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.

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: ). 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.

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Secondary metabolites produced by (Davis & Bowen 1994). By using oviposition attractants, vector mosquitoes could be attracted to chosen sites for laying eggs. Various chemicals serve as oviposition attractants for mosquitoes even when present in relatively small quantities (Beehler & Mulla 1993). Oviposition attractants are known to be produced by microorganisms also (Hazard et al. 1967, Rockett 1987, Hasselschwert & Rockett 1988, Beehler et al. 1994. Among deuteromycetes fungi, Trichoderma species are known to produce aromatic/volatile secondary metabolite(s) (Kikuchi et al. 1974, Keszler et al. 2000, SarhyBagnon et al. 2000, Kalyani et al. 2000. In the present study secondary metabolite(s) produced by the fungus Trichoderma viride was evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus and the results are presented in this paper. MATERIALS AND METHODSA natural strain of T. viride (F24), obtained from the culture collection of Vector Control Research Centre (VCRC) was subcultured on Sabouraud Dextrose Agar (SDA) (glucose 40 g; peptone 10 g; agar 20 g; distilled water 1000 ml; pH 4.5-5.0) slants and incubated for 5 days to attain sporulation. Then a loopful of spores were inoculated to 50 ml Sabouraud Dextrose Broth (SDB) (glucose 40 g; peptone 10 g; agar 20 g; distilled water 1000 ml; pH 4.5-5.0) in a 250 ml conical flask and incubated on a rotary shaker (New Brunswuck Scientific Co., New Jersey, USA) at 110 rpm and at 30°C for 3 days. From the seed culture, 2% of the inoculum was transferred to 200 ml of SDB and incubated for 15 days at the above mentioned conditions. The culture was filtered through Whatman no.1 filter paper, the mycelial mass was discarded, and the culture filtrate was lyophilized. The residue was dissolved in 50% ethanol at a ratio of 500 mg of residue in 10 ml of ethanol. This formulation was code named as VCRC F24 and used for oviposition attractancy tests.Determination of optimum concentration of VCRC F24 and p-cresol for oviposition attractancy test -Threeday-old Cx. quinquefasciatus Say female mosquitoes, an indigenous strain obtained from a laboratory colony maintained at VCRC, were fed on fowl blood and maintained for two days on raisins at 28 ± 2 o C and 70-80% RH. Gravid female mosquitoes were used for the determination of oviposition attractancy using standard procedure (WHO 1963). Different concentrations (5 µg ml -1 , 10 µg ml -1 and 15 µg ml -1 ) of VCRC F24 and a known oviposition attractant, p-cresol (a volatile pentane), was prepared in tap water. In the case of p-cresol, a stock solution of 1mg ml -1 was made in ethanol and diluted to the required levels with tap water. Tap water with equal amount of ethanol served as control for VCRC F24 and p-cresol. Two hundred ml of each of the test/control solutions held in disposable bowls (capacity, 250 ml) were placed in the floor of a mosquito cage (size, 55 x 55 x 55 cm). One hundred numbers of fully gravid female mosquitoes were released into the cage. For each test at any given time, not more...

BACKGROUND: A biosurfactant, surfactin, produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471), was effective in killing mosquito larval and pupal stages. As it was lethal to the non‐feeding pupal stage, it was presumed that it could kill the adult mosquitoes also. In this study, the adulticidal effect of the biosurfactant was assessed in the laboratory against a malaria vector, Anopheles stephensi.RESULTS: The biosurfactant surfactin, separated from the culture supernatant of the production strain, showed mosquito adulticidal activity when tested as ultralow‐volume (ULV) spray in a Peet‐Grady chamber. Knockdown activity and mortality were found to increase with increasing surfactin dosage. Knockdown dosage (KD) and lethal dosage (LD) were calculated by statistical analysis. The KD50 and KD90 dosages were 10.73 and 26.39 mg m−3 respectively. The LD50 and LD90 dosages were 16.13 and 39.21 mg m−3. The average droplet size of B. subtilis surfactin was 17.5 ± 1.07 µm.CONCLUSION: The present study indicates that the biosurfactant surfactin, produced by B. subtilis subsp. subtilis (VCRC B471), is a potential bioadulticide for ULV spray against malaria‐transmitting Anopheles stephensi mosquitoes. This is the first report of a mosquito adulticide from a microbial source. Copyright © 2012 Society of Chemical Industry

Bacillus sphaericus is a bio-control agent effective against Culex quinquefasciatus, the vector of bancroftian filariasis. Apart from its larvicidal effect, there are reports of reduced infection of filarial parasites in mosquitoes exposed to it. In the present study, adults of Cx. quinquefasciatus emerged from B. sphaericus treated larvae were fed on blood samples positive for microfilariae of Wuchereria bancrofti and examined at various time intervals to assess the infection level. The rate of infection was reduced from 95% on day 1 post-feeding to 75% on day 13, when fed with blood sample containing 41 mf/20 μl. The mean parasite burden was also reduced from 4.9 per mosquito on day 1 to 2.15 on day 13. When fed with another sample (30 mf/20 μl), the infection was reduced from 100% on day 1 to 80% on day 13. Reduction in parasite burden was 4.0 to 1.75. Abnormally developed second-stage larvae of the parasite were seen in treated mosquitoes. Thus, the results indicated adverse effect of B. sphaericus treatment on infection and development of the filarial parasite in mosquitoes. The possible reason for the parasite regulation was studied through the assessment of the carryover of the bacterium as well as its toxins to the surviving mosquitoes. The presence of B. sphaericus was determined through plating of homogenate of survived mosquitoes on NYSM agar. Toxic protein was detected through immunoblotting. The bacterium as well as its 41.9-kDa toxic protein was found to be transmitted from larvae to adults and affected the parasite development, directly by the toxin or indirectly by eliciting humoral immune response of the mosquito.