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.

Utility of the rDNA-ITS2-PCR assay in detecting the life stages of species S of Anopheles fluviatilis complex.

Background & objectives: Anopheles fluviatilis, which ranks second among the major malarial vectors in India occurs as a complex of three morphologically identical species (species S, T and U) of which only species S is a vector. Hence, it becomes pertinent to have a method for the detection of this vector species under field conditions to map the distribution of this vector. An rDNA-ITS2-PCR assay has been developed earlier for species S of this complex using female adult specimens. In order to widen the range of samples on which this technique can be employed, the utility of this PCR assay in detecting different life stages/gender/parts of the vector species was studied. Also, its reliability in detecting a single species S in pools of species T was studied. Methods: Mosquitoes were collected from Malkangiri and Koraput districts of Orissa State where species S and T of this complex are reported. The wild caught fed females, after egg laying were subjected to PCR assay for species identification. The F1 progeny of a few PCR identified specimens was raised and samples at larval, pupal and adult stages were used for PCR assay. Single adult specimen of species S was added to pools containing different numbers of adults of species T and the pools were subjected to DNA extraction and PCR assay. Results: The PCR assay could detect species S from pure DNA extracts of the immature stages and crude DNA extracts of parts of adult/whole adult mosquito of either gender. Crude DNA extracts of pools of mosquitoes had to be diluted and used in order to obtain the species diagnostic fragment. Interpretation & conclusions: The rDNA-ITS2-PCR assay producing an amplicon of 350 bp. diagnostic for species S, could detect all stages/gender. Any part of the adult can be used for species identification. Further, a single adult of species S in pools of as many as 99 adults of species T could be detected. Application of this PCR assay will be useful in mapping the distribution of species S, an important malarial vector.

Utility of the rDNA-ITS2-PCR assay in detecting the life stages of species S of Anopheles fluviatilis complex.

BACKGROUND & OBJECTIVE: Anopheles fluviatilis, which ranks second among the major malarial vectors in India occurs as a complex of three morphologically identical species (species S, T and U) of which only species S is a vector. Hence, it becomes pertinent to have a method for the detection of this vector species under field conditions to map the distribution of this vector. An rDNA-ITS2-PCR assay has been developed earlier for species S of this complex using female adult specimens. In order to widen the range of samples on which this technique can be employed, the utility of this PCR assay in detecting different life stages/gender/parts of the vector species was studied. Also, its reliability in detecting a single species S in pools of species T was studied. METHODS: Mosquitoes were collected from Malkangiri and Koraput districts of Orissa State where species S and T of this complex are reported. The wild caught fed females, after egg laying were subjected to PCR assay for species identification. The F1 progeny of a few PCR identified specimens was raised and samples at larval, pupal and adult stages were used for PCR assay. Single adult specimen of species S was added to pools containing different numbers of adults of species T and the pools were subjected to DNA extraction and PCR assay. RESULTS: The PCR assay could detect species S from pure DNA extracts of the immature stages and crude DNA extracts of parts of adult/whole adult mosquito of either gender. Crude DNA extracts of pools of mosquitoes had to be diluted and used in order to obtain the species diagnostic fragment. INTERPRETATION & CONCLUSION: The rDNA-ITS2-PCR assay producing an amplicon of 350 bp. diagnostic for species S, could detect all stages/gender. Any part of the adult can be used for species identification. Further, a single adult of species S in pools of as many as 99 adults of species T could be detected. Application of this PCR assay will be useful in mapping the distribution of species S, an important malarial vector.

Association of the level of mosquito larvicidal activity with the growth & sporulation in Bacillus sphaericus H-5a5b strains.

The role of growth and sporulation in the production of mosquito larvicidal factors in B. sphaericus H-5a5b strains was investigated using 6 strains that differed in their larvicidal activity. Among these, strain B64 produced maximum biomass (15.5 g/l by 29th h) while B45 and B85 yielded the least (12.8 g/l by 41st and 37th h respectively). Strains B43 and B42 reached the peak of viable cell count (4-6 x 10(10) cells/ml) 4 h earlier than B64 and 12 h earlier than the rest of the strains. Strains B42 and B43 produced higher number of heat resistant spores (4 x 10(8) spores/ml), while strains B45 and B57 produced the lowest numbers (2-4 x 10(5) spores/ml). Mosquitocidal toxin synthesis was noticed as early as the 5th and 9th h in the cultures of the strains B42 and B64 respectively while in those of other strains it was detected by the 13th h or later. The results indicated that generally the highly and moderately toxic strains grew faster and sporulated better than the poorly toxic ones.

In vitro cultivation of third stage larvae of Wuchereria bancrofti to fourth stage: influence of some physico-chemical factors.

It has been reported that third stage larvae (L3) of Wuchereria bancrofti strain from Jakarta, molted to the fourth stage (L4) in vitro, in a simple culture medium supplemented with 10% human serum. In the present study, this culture medium has been used to examine the effects of some physico-chemical parameters on larval growth, development and molting of Wuchereria bancrofti from India. Lymph at 10% concentration enhanced the in vitro survival time of larvae. Molting of larvae from L3 to L4 stage has been obtained using human fetal lung cells in cellular co-culture and as a source of conditioned medium. Given these improvements in the medium supplementation, it has been observed that the age of L3s (duration of L3s maintenance within the mosquitos) is one of the most important parameters for the development of L3s in vitro. No molting was observed when one day L3s were used whereas, molting occurred with one or two weeks old L3s. On the contrary, when more than 3 weeks old L3s were used molting failed to occur even though duration of survival of L3s was improved and in this case, most of the larvae were degenerated.

Isolation of mosquito-pathogenic Bacillus sphaericus & B. thuringiensis from the root surface of hydrophytes.

Attempts were made to isolate B. sphaericus and B. thuringiensis active against mosquito larvae from the root surface of hydrophytes. Out of 139 samples processed, 86 B. sphaericus and 23 B. thuringiensis isolates were obtained. Sixty two of the B. sphaericus isolates belonged to the serotype H5a5b, 2 to H6 and 22 isolates did not agglutinate with any of the 6 antisera tested. Twenty of the B. thuringiensis isolates belonged to the H14 serotype, 1 each to the H10 and H17 serotype(s) and 1 to an unknown serotype. Fifty nine of the B. sphaericus and 20 of the B. thuringiensis isolates fall under highly toxic category with the LC50 dose of 1-50 ng/ml for Culex quinquefasciatus third instar larvae.

Characterization & larvicidal activity of indigenous isolates of Bacillus sphaericus from natural breeding habitats.

Soil, water and moribund/or dead mosquito larval samples collected from various mosquito breeding habitats of Pondicherry and Tamil Nadu were screened for the presence of mosquito pathogenic B. sphaericus isolates. From 1892 samples 21 isolates were obtained. All these isolates fell under 3 serogroups viz., H5a5b, H6 and H45, the latter two serotypes not reported hitherto as toxic to mosquito larvae and three phage-groups namely, phage group 2, phage group 3 and an unknown one. Twelve of the isolates were highly toxic and superior to the standard strains 1593, 2297 and 2362 supplied by Pasteur Institute, Paris when tested against Culex quinquefasciatus larvae. Out of these 12 strains 11 belonged to the serotype H5a5b and the phage-group 3 and 1 belonged to the serotype H45 and an unknown phage group.

Establishment of a standard test method for determining susceptibility of Mesocyclops to different insecticides.

A method to determine the susceptibility of Mesocylops sp. to insecticides was devised. Matured adults were used for the bioassays. Mesocyclops sp. (25 in number) were placed in 24.9 ml of sterilized paddy field water in 5 cm diameter petri dish and 0.1 ml of appropriate stock solution of the insecticide was added. Mortality was scored 24 h after treatment. Among the insecticides tested, Permethrin, Dichlorvos, Temephos, DDT, Carbendazim, Fenitrothion, Zolone and Aldrin were effective against Mesocyclops sp.