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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