution kit (Qiagen Corp., Hilden, Germany), and sequenced (Microsynth AG, Switzerland). 2.5.2. PCR of cry and cyt genes of B. thuringiensis (B474) The NYSM grown culture (1 ml) was centrifuged at 10,000 rpm for 1 min at 4 °C, the cells were separated, and re-suspended in 0.1 ml of sterile water. It was frozen for 20 min at 70 °C and boiled for 10 min to lyse the cells, briefly spun for 1 min at 10,000 rpm, and the supernatant was used as template. The dipteran-specific cry genes (cry IVA and cry IVB) were detected using primers and PCR conditions as reported by Carozzi et al. (1991). Whereas the mosquito-specific cry genes (cry 2, cry 4A, cry 4B, cry 10, cry 11, cry 17, cry 19, cry 24, cry 25, cry 27, cry 29, cry 30, cry 32, cry 39 and cry 40) and cyt genes (cyt 1 and cyt 2) were detected according to Ibarra et al. (2003). The PCR ingredients, PCR protocol and gel electrophoresis were as described above, except that 5 ll of template DNA was used. After confirmation of the size of the amplicon, the amplified PCR products was purified using Qiagen QIA quick gel elution kit (Qiagen Corp., Hilden, Germany), and sequenced (Microsynth AG, Switzerland).

assay with bovine serum albumin (Sigma, St. Louis, USA) as standard (Bradford, 1976). The lyophilised SCC was bioassayed only against larvae of the three mosquito species. Bioassays were conducted in 300 ml wax coated paper cups containing 50 third instar larvae or pupae in 250 ml of chlorine-free tap water. Laboratory reared larvae and pupae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti obtained from the rearing and colonization facility of the Centre were used in the experiments. For each test, six doses of the SCC (ng/ml) or CS (ll/ml) were added. Four replicates per dose and appropriate controls (chlorine-free tap water + same dose of un-inoculated NYSM broth) were maintained. All the bioassay experiments were conducted at a room temperature of 28 ± 2 °C, and at 80% relative humidity. The mortality of the larvae/pupae was scored after 24 h of exposure by counting the number of live ones present in the bioassay cup. Probit regression analysis was carried out with SPSS 10.0 for windows software and LC50 and LC90 as well as their 95% fiducial limits were determined. 3. Results The mangrove habitat collection consisted of 341 soil, 70 leaf and 49 water samples from which bacteria were isolated. A total of 857 bacteria were selected randomly from the isolates and screened for mosquitocidal activity. Preliminary screening with 1 ml of culture supernatant and/ or 1 mg of SCC showed that 12 bacterial strains had mosquito pupicidal/larvicidal properties. Subsequently, all the 12 strains were bioassayed at different concentrations to find out their dosage dependent effect and upon this, three strains showed activity even at low concentrations. These three were from soil samples. They were selected, code named as B469, B471 and B474, and studied further. Whereas the CS of B469 and B471 showed mosquito larvicidal and pupicidal activity, the SCC of B474 showed only larvicidal activity.

2.6. Laboratory bioassay against different species of mosquitoes

3.1. Characterisation of the potential mosquitocidal bacterial strains

The three strains characterised such as B469, B471 and B474, were further studied for their dosage dependent effect on mosquito larvae and pupae. A loopful of bacteria from the NYSM slant was inoculated to 10 ml of NYSM broth and incubated overnight at 30 °C on a rotary shaker at 200 rpm. From this, 2 ml was inoculated to 100 ml of NYSM broth and incubated as above for 6 h. Subsequently, 25 ml (5% v/v) was transferred to 500 ml of NYSM broth and incubated as above for 72 h. The culture was centrifuged at 8000 rpm for 20 min to separate the cell mass from CS. The CS of the bacterial strains (B469 and B471) was bioassayed against third instar larvae and pupae of An. stephensi, Cx. quinquefasciatus and Aedes aegypti. The spore–crystal complex (SCC) from B474 was lyophilised and protein concentration was measured by Bradford

All the three mosquitocidal bacterial isolates were found to be aerobic, gram-positive, motile, rod-shaped bacteria, and formed cylindrical spores centrally or para-centrally in non-swollen sporangia. The morphological and biochemical characteristics of the three isolates are presented in Table 1. Based on the morphological and physiological characters, strains B469 and B471 were identified as B. subtilis, and B474 as B. thuringiensis. Further characterisation of the B. thuringiensis strain, through serotyping, revealed it as an unusual serotype, viz., B. thuringiensis subsp. israelensis/tochigiensis (H14/19), having the flagellar antigens of two different serotypes. PCR amplification of the hyper variable region of 16 S rRNA gene of B469 and B471 yielded amplicons of the size of 595 bp. When the nucleotide sequences were blasted

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Table 1 Morphological and biochemical characteristics of mosquitocidal bacterial strains Property

B471

B469

B474

Colony morphology Cell size Presence of crystal Spores Utilization of D-Glucose L-Arabinose D-Xylose Mannitol Citrate Phenyl alanine Arginine Lysine Ornithine Malonate Tyrosine Hydrolysis of Starch Decomposition of Urea Growth in the presence of 2% NaCl 5% NaCl 7% NaCl 10% NaCl Nitrate reduction Growth at 5 °C 30 °C 50 °C 60 °C Catalase Oxidase Acetyl methyl carbinol Indole Identified as

Dull white, irregular, convex, mucoid Length 2–3 lm, width less than 1 lm  Cylindrical

Dull white, irregular, convex, mucoid Length 2–3 lm, width less than 1 lm  Cylindrical

White, irregular, flat Length 3–4 lm, width 1 lm + Cylindrical

+ + + +    + +  

+ + + + + + -

+   +   +    +

+

+

+

+

+

+

+ + + + +

+ + + + +

W    +

+ + + + + + + + B. subtilis

+ + + + + + + + B. subtilis

 +   + + + + B. thuringiensis

+, positive; , negative; W, weak.

against the NCBI database using BLASTN, it showed the highest identity score to B. subtilis (99%) (Altschul et al., 1997). The sequences have been submitted to the GenBank (Accession Nos. DQ 133460 and DQ 133461). Amplification of cry and cyt genes of B474 showed the presence of cry 4A, cry 4B, cry 11, cry 10, cyt 1 and cyt 2 (Fig. 1). The sequences showed 100% identity with the same genes found in B. thuringiensis subsp. israelensis. The sequences have been submitted to the GenBank (Accession Nos. EF 182766, EF 182767, EF 182768, EF 182769 and EF 182770).

to that of B469. The larvae and pupae of Ae. aegypti were more susceptible to the CS of B471. Against this mosquito species, the larvicidal activity was 1.2 times and pupicidal activity was 2.1 times higher than that of B469. The SCC of B474 was bioassayed against the larvae of An. stephensi, Cx. quinquefasciatus and Ae. aegypti and the results are presented in Table 4. It was more toxic to the larvae of Cx. quinquefasciatus with an LC50 of 4 ng/ml. To elicit the same response, the larvae of Ae. aegypti required 1.8 times higher dose. The larvae of An. stephensi required 4.64 times higher dose than that of Cx. quinquefasciatus.

3.2. Mosquitocidal potential of the bacterial strains

4. Discussion

The CS of B469 was three times more toxic to the larvae of An. stephensi than that of B471. Also, it was 1.7 times more toxic to the larvae of Cx. quinquefasciatus (Tables 2 and 3). Whereas, the CS of B471 was 3.5 times more toxic to the pupae of An. stephensi. Similarly it was 2.8 times more toxic to the pupae of Cx. quinquefasciatus, compared

The Bacillus strains, code named as B469 and B471, isolated from soil samples from mangrove forests of Andaman–Nicobar Islands were identified as B. subtilis by their morphological and biochemical features. Identification of B. subtilis like organisms is difficult and laborious as they cannot be distinguished from each other by

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I. Geetha et al. / Biological Control 42 (2007) 34–40 Table 4 Bioassay results of SCC (ng/ml) of B. thuringiensis subsp. israelensis/ tochigiensis (B474) against third instar larvae of three species of mosquitoes

Fig. 1. Agarose gel electrophoresis of the PCR products obtained with B. thuringiensis subsp. israelensis/tochigiensis (H14/19) showing the cry and cyt genes [Lane 1: cry 10 (348 bp); 2: 100 bp Ladder; 3: cry 4A (797 bp); 4: cry 4B (321 bp); 5: cry 11 (342 bp); 6:- cyt 2 (355 bp); 7: cyt 1 (477 bp)].

conventional phenotypic tests (Chun and Bae, 2000). The sequence of 16 S rRNA gene has been widely used to identify an unknown bacterium to the genus or species level (Gray and Herwig, 1996; Kuske et al., 1997; Wise et al., 1997; Siefert et al., 2000; Sacchi et al., 2002), and in the present study the 16 S rRNA sequence analysis confirmed the identity of both the mosquitocidal isolates as B. subtilis.

Mosquito species

LC50 (95% FL)

LC90 (95% FL)

An. stephensi Cx. quinquefasciatus Ae. aegypti

18 (16–21) 4 (3.4–4.8) 7.5 (6.2–9.2)

44 (36–54) 16 (10–24) 28 (21–38)

As early as in 1989, Gupta and Vyas reported a strain of B. subtilis capable of infecting and causing mortality of larvae of Anopheles culicifacies, the primary vector of malaria in central India. Recently, Das and Mukherjee (2006) have reported two B. subtilis strains active against third instar larvae of Cx. quinquefasciatus. However, the only bacterial biocontrol agent known to exhibit mosquito pupicidal activity is a gram-negative bacterium Pseudomonas fluorescens (Prabakaran et al., 2003). Serotyping of the mosquito larvicidal B474 showed that the strain shared flagellar antigens of two serotypes i.e., israelensis and tochigiensis (H14/H19). Isolates of B. thuringiensis subsp. israelensis (H14) are well known, since 1977 (Goldberg and Margalit, 1977), for their activity against mosquitoes. In the present study, the protein concentration of LC50 dosage of B. thuringiensis subsp. israelensis/tochigiensis SCC was 0.001, 0.0046 and 0.002 lg/ml, respectively, against the larvae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti. Yu et al. (1991) reported an LC50 dosage of 0.0049 and 0.0043 lg/ml of solubilised crystal proteins of B. thuringiensis subsp. israelensis against the larvae of Cx. quinquefasciatus and Ae. aegypti, respectively. Being a different serotype, B. thuringiensis subsp. israelensis/tochigiensis is apparently more toxic than B. thuringiensis subsp. israelensis and could be a potential alternative for biological control of mosquitoes. Strains like B. thuringiensis subsp tochigiensis (H19) and B. thuringiensis subsp. israelensis/tochigiensis (H14/H19) are known mosquito pathogens (Lonc et al., 2001; Maeda et al., 2001).

Table 2 Bioassay results of CS of B. subtilis (B469) against third instar larvae and pupae of three species of mosquitoes Mosquito species

An. stephensi Cx. quinquefasciatus Ae. aegypti

Larva (ll/ ml)

Pupa (ll/ ml)

LC50 (95% FL)

LC90 (95% FL)

LC50 (95% FL)

LC90 (95% FL)

5 (4–6) 16 (14–18) 25 (24–26)

11 (10–13) 36 (32–40) 43 (41–45)

2 (1–3) 10 (7–12) 22 (19–24)

6 (5–6.3) 37 (34–40) 56 (52–61)

Table 3 Bioassay results of CS of B. subtilis (B471) against third instar larvae and pupae of three species of mosquitoes Mosquito Species

An. stephensi Cx. quinquefasciatus Ae. aegypti

Larva (ll/ml)

Pupa (ll/ml)

LC50 (95% FL)

LC90 (95% FL)

LC50 (95% FL)

LC90 (95% FL)

15 (14–16) 27 (25–30) 20 (19–21)

22 (20–25) 59 (50–73) 46 (40–54)

0.6 (0.4–0.7) 4 (3–5) 10 (9–11)

3.5 (2–4) 15 (13–19) 24 (20–32)

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Interestingly, the B. thuringiensis subsp. israelensis/tochigiensis reported earlier was also isolated from mangrove sediments. However, it is the first time that the cry and cyt gene profile of B. thuringiensis subsp. israelensis/tochigiensis (H14/H19) is studied. It is interesting to note that the strain harbors cry and cyt genes, and also spherical parasporal crystal, which are specific to the serotype israelensis unlike the rhomboidal crystals of B. thuringiensis subsp. tochigiensis (H19) (Ohba et al., 1981). This is the first report wherein the CS of B. subtilis is shown to have activity against pupal stages of mosquitoes. It is very clear that the CS has not acted on mosquito ingestion, as the pupa which is a non-feeding stage also is killed in addition to larvae. Though both the B. subtilis strains were morphologically and physiologically similar, they differed in their mosquitocidal activity and the difference may be at the level of expression of the mosquitocidal metabolite(s). Since, the novelty of the B. subtilis strains is their pupicidal activity, B471 appears to be more promising than B469. It is not possible to compare the activity of the CS of B. subtilis with the SCC of B. thuringiensis, because the nature and mode of action of the toxins should be different. Though the mode of action is not known, the activity of B. subtilis on larval stage has been reported to be due to the cyclic lipopeptides present in the CS (Das and Mukherjee, 2006). Whereas, in the case of B. thuringiensis, upon ingestion by susceptible insect larvae, toxin inclusions are released from disrupted bacterial cells and then solubilised in the larval midgut lumens that are generally alkaline pH. The soluble protoxins are subsequently activated by gut proteases to yield toxic fragments that are relatively resistant to further proteolysis (Schnepf et al., 1998). The extremely high dose requirement of the CS of B. subtilis for mosquitocidal effect can be brought down by purifying the mosquito active principle(s) and incorporating it into suitable formulation. The isolation of B. subtilis showing mosquito pupicidal activity assumes greater importance as they can be preserved as spores for long periods. As B. subtilis are a non-pathogenic species, normally found in soil, exhibiting a wide range of physiological and nutritional requirements (Blackwood et al., 2004), they can be cultured easily and used for mosquito control safely. Hence, further studies on identification and characterisation of the active principle(s) present in the CS of B. subtilis would help in increasing our arsenal to combat vector mosquitoes through the development of appropriate formulations. Acknowledgements We are grateful to Dr. P.K. Das, Director, Vector Control Research Centre for the encouragement and the facilities provided throughout the study. The technical assistance of Mr. S. Venugopalan is gratefully acknowledged. We thank Dr. Michio Ohba, Kyushu University, Japan for flagellar serotyping of B. thuringiensis subsp. israelensis/tochigiensis.

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