Irudayaraj Geetha, Unit of Microbiology and Immunology, Vector Control Research Centre, Indian Council of Medical Research (ICMR), Indira Nagar, Puducherry 605 006, India. E-mail: firstname.lastname@example.org
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.
The current interest in the development of biological agents for the control of vectors, especially mosquitoes, is an indication of concern about the recrudescence of mosquito borne diseases like malaria and dengue in epidemic proportions. Biological control of obnoxious mosquitoes relies mainly on the use of biolarvicides based on two entomopathogenic bacteria viz. Bacillus thuringiensis and Bacillus sphaericus. These biolarvicides are being used effectively both in the laboratory and field conditions for the control of mosquitoes. Though these biolarvicides are eco-friendly, they have some disadvantages. Under field conditions, its efficacy was found to be influenced by various abiotic factors such as pH of water, temperature and sunlight (Lacey and Inman 1985; Becker et al. 1992; Mittal et al. 1993; Lacey 2007). Above all, recent reports on the development of resistance among the mosquitoes vectors to B. sphaericus toxins (Nielsen-Leroux et al. 2002; Su and Mulla 2004) warrant the development of newer biocontrol agents or toxins to combat mosquito borne diseases.
Recently, a Bacillus subtilis strain producing mosquitocidal (larvicidal and pupicidal) toxin was isolated from mangrove forests of Andaman and Nicobar islands of India. The culture supernatant of this bacterium was found to kill the larval and pupal stages of three species of mosquitoes viz., Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti (Geetha et al. 2007). It is the first Gram-positive bacterium highly toxic to mosquito pupae. Mosquitocidal activity of B. subtilis is associated with the exotoxin unlike the other biolarvicides where the toxicity is associated with endotoxins. Further, mosquitocidal toxin production was found to be associated with vegetative growth of the organism rather than with sporulation (Geetha and Manonmani 2008). The mosquito pupicidal toxin produced by B. subtilis ssp. subtilis was identified as surfactin, a cyclic lipopeptide (Geetha et al. 2010). It is a mixture of several β-hydroxy fatty acids with chain lengths of 13–15 carbon atoms as its lipid portion. The main component is 3-hydroxy-13-methylmyristic acid, which forms a lactone ring system with an anionic heptapeptide. Surfactin is an antibacterial, antifungal, hypocholesteremic and protoplast-bursting agent, but the mosquito pupicidal activity is novel (Tsukagoshi et al. 1970; Kameda et al. 1974; Imai et al. 1971; Tendulkar et al. 2007).
The mosquitocidal strain (VCRC B471) was identified as B. subtilis by 16S rRNA (Geetha et al. 2007) and as B. subtilis ssp. subtilis, based on gyrA gene sequencing (Geetha and Manonmani 2008). However, the taxonomy of Bacillus and allied taxa requires a polyphasic approach rather than relying on one method. rpoB gene is reported as one of the molecular taxonomy probe for bacterial species identification. rpoB gene, coding for beta subunit of RNA polymerase, is described as possessing the same key attributes as 16S rRNA, in that it is common to all bacteria and is a mosaic of conserved as well as variable sequence domains (Dahllof et al. 2000). Most importantly, the rpoB gene exists as a single copy in bacterial genomes (Mollet et al. 1997). Therefore, rpoB gene sequencing was carried out in this study to further confirm the identity of the mosquitocidal bacterium. It is a prerequisite for any biocontrol agent intended to be developed commercially, to be effective in different breeding habitats under natural conditions. Because the mosquito pupicidal toxin is novel, its efficacy under natural conditions is not known. Hence, in this study, the impact of abiotic factors like pH, temperature of water and sunlight on the efficacy of the toxin was studied under laboratory conditions.
Materials and methods
Partial sequencing of rpoB gene
An rpoB fragment corresponding to B. subtilis rpoB positions 6–585 was PCR amplified by using primers rpoB-f (5′-AGGTCAACTAGTTCAGTATGGAC-3′) and rpoB-r (5′-AAGAACCGTAACCGGCAACTT-3′) reported by De Clerck et al. (2004). The reactions were carried out with a 50-μl reaction mixture containing 20 pmol of each primer, 10 nmol of each deoxynucleoside triphosphate, 5 μl of 10× PCR buffer (Applied BioSystems, Foster City, CA), 1 U of Taq polymerase (Applied BioSystems) and 50 ng of template DNA. The PCR profile consisted of denaturation at 94°C for 2 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 45 s, and extension at 68°C for 50 s; and a final extension at 68°C for 90 s. The resultant amplicons were purified with the Qiagen QIA quick gel elution kit (Qiagen Corp., Hilden, Germany) and sequenced in both directions using the same primers (Microsynth AG, Balgach, Switzerland).
Source of bacterium
The B. subtilis ssp. subtilis strain isolated from soil samples collected from the mangrove forests of Andaman and Nicobar islands was used for production of the pupicidal toxin. The production strain has been deposited in Microbial Type Culture Collection (MTCC), Chandigarh, India (Accession Number 5368). Stock cultures were stored in 15% glycerol (v/v) (Sigma, St Louis, MO, USA) at −80°C.
Source of mosquito
Freshly emerged pupae of A. stephensi were obtained from a laboratory colony maintained at the Rearing and Colonization Unit of the Vector Control Research Centre and used for bioassay.
Bacterial growth conditions
For the production of crude surfactin, the bacterium was grown aerobically on nutrient yeast salt mineral medium (NYSM). The medium contained 5 g glucose, 5 g peptone, 5 g NaCl, 3 g beef extract, 5 g yeast extract (Hi-Media, Mumbai, India) and 10 ml of trace elements solution per litre. The stock solution of trace elements contained 203 mg MgCl2, 10 mg MnCl2 and 103 mg CaCl2 per litre. The pH of the medium was 7·0 ± 0·2. Tubes containing 10 ml NYSM broth were inoculated with a loopful of bacterial cells from the slant culture. The tubes were incubated overnight on a rotary shaker (New Brunswick Scientific Co. Inc., Edison, NJ, USA) at 28 ± 2°C and 250 rev min−1. After incubation, cultures were inoculated to fresh 50 ml of NYSM broth at 1% v/v and incubated again for a further period of 7 h to synchronize the growth. From this young culture, 5% inoculum was added to 2 l Erlenmeyer flasks containing 600 ml of the medium and incubated with shaking for 72 h as mentioned above. Bacterial cells were removed from the medium by centrifugation at 9000 g for 25 min at 4°C in a Sorvall Evolution RC superspeed centrifuge (Kendro Lab. Products, Asheville, NC, USA) using SLA-1500 rotor. The culture supernatant (CS) obtained was precipitated with 6 N HCl for the separation of crude mosquitocidal metabolite (CMM) and was collected by centrifugation at 9000 g for 25 min at 4°C. MALDI-TOF analysis of CMM confirmed that the main component is surfactin (A.M.M. and I.G., unpublished data), and the mosquitocidal metabolite will be referred as crude surfactin henceforth.
Crude surfactin obtained from the culture supernatant was bioassayed against freshly emerged pupae of A. stephensi. The pupicidal bioassay followed the WHO standard protocols (WHO 2005). For experimental treatment, 100 mg of crude surfactin was dissolved in 10 ml of distilled water and used as the stock solution. The concentrations of crude surfactin tested were 0·8, 1·6, 3·2, 4·0, 4·8 and 6·4 μg ml−1. To 150 ml capacity, disposable wax-coated paper cups, 100 ml of chlorine-free tap water was added, and 25 freshly emerged pupae of A. stephensi were transferred to each cup. Each experiment was performed using four replicates, and an equal number of controls were set up simultaneously. The treated and control cups were held at 27 ± 2°C, 80–90% relative humidity and a photoperiod of 12 h of light followed by 12 h of dark. All the bioassay cups were covered with mosquito-net cloth to prevent the escape of emerging adults, if any. The moribund and dead pupae in four replicates were combined and expressed as a percentage of pupal mortality of each concentration. Dead pupae were identified when they were found at the bottom of the bioassay cups as straightened pupae by losing their typical comma shape as well as the dead late stage pupae which were moulted to adult but were unable to come out of the pupal exuvia were also recorded. The mortality of the pupae was scored after 24 h of exposure by counting the number of live ones present in the bioassay cup. The experiments were repeated twice. In cases where the control mortality was between 5 and 20%, the observed percentage mortality was corrected using Abbott’s formula (Abbott 1925). Data from all replicates was pooled for analysis. LC50 and LC90 values were calculated from a log dosage-probit mortality regression line using spss 10.0 for windows software (SPSS Inc., Chicago, IL) yielding a level of effectiveness at 50 and 90% mortality and 95% confidence intervals (95% CI). The LC50 values obtained with different parameters were compared using 95% confidence interval. LC50 values with nonoverlapping confidence interval were considered to be significant at P < 0·05.
Influence of physico-chemical factors on the efficacy of crude surfactin
Influence of physico-chemical factors on the efficacy of crude surfactin was tested as per the method described by Becker et al. (1992).
Effect of water temperature. For testing the effect of water temperature on crude surfactin, the pupae were acclimatized at 25 and 37°C for 2 h in a temperature-controlled unit, prior to their transfer to bioassay cups. The acclimatized pupae were bioassayed against crude surfactin with un-acclimatized pupae as control.
Effect of water pH. To test the effect of pH on the pupicidal effect of crude surfactin, the pH of the bioassay water was adjusted to 3, 5, 7, 9, 11 with 1 N HCl or 1 N NaOH. Pupae were acclimatized for 2 h in rearing water with different pH and were subjected to bioassay with unexposed pupae as control. After 24 h of exposure to different concentrations of crude surfactin, LC50 value at each pH was determined.
Effect of sunlight. Solar radiation may be transmitted through a transparent object such as air, water or glass with a change in speed and direction. Glass containers are reported to transmit light in the near-ultraviolet region (A), which is the most lethal range, as well as in the visible range of the spectrum (Acra et al. 1984). Two sets of four 250-ml glass beakers with 125 ml of water were used for this experiment. Both the sets were treated with a single dose i.e. LC90 dosage of crude surfactin. One set was exposed to direct sunlight for 8 h, and after every 2 h of exposure, the temperature of the water was recorded. The other set was kept under laboratory conditions for the same time period. Freshly moulted pupae were released into sunlight exposed and unexposed beakers, and pupal mortality was scored after 24 h of treatment.
Thermostability of crude surfactin. The crude surfactin was subjected to moist heat at 121°C for 15 min (Munimbazi and Bullerman 1998), and its efficacy was tested by bioassay against pupae. Untreated crude surfactin was tested for comparison.
Detection of surfactin production
Surfactin production was detected by haemolysis of erythrocytes. Cells of mosquito pupicidal strain, B. subtilis ssp. subtilis (VCRC-B471), were streaked on blood agar plates containing defibrinated sheep blood (5% v/v) and incubated at 37°C for 24–48 h.
Partial sequencing of rpoB gene
The rpoB gene amplicon of VCRC B471 (c. 570 bp of) was sequenced, and the sequence was submitted to the GenBank (accession number: EU057603). The rpoB gene sequence of VCRC B471 was subjected to blast and was found to be 100% similar to B. subtilis ssp. subtilis and 99% similar to B. subtilis ssp. spizizenii. Therefore, to study the relatedness of this species to other members of the genus Bacillus, rpoB gene sequences of ten Bacillus sp. viz., Bacillus sp. (gi 116563503), two strains of B. subtilis ssp. subtilis (gi 46019147, gi 116563649), B. subtilis ssp. spizizenii (gi 116563633), Bacillus mojavensis (gi 116563585), Bacillus vallismortis (gi 116463669), Bacillus atrophaeus (gi 116563659), Bacillus amyloliquefaciens (gi 161345230), Bacillus licheniformis (gi 156181153) and Bacillus sonorensis (gi 115663671) were recovered from the GenBank database and aligned with the gene sequence of VCRC B471 using the software Clustal-W (Thompson et al. 1994). Tree construction was performed with the software mega 4.0 (Tamura et al. 2007) (Fig. 1).
Influence of physico-chemical factors on pupicidal efficacy of crude surfactin
Influence of temperature and pH of the rearing water on the pupicidal activity of the crude surfactin of VCRC B471 is presented in Table 1. The pupicidal activity (LC50) of crude surfactin was found to be 2·2 and 2·3 μg ml−1 at 25 and 37°C, respectively. The efficacy of mosquito pupicidal property of crude surfactin was not affected by increasing the temperature. The susceptibility of pupae of A. stephensi to crude surfactin was not affected when the pH of the rearing water ranged from 3 to 9. At pH 11, the LC50 dose was found to be 1·3 times higher than that required at pH 7·0. However, the result is not significant because the 95% confidence interval between pH 7 and pH 11 is overlapping. The pupicidal potency of crude surfactin was not affected by exposure to sunlight as complete mortality of all the pupae was observed in exposed and unexposed crude surfactin unlike control where all the pupae were alive. The maximum water temperature recorded during exposure to sunlight was 42°C which in turn did not affect the pupicidal activity of the crude surfactin.
Table 1. Influence of physico-chemical factors on the pupicidal efficacy (LC50) of crude surfactin produced by Bacillus subtilis ssp. subtilis (VCRC B471)
LC50 (μg ml−1)
pH 7·0, Temp. 30 ± 1°C
Bioassay at varied temperatures of rearing water
Bioassay after subjecting the crude surfactin to 121°C for 20 min
Different pH of the rearing water
Temperature stability and haemolytic activity of surfactin
The pupicidal activity of the crude surfactin which was subjected to 121°C for 15 min remained undiminished (LC50 2·8 μg ml−1) when compared to control (LC50 2·6 μg ml−1). This indicates that the pupicidal crude surfactin of VCRC B471 is highly thermostable. NYSM plates (24 h) showed mucoid colonies, indicative of biosurfactant production, and blood agar plates (24 h) showed a clear halo around the cells as a result of haemolysis (Fig. 2).
To confirm the identity of VCRC B471, rpoB gene sequencing was carried out, and its identity was confirmed as B. subtilis ssp. subtilis from the phylogenetic tree. The rpoB protein has been used to infer relationships between archaeal orders (Matte-Tailliez et al. 2002), Gram-positive and Gram-negative bacteria (Morse et al. 2002). Although the mosquitocidal strain was identified as B. subtilis by classical biochemical tests and 16SrRNA sequencing (Geetha et al. 2007) and as B. subtilis ssp. subtilis by gyrA (Geetha and Manonmani 2008), the rpoB gene sequence further confirmed its identity which is essential in the taxonomy of Bacillus sp.
The mosquitocidal activity of surfactin produced by B. subtilis is novel; and therefore in the present study, the influence of pH, temperature and sunlight on its efficacy was studied. The optimum range of pH at which B. subtilis toxins exhibited maximum larvicidal efficacy was between 3 and 10. The pupicidal crude surfactin was found to be stable at different temperatures (25 and 37°C) and when exposed to sunlight. Unlike the toxins of the commonly used biolarvicides, B. thuringiensis and B. sphaericus, crude surfactin produced by VCRC B471 is not affected by sunlight/UVradiation. Our results corroborate with the results of Das and Mukherjee (2006) where the larvicidal efficacy of the cyclic lipopeptides produced by B. subtilis strains was unaffected by physico-chemical factors such as pH of water, temperature, heating and exposure to sunlight. The crude surfactin produced by B. subtilis ssp. subtilis, being able to withstand a wide range of temperature, water pH, exposure to long hours of sunlight/UV radiation and able to tolerate high temperature (121°C), can be used year round in mosquito breeding habitats of tropical countries like India for the control of mosquito immatures.
Haemolysis is used as one of the methods to screen strains of bacteria producing biosurfactants and also for the detection of surfactin production (Nakano et al. 1988; Youssef et al. 2004). In the present study, the production of biosurfactant, surfactin by the mosquitocidal strain was indicated by haemolysis. Surfactin, produced by B. subtilis, was first identified as a potent inhibitor of fibrin clotting (Arima et al. 1968) and later found to lyse erythrocytes as well as spheroplasts and protoplasts of some bacterial species (Bernheimer and Avigad 1970). To reduce the haemolytic activity, recently, linear surfactin molecules were synthesized which are to be devoid of haemolytic activity (Dufour et al. 2005). Surfactin is one of the most powerful biosurfactants known for reducing the surface tension of water from 72 to 27 mN m−1 (Cooper et al. 1981), and reduction to levels 41–31 mN m−1 have been found to result in total pupal mortality (Singh and Micks 1957). In addition to surface tension reduction, the possibility of its action on the cuticle of the pupae cannot be ruled out as there are reports on the action of surfactin on biological membranes (Vollenbroich et al. 1997a,b; Buchoux et al. 2008).
In conclusion, the mosquitocidal toxin, surfactin produced by B. subtilis ssp. subtilis (VCRC B471) being highly active at acidic and basic pH, temperatures between 25 and 42°C, and UV stability renders this molecule an interesting lead to be developed as a mosquitocidal agent. Preliminary mammalian toxicity studies viz. acute oral toxicity/pathogenicity tests and primary skin irritation test carried out with crude surfactin showed that it is nontoxic to mammals (VCRC, unpublished data). For the past three decades, only larvicidal bacteria were used in mosquito control programmes and the arsenal of biocontrol agents is presently augmented with this potential bacterium and the unique pupicidal toxin, surfactin. Further studies are underway to find its safety to nontarget fauna and elucidate the mechanism of action of surfactin on immatures of mosquitoes.
We are grateful to Dr P. Jambulingam, Director, Vector Control Research Centre and Dr S.L. Hoti, Scientist ‘F’ for the encouragement and the facilities provided throughout the study. The technical assistance of Mr S. Venugopalan is gratefully acknowledged.