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.

Cost-effective medium for the production of mosquito pupicidal lipopeptide from Bacillus subtilis subsp. subtilis (VCRC B471).

BACKGROUND & OBJECTIVES: A cyclic lipopeptide (CLP), surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit mosquitocidal activity. The present study was carried out to enhance the surfactin level using low cost material in the production medium. METHODS: Two carbon sources, glucose and common sugar, and two nitrogen sources, ammonium nitrate and soya were used in the study. Different concentrations of 'C' and 'N' sources were used in the production medium to enhance the production of surfactin. RESULTS: A new medium (SS7) containing 2% sugar, 6% soya and 0.5% common salt with micronutrients was designed which was found to enhance the production of surfactin. The crude mosquitocidal metabolite (CMM) produced in this medium was 3 g/l which was two times higher than that obtained using synthetic medium NYSM. The LC50 dosage of the CMM to the pupal stages of An. stephensi (2.3 µg/ml) was comparable to that obtained with CMM from the conventional medium. INTERPRETATION & CONCLUSION: The newly designed cost-effective medium designated as sugar soya medium (SSM) enhanced the production of surfactin and the cost of production was estimated as [symbol: see text] 6 per litre, which is six times lesser than that of the conventional medium. Replacement of sodium chloride with cooking salt further reduced the cost of the medium.

Fly ash based Bacillus thuringiensis var. israelensis formulation for use against Culex quinquefasciatus, the vector of filariasis in natural ecosystems.

BACKGROUND & OBJECTIVES: Fly ash is produced in huge quantities by the various thermal power stations in India. This thermal waste has been employed as a carrier material in the preparation of a biopesticidal water dispersible powder (WDP) formulation for use against mosquitoes. In the present investigation, this newly developed fly ash based WDP formulation was evaluated in natural breeding habitats of mosquito. METHODS: Fly ash based WDP formulation of Bacillus thuringiensis var. israelensis (VCRC B17) was evaluated for its efficacy and residual activity in aquatic habitats supporting breeding of Culex quinquefasciatus, the vector of lymphatic filariasis in Neyveli Township, Neyveli Lignite Corporation, India for a period of one month. RESULTS: At an application rate of 10 kg/ha, the WDP was effective for five days regardless of the habitat, and provided 80-100% reduction in larval abundance of Cx. quinquefasciatus. INTERPRETATION & CONCLUSION: The study indicates that for continued control of immature density and prevention of adult emergence, a weekly application of this formulation is necessary. This study also showed that fly ash based formulations can be used for immediate control of mosquitoes in different types of habitats and has also brought out a new avenue for the utilization of coal ash.

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 (LT 50 ) was found to be 1.23 h when the pupal stages of the above species were exposed to lethal concentration LC 90 (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 ß-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.

Species-diagnostic polymerase chain reaction assays for Phlebotomus argentipes and Phlebotomus papatasi, vectors of Leishmania.

Species-specific differences encountered in the nucleotide sequences of a highly variable region of the 18S rRNA gene were used to design a multiplex polymerase chain reaction (PCR) assay for the identification of Phlebotomus papatasi Scopoli and Phlebotomus argentipes An-nandale & Brunetti, vectors of Leishmania. This multiplex PCR assay uses a common forward primer and two reverse primers, which are specific for the two species. Amplification of a PCR product of size 788 bp indicates the presence of P. papatasi, whereas a product of size 677 bp indicates the presence of P. argentipes. The assay was found to be highly specific and sensitive.

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain.

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC(50) dose of 1 microg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13-C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

Surfactin: a novel mosquitocidal biosurfactant produced by Bacillus subtilis ssp. subtilis (VCRC B471) and influence of abiotic factors on its pupicidal efficacy.

AIM: The rpoB gene of the mosquito pupicidal isolate Bacillus subtilis (VCRC B471) was amplified to confirm the subspecies as subtilis. The mosquito pupicidal activity expressed by the biosurfactant surfactin is novel, and hence, the influence of abiotic factors like pH, temperature of water and sunlight on its efficacy was studied under laboratory conditions. METHODS AND RESULTS: The rpoB gene amplicon of the bacterium (c. 570 bp of) was sequenced (accession number: EU057603). The relatedness of the bacterium to other members of the genus Bacillus was studied by tree construction, and the identity of VCRC B471 was confirmed as B. subtilis ssp. subtilis. The mosquito pupicidal activity exhibited by surfactin was found to be unaffected between pH 3-9, temperatures 25 and 37 °C and exposure to sunlight/UV radiation. Further, the pupicidal activity of surfactin was not diminished after exposure to 121 °C for 15 min, indicating its thermostable nature. CONCLUSIONS: VCRC B471 is confirmed as a strain of B. subtilis ssp. subtilis. The mosquitocidal toxin, surfactin produced by this bacterium being stable to UV and varied temperature, active at acidic and basic pH and temperatures between 25 and 42 °C renders this molecule an interesting lead to be developed as a mosquitocidal agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The mosquitocidal toxin, surfactin produced by B. subtilis ssp. subtilis (VCRC B471), being a biodegradable biosurfactant, exhibiting high stability to varied environmental conditions, can be used year round in breeding habitats and will be a prospective microbial toxin for use against mosquitoes.

Retention of mosquito larvicidal activity of lyophilized cells and WDP formulation of Bacillus thuringiensis var. israelensis on long-term storage.

Lyophilized cells (sealed and unsealed) and water dispersible powders (WDPs) of two indigenous isolates of Bacillus thuringiensis var.israelensis were stored at -10, 4, 30 and 40 degrees C for up to 4 years and checked for activity. Lyophilized cells stored in sealed condition at -10, 4 and 30 degrees C and WDPs stored at -10 and 4 degrees C were found to maintain the activity fairly well. The lyophilized cells stored at 30 degrees C and WDPs stored at 4 degrees C maintained their larvicidal property for up to 20 years. Hence, lyophilization and WDP formulations are reliable methods for long-term storage of B. t. var. israelensis.

Coconut water as a cheap source for the production of delta endotoxin of Bacillus thuringiensis var. israelensis, a mosquito control agent.

Bacillus thuringiensis var. israelensis (B. t. i.) is being widely used in mosquito control programs. However, the large-scale production of this bacillus is expensive due to the high cost of the production medium. In this study, we attempted to develop a cost-effective medium, based on a locally available raw material namely coconut water which is available in plenty as waste product from coconut oil industry. The yield of cell mass, sporulation and mosquito larvicidal activity were studied by growing this bacterium in this waste product and in comparison with the conventional medium (NYSM). Cell mass yield of 3.1g/L, spore count of 3.4x10(11)spores/mL and mosquito larvicidal activity (LC(50)) of 14.85ng/mL (against early fourth-instar larvae of Aedes aegypti) were obtained with a 30h old culture of this bacterium grown in coconut water. This is almost similar to that obtained with NYSM medium. Hence, coconut water-based culture medium is economical for the production of B. t. i.

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.

rDNA-ITS2-PCR assay for grouping the cryptic species of Anopheles culicifacies complex (Diptera: Culicidae).

Anopheles culicifacies, a predominant vector of malaria in India exists as a complex of five sibling species A, B, C, D and E, of which, except species B, all the rest are vectors with varying vectorial capacities. With a combination of PCR assays, it is possible to identify all the five members of this species complex. These assays include amplification of the rDNA-ITS2 region followed by digestion of the ITS2 amplicon using restriction enzyme, Rsa I which groups the five members of the An. culicifacies complex into two categories: species A and D forming one category and species B, C and E forming another. The samples grouped thus are then subjected to two allele-specific PCR assays (AD-PCR and BCE-PCR), which has been designed using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C and E. In the present study, the differences in the ITS2 region of the five species was used to design a PCR assay which groups the five members into the same two categories as obtained after digestion of the ITS2-PCR product. This assay uses a common forward primer based on the 5.8S region and two reverse primers, which is specific for the two categories. Amplification of a PCR product of size 253bp indicates the presence of species A/D, while a product of size 409bp indicates the presence of species B/C/E. By using this ITS2 PCR assay, the three-step procedure is reduced to two cutting down the time and cost involved. The ITS2 PCR assay has been validated on specimens collected from different regions of India and the results confirm to the earlier reports on the distribution of the members of the An. culicifacies complex.

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.

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.

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.

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.

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.

Bacillus amyloliquefaciens: a mosquitocidal bacterium from mangrove forests of Andaman & Nicobar islands, India.

Samples collected from the mangrove forests of Andaman & Nicobar islands yielded a mosquitocidal bacterium, whose extracellular metabolite(s) exhibited mosquito larvicidal and pupicidal activity. The bacterium was isolated using standard microbiological methods and identified using classical biochemical tests and rpoB gene sequences. The mosquitocidal bacterium was identified as Bacillus amyloliquefaciens. Mosquitocidal metabolite(s) was separated from the culture supernatant of the bacterium and its efficacy against the larval and pupal stages of different species of mosquitoes was determined in terms of LC(50) and LC(90). Mosquito larvicidal activity in terms of LC(50) against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was respectively, 26.4µg, 22.2µg and 20.5µg/ml and its pupicidal activity was 4.4µg, 8.2µg and 14.5µg/ml respectively. The mosquitocidal metabolite(s) was found to be a biosurfactant. This is the first report of the mosquitocidal activity of B. amyloliquefaciens and it is a new weapon which can be added to the array of microbial agents for use against mosquitoes.