SUMMARY SUMMARY

Transcription

1 SUMMARY SUMMARY It has been the practice in the past to control mosquitoes and other insects of public health importance mainly by the use of chemical insecticides. These chemical insecticides have produced undesirable side effects such as air pollution, water pollution, soil pollution, accidental poisoning of human and above all the emergence of insect resistance. Under these circumstances, the interest in biological control technology especially the use of microbial agents has emerged in recent times. Nowadays, the larval control of mosquitoes either by source reduction or by use of larvicides or a combination of these is a preferred method of reducing the population of adult mosquitoes. The microbial control of mosquitoes is a rapidly developing area of research and has assumed significance in the light of mounting insecticidal resistance in mosquitoes, particularly the disease vectors and henceforth, it is a matter of serious concern about environmental problems caused by the excessive use of insecticides against these vectors. After screening the literature, it is evident that there is almost no work on the isolation and evaluation of strains of biocides against mosquitoes inhibiting different habitats in Garhwal Himalaya. Hence to fill up the void in the existing

2 SUMMARY knowledge, it has been proposed to undertake the work on Assessment of biocides against mosquitoes in Submontane ecosystem of Garhwal Himalaya with the following specific objectives * to select virulent strains of mosquito pathogen from various habitats * to evaluate selected strains for insecticidal activity, and * preliminary evaluation of insecticidal activity in mosquito breeding sites. The entire work on the assessment of biocides against mosquitoes in submontane ecosystem of Garhwal Himalaya has been summarised as follows - 1. The samples of soil, aquatic weeds, water and dead larvae were collected mainly from the various mosquito breeding habitats, which included forests, streams, pools, rivers, swamps, ditches, etc., for isolation and evaluating indigenous microbial biocides, especially the Bacillus group. For isolation of bacteria, 10 fold serial dilutions were made and plated onto Nutrient Yeast Extract Mineral Salt Medium (NYSM) agar medium. Thereafter, the petri plates were incubated for development of bacterial colonies. 2. The bacterial colonies which have appeared on the NYSM agar plates were differentiated on the basis of their morphology (colour, texture and type of haemolysis) and were purified further by streaking them individually on NYSM agar plates. These plates were incubated and clear well defined pure colonies were picked up and transferred to NYSM slants or preserved as lyophilised stocks or spores in glycerol. 3 For screening the larvicidal activity, a loopful of each type of colony was then emulsified in distilled water in bioassay cups containing 20 II-III

3 SUMMARY instars larvae of Anopheles culicifacies and Aedes aegypti. The mortality of the larvae was then recorded at 24 h. post-treatment. Any bacterial colony that induced 50% or more mortality to either or both of the larvae was then considered as larvicidal. 4. Each bacterial colony was subjected to preliminary test for toxicity. NYSM broth (50 ml) in 250 ml. capacity Erlenmeyer flask was inoculated with a loopful of purified culture of an isolate and incubated on a rotary shaker till sporulation. Then, the biomass termed spore crystal complex (SCC), was harvested by centrifugation at 6,000 rpm. One ml. of this suspension was treated to 50 numbers of An. culicifacies and Ae. aegypti larvae placed in cups containing 250 ml. of tap water in quadruplicate. For serotyping of isolates, the procedure of de Barjac and Bonnefoi (1962) was followed. 5. For purification of active isolates, the selective bacteria were plated on to selective nutritive yeast of the medium containing the antibiotics for the selective isolation of specific bacteria. The purified isolates were then sub cultured onto nutrient agar slants for further purification and incubated before storing in cold room. 6. All the isolates of Bacillus thuringiensis and Bacillus sphaericus which were selected after the preliminary tests for toxicity were inoculated to 200 ml NYSM broth in 11 Erlenmeyer flask and incubated till complete sporulation. A few strains of B. sphaericus which sporulated poorly in NYSM broths were grown in the standard medium. The spores were harvested by centrifugation at 6,000 rpm for 10 min. distributed in 100 mg. in sterile glass ampoules and lyophilised. The ampoules after sealing hermetically were stored at room temperature.

4 SUMMARY 7. Spores of different isolates obtained as mentioned above, were washed twice with distilled water and transferred to vials containing 50% glycerol. Whenever necessary, the spore suspension was centrifuged, the spore pellets washed and resuspended in NYSM broth. This suspension was heat treated at 80 C for 10 min. and inoculated into the desired medium. 8. For serotyping of isolates, the procedure of de Barjac and Bonnefoi (1962) was followed by preparing H-antigens, antisera procurement and tube agglutination test. 9. Six strains each of B. thuringiensis H-14 and B. sphaericus H-5a5b with varying levels of larvicidal activity were chosen for dynamics of growth and sporulation. Each strain was grown in 400 ml. of NYSM both in EM flasks, inoculated with overnight culture at 5% level, and incubated on a rotary shaker. The growth was determined by reading the absorbance of culture samples at 470 nm in a spectrophotometer. The biomass yield was estimated after centrifuging the samples and weighing the pellet, and the total cell and spore counts were computed by plating 10 fold serial dilutions of samples, before and after heat treatment on NYSM agar plates. 10. Susceptibility status of mosquitoes to bacterial strains was worked out by fixing the optimum dose against each selected mosquito species breeding in natural habitats representing three levels of toxicity viz., toxic, moderately toxic and poorly toxic. Their biomass was harvested by centrifugation and formulated into water dispersible powders. The water dispersible powders were tested against III instars larvae of mosquitoes for larvicidal activity. 11. Once the larvicidal strains were obtained, they were produced on a large scale for laboratory testing. Two strains of B. thuringiensis H-14 (B 113 &

5 SUMMARY B 77) and B. sphaericus H-5a5b (B 42 and B 64), were grown in jaggery broth for 48 h. Then their biomass were harvested and lyophilised. Its LC 50 value determined against III rd instars mosquito larvae and then dispersed into sterilized screw capped vials. 12. Impact of certain environmental factors like - indoor and outdoor, temperature, sunlight and larval density on the activity of bacterial strains was assessed under laboratory conditions. 13. Field trials were carried out to test the efficacy of B. thuringiensis and B. sphaericus for the control of Ae. aegypti and An. culicifacies. The strains selected for the present study were B-17, B-300 and B-325 of B. thuringiensis H-14 and B-42, B-288 and B-381 of B. sphaericus H-5a5b. For comparison purpose, a reisolate of IPS-80 standard preparation of B. thuringiensis H-14 and strain 2362 of B. sphaericus H-5a5b were used. 14. Larvicidal efficacy of Bacillus thuringiensis israelensis H-14 in different habitats was evaluated in Litchi garden pits harboring immature of Ae. aegypti and An. culicifacies. The density of the immature mosquitoes was monitored before and after the treatment at regular intervals. 15. Larvicidal efficacy of Bacillus sphaericus H-5a5b was evaluated in Litchi garden pits harboring Ae. aegypti and An. subpictus larvae. Larval population in these pits was monitored by taking 5-dipper samples/pit prior to the treatment and on 2, 6, 11, 16, 21, 26 and 31 day after the treatment. 16. Two strains each of B. thuringiensis H-14 (B 300 & B 325) and B. sphaericus H-5a5b (B-288 & B-381), which were found to be extremely toxic to mosquito larvae than IPS-80 and 2362 were tested further in the

6 SUMMARY field. Each test was replicated thrice and an equal number of pits were left untreated as controls. 17. In all, 1942 samples covering soil, water, larval and roots of aquatic plants were collected from 16 different habitats. Out of these, 807 (41.55%) were soil samples, 409 (21.06%) water samples, 586 (30.17%) larval samples and 140 (7.2%) aquatic marshy plants. As many as 312 isolates of Bacillus thuringiensis and Bacillus sphaericus were obtained from these samples. There were 190 colonies of B. thuringiensis and 122 colonies of B. sphaericus. 18. Out of 280 soil samples collected from paddy fields, yielded 63 larvicidal bacilli, while 145 soil samples from transient pools yielded 40 larvicidal bacilli, all belonging to B. thuringiensis. More number of isolates of B. thuringiensis were obtained from paddy fields (48) and transient pools (40), while less isolates were encountered in samples of seepage pools, stream bed and farm yard. Lakes, grassland pools, burrow pits, cesspits, wells, termatarium and drainage were found negative for B. thuringiensis. Considering the composition of different serotype of B. thuringiensis in soil samples, the maximum belonged to H-14 (92.3%) followed by untesteable (4%), untypeable (3%) and H-20 (1%). As regards to toxicity grouping, the poor toxic isolates were more (60%) than moderately toxic (14%), highly toxic (8%), toxic (6%), non-toxic (4%) and extremely toxic (2%) in succession. Among the 27 isolates of B. sphaericus, 15 were obtained from paddy fields, 6 from ponds and 4 from streambeds, 1 each was obtained from grassland pools and termatarium. Two of B. sphaericus H-5a5b isolates from ponds were extremely toxic and one each isolate from pond and paddy field were highly toxic.

7 SUMMARY 19. Out of 399 water samples, 51 were found belonging to larvicidal bacilli 44 (11.02%) were positive for B. thuringiensis and 7 (1.75%) for B. sphaericus. Among different habitats, water samples from transient pools yielded 33 isolates. The samples collected from ponds, lakes, canals, seepage pools and wells were also found positive while those collected from riverbeds, paddy field, garden land, burrow pits, streambeds, cesspits, drainage, waterfall and tree holes were negative. Water samples from seepage pools, lake and wells yielded only B. thuringiensis isolates, while, from ponds, canals and transient pools both B. thuringiensis and B. sphaericus were recorded. The larvicidal bacilli from water samples constituted 86.27% of B. thuringiensis and B. sphaericus were 13.72%. 20. The distribution of the larvicidal bacilli in the mosquito larval samples collected from different habitats revealed availability of 28 larvicidal bacilli. Among different habitats, samples from ponds, canals, seepage pools, transient pools and drainage yielded (17.8%), (3.57%), (14.2%), (32.14%), (17.8%) and (14.2%), isolates respectively while grasslands, paddy field, cesspits, wells and riverbeds did not yield any isolate. Out of the total larvicidal bacilli obtained, B. thuringiensis contributed 75.0% and B. sphaericus 25.0%. 21. Aquatic and marshy plants collected from five habitats (ponds, canals, river bed, transient pools and cesspits) revealed distributional positivity of larvicidal bacilli. Both B. thuringiensis and B. sphaericus were yielded from all the chosen habitats, majority of them were from ponds (54.9%) and canals (35.29%). A total of 72.8% of the 140 samples were found positive for B. thuringiensis and B. sphaericus. The number of samples positive for B. thuringiensis were much lower (15%) than the B. sphaericus (57.8%).

8 SUMMARY 22. Sporulation in the bacterial strains is witnessed by 5 stages-ovo-elongate (germinative cells), rod shaped cells (vegetative form), cell with one end swollen, cells in tennis racket stage and matured cells (spores). The tennis racket stage was observed at 9 th h of growth in B-42, 13 th h in B-64 and B- 43, 17 th h in B-85 and 25 th h in the other 2 strains. 23. Susceptibility of different species of mosquitoes viz., Culex mimeticus, Cx. quinquefasciatus, An. subpictus, An. culicifacies and Ae. aegypti to Bacillus thuringiensis H-14 and B. sphaericus H-5a5b was carried out by considering LC 50 values. The order of susceptibility of different mosquito species to different Bacillus thuringiensis H-14 strains was recorded as Cx. mimeticus > Cx. quinquefasciatus > An. subpictus > Ae. aegypti > An. culicifacies. Among different strains of B. sphaericus, H-5a5b, B-42 and B-64 were moderately toxic to An. subpictus, toxic to Cx. mimeticus, moderately toxic to Cx. quinquefasciatus and poorly toxic to Ae. aegypti and An. culicifacies. The order of susceptibility of the larvae of different mosquito species to Bacillus sphaericus strains was An. subpictus > Cx. mimeticus > Cx. quinquefasciatus > An. culicifacies > Ae. aegypti. 24. Shelf life of bacterial strains is based on LC 50 values of the lyophilized cells and water dispersible powder of B. thuringiensis H-14 strain B-113 at -10 C, 8 C, 30 C and 40 C. The strain B-113, when stored as water dispersible powder at -10 C, it lost only 5% of its activity after 24 months and at 8 C it lost 18% of activity. At 30 and 40 C, it lost about 21% and 36% activity respectively at 24 months. 25. Two strains (B-42 and B-64) of B. sphaericus H-5a5b were taken for studying shelf life at different temperatures (-10 C, 8 C, 30 C and 40 C). The LC 50 values of lyophilised cells and water dispersible powder of B.

9 SUMMARY sphaericus strains of B-42 and B-64, prior to storage were 6.0 and 106 and 6.4 and 112 mg/ml respectively. The strain B-64 of B. sphaericus H5a5b, when stored as water dispersible powder, it lost only 15% of activity after 18 months at -10 C and within 32-35% activity after 24 months at 8 and 30 C and 50% activity after 11 months at 40 C. When stored as lyophilised powder under hermatically sealed condition, it lost 38-40% activity after 24 months at 10 C and 8 C. 26. Environmental factors have been found to influence the activity of bacterial strains on mosquito larvae. In the case of An. culicifacies, 100% mortality was observed both indoors and outdoors within 2h for 10 6 and 10 5 concentrations and within 24h for 10 4 concentrations, when B. sphaericus was used. There was a similarity in the results for both indoors and outdoors, when B. thuringiensis was used. A strong sunlight has been found to have a negative effect on the activity of B. thuringiensis israelensis. The activity of B. thuringiensis israelensis at 8 C and 15 C was found to be about 4 times less than at 25 C. There was no significant difference in the activity of B. thuringiensis israelensis between 8 C and 15 C. In both the mosquito species - Ae. aegypti and An. culicifacies, the LC 50 value towards II nd and IV th instars was almost in similar pattern at 8 C, 15 C and 25 C. 27. It was observed that both LC 50 and LC 90 values increased as the density of Daphnia species increased. In the tests with B. sphaericus H-5a5b, the LC 50 and LC 90 values were 5 to 6 times higher at a density of 90 Daphnia per 150 ml. than those without Daphnia. 28. To evaluate the larvicidal efficacy of selected strains of B. thuringiensis and B. sphaericus, the experiments were carried out in Litchi garden pits,

10 SUMMARY where the breeding of selected mosquito species (An. subpictus and Ae. aegypti) was maintained under controlled conditions. The strain B-77 of Bacillus thuringiensis H-14 was effective in reducing the larval density of Anopheline and Culicine larvae within a period of 24 h after application. 29. The strain B-42 of B. sphaericus H-5a5b has been found effective in reducing the immature density of both An. subpictus and Ae. aegypti within a period of 24 h after the treatment. 30. Comparative efficacy of highly potent strains of B. thuringiensis B-325, B-300 and IPS-80 and of B. sphaericus - B-288, B-381 and 2362 has been observed on the density of immature of An. subpictus breeding in Litchi garden pits. The treatment days were 1 st, 5 th and 10 th and the next consecutive days were the observation days. Further, the observations were made separately on early (I & II) and late (III & IV) instars. 31. Bacillus thuringiensis (H-14) has been represented by three bacterial strains - B-325, B-300, IPS-80. There was cent percent reduction of late instars of An. subpictus for all the three bacterial strains. In the Litchi pits treated with B-325, the density of early instars increased from 6 on 2 nd day and 40 on the 10 th day and that of late instars from 0 to 23. As far as, the average percentage of reduction of both early and late instars against the efficacy of different bacterial strains is concerned, the maximum reduction was observed in B-300 followed IPS-80 and B The efficacy of three bacterial strains (B-288, B-381 and 2362) against the early and late instars of An. subpictus was assessed. The days of treatment were the same as chosen for B. thuringiensis strains. After the 1 st treatment i.e., on the 2 nd day, there was a reduction in the density of early instars by 65.6%, 100% and 92.7% respectively against the efficacy of B-288, B-381

11 SUMMARY and However, there was absolute reduction in the density of late instars against all the selected strains of B. sphaericus (H-5a5b). The efficacy of B-381 was most efficient than 2362 and B-288 in succession towards both early and late instars. Conclusively, it can be said that the main sources of the wild type of strains are soils/sediments, plants, sick/moribound insects and water. The toxicity of the strains within a subspecies / serotype varied widely. And, unlike insecticides, bio-control agents are host specific, safer to the environment, find easy application in the field, are cost-effective in production, lack infectivity and pathogenicity in mammals including man and has little evidence of resistance development in target mosquito species. More emphasis be given to characterize the bacterial strains that could be used to control mosquito larvae to a minimum level.