Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi

Background & objectives: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India. Methods: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations. Results: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2. Interpretation & conclusion: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2 . The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria

Mosquitocidal Bacillus amyloliquefaciens: Dynamics of growth & production of novel pupicidal biosurfactant.

Background & objectives: A strain of Bacillus amyloliquefaciens (VCRC B483) producing mosquito larvicidal and pupicidal biosurfactant was isolated from mangrove forest soil. The present study was aimed at studying the kinetics of growth and production of the mosquitocidal biosurfactant by this bacterium. Methods: Dynamics of growth, sporulation and production of mosquitocidal biosurfactant were studied by standard microbiological methods. The mosquitocidal biosurfactant was precipitated from the culture supernatant and bioassayed against immature stages of   mosquito vectors to determine lethal dose and lethal time. The activity, biological and biochemical properties of the biosurfactant have also been studied. Results: The pupal stages of mosquitoes were found to be more vulnerable to the biosurfactant produced by this bacterium with Anopheles stephensi being the most vulnerable species. The median lethal time (LT50) was found to be 1.23 h when the pupal stages of the above species were exposed to lethal concentration LC90 (9 μg/ml) dosage of the biosurfactant. Production of biosurfactant was found to increase with incubation time and maximum biomass, maximum quantity of biosurfactant (7.9 mg/ml), maximum biosurfactant activity (6 kBS unit/mg) and maximum mosquitocidal activity (5 μg/ml) were attained by 72 h of growth. The lipopeptide nature of the biosurfactant was confirmed by β-haemolysis, lipase activity, biofilm forming capacity, thermostability and biochemical analysis. Interpretation & conclusions: The mosquitocidal biosurfactant produced by B. amyloliquefaciens (VCRC B483) may be a prospective alternative molecule for use in mosquito control programmes involving bacterial biopesticides.

Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis.

Background & objectives: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Methods: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. Results: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. Interpretation & conclusions: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.

Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride

Secondary metabolites produced by Trichoderma viride, a deuteromycetes fungus, under submerged culture condition were formulated and evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus mosquito. At a concentration of 10 æg ml-1 the formulation showed remarkable attractancy with an oviposition active index (OAI) of +0.52. When the oviposition attractancy of the formulation was compared with a known oviposition attractant, p-cresol, both at 10 æg ml-1, the former was found to be more attractive to result in 70 percent egg laying than the later with 30 percent egg laying. Thin layer chromatography fractions of the secondary metabolites showed that a fraction with Rf value of 0.88 was highly active as oviposition attractant with an OAI of +0.65. Further work on identification of the active principle(s) of the microbial formulation might lead to an oviposition attractant useful in mosquito vector management

Effect of entomopathogenic fungus, Beauveria bassiana on larvae of three species of mosquitoes.

Beauveria bassiana, an entomopathogenic fungus, was evaluated for its potential against second and third instar larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. Conidiospores of this fungus were effective in causing infection leading to mortality of different larval instars. Larvae of Cx. quinquefasciatus were more susceptible to infection than An. stephensi and the second instar larvae of these two species were more susceptible than third instar larvae. Larvae of Ae. aegypti were resistant to infection by B. bassiana.