1 Introduction

Mosquitocidal bacteria are eco-friendly alternatives to chemical insecticides for controlling vector mosquitoes and therefore there have been tremendous world-wide efforts to isolate novel mosquitocidal bacteria with improved efficacy (Federici et al., 2006; Park et al., 2007). Despite these efforts, however, a few, such as, Bacilus thuringiensis, B. sphaericus, Brevibacillus laterosporus, Clostridium bifermentans are known to be mosquitocidal (Federici et al., 2006; Thiery et al., 1992; Orlova et al., 1998; Park et al., 2006). These bacteria produce parasporal endotoxin crystals during sporulation and which are believed to be responsible for the mosquitocidal activity. Mosquito-borne diseases, notably filariasis, dengue, malaria and Japanese encephalitis remain endemic in many tropical areas. Chemical pesticides such as DDT, malathion etc. applied with the aim of eliminating mosquitoes (Phillips, 2001) have given rise to other serious problems. Not only resistance against these chemicals has been reported, but also the pesticides themselves present threats to both human health and the ecosystem (Phillips, 2001; World Health Organization, 1995). In these circumstances, the possibility of utilizing biopesticides as alternatives to chemicals is being examined in a regular fashion to mitigate the need of the hour. At present, B. thuringiensis israelensis (Bti) and B. sphaericus (Bs) are being used worldwide in field to control the populations of Aedes, Culex, and Anopheles larvae (Phillips, 2001). However, utilization of these bacteria has been limited by several disadvantageous biological properties that they exhibit. The mosquito larvicidal crystals of the bacteria do not persist for long periods in the environment due to their rapid inactivation by sunlight or other degradation agents, while crystals and spore-crystal complexes rapidly sediment from the water surface, which is the predominant larval feeding zone (Schnepf et al., 1998; Porter et al., 1993). Therefore, the isolation and/or development of a bacterial strain with (i) larvicidal activity having a broad host range specificity, (ii) stable habitation, and (iii) non-hazardous properties, is of utmost importance.

During the present study, an attempt was made to isolate and identify a soil bacterial stain (N) and to evaluate its larvicidal properties, if any against Culex quinquefasciatus mosquito, the principal vector of filariasis throughout tropics and subtropics, both in laboratory and field conditions. Effects on the health of aquatic non-target organisms such as Daphnia, Chironomus circumdatus, Poecilia reticulata and tadpole of Bufo sp. were also analyzed.

2 Materials and Methods

2.1 Collection of larvae

Early (1st and 2nd ) and late (3rd and 4th) instars larvae of Culex quinquefasciatus used in the study were taken from Mosquito, Microbiology and Nanotechnology Research Units, Parasitology Laboratory, Department of Zoology, Burdwan University, maintained at 27±1°C temp, 85% RH and normal photoperiod of 14L: 10D cycle.  

2.2 Isolation of bacterium from soil

The bacterium was isolated from the garbage soil samples collected randomly from four different sites of Burdwan, West Bengal in 2012. Samples were then packed in a sterilized container and transported to laboratory and air-dried for 2-3 days. One gram of air-dried powdered soil was taken into 100 ml sterilized distilled water, vigorously and subsequently decimally diluted following the technique of Alexander (Alexander, 1965). For total heterotrophic count in nutrient agar plate media, 0.1 ml of diluted sample taken from 10-5 dilution was poured into nutrient agar plate by spread plate technique. The plates were incubated for 24 hrs at 37±1°C.

Colonies appeared in the plates with different morphological characters were isolated and subsequent repeated cultures were made to obtain pure culture by streaking method. After overnight incubation, the total number of bacteria was counted based on their colour and morphology of the colonies. Each pure culture was identified by various taxonomical tests.

2.3 Identification

For preliminary identification morphological characteristics of each isolated bacterium were examined. Biochemical tests were also performed according to Bergey’s Manual of Systematic Bacteriology (Williams et al., 1986). Identity of the species of the selected bacterium was confirmed by 16S rDNA sequence study.

Antibiotic sensitivity of the seleted isolate was assessed by the disc diffusion method (Bauer et al., 1966) using Müller-Hinton agar (BD). High – potency bio-discs were procured from Himedia, Mumbai, India. The concentrations (μg disc-1) of different antibiotics used in the test were chloramphenicol (30), oxytetracycline (30), ampicillin (10), norfloxacin (10), ciprofloxacin (5), oxolinic acid (2), kanamycin (30) and erythromycin (15). Diameters of the inhibition zone were measured following 24 h of incubation at 25°C.

2.4 16S rDNA sequence determination

The DNA of the isolated strain was extracted using a Genomic DNA extraction kit (GeNei™, Elpho kit, Category# 107070, Bengaluru, India) following the manufacturer’s protocol. The DNA templates were amplified by a polymerase chain reaction (PCR) thermocycler (Gene Amp 2400, Perkin Elmer, Chicago, IL, USA). A total volume of 100 μl PCR reaction mixture containing PCR buffer, 0.2 mM deoxynucleoside triphosphate (dNTP), 1.0 mM MgCl2, 20 pmole of bacterial primer 8f (5′-AGA GTT TGA TCC TGGCTC AG-3′) and 1492r (5′-GGT TAC CTT GTT ACG ACT T-3′) was used for the study. This mixture was used to denature 0.1 μl of fresh cells at 95°C for 4 minutes. Then, 1 unit of Taq DNA polymerase was added to the reaction. This was followed by 30 cycles of denaturation at 94°C for 30s, annealing at 48°C for 30s and further extension at 72°C for 1 min. The final cycle was extended at 72°C for 2 min.

The reaction product was purified using PCR purification kits (GeneiPure™, Quick PCR purification kit, Category# 117309, Bengaluru, India). The analysis of the sequences was performed using a genetic analyzer (ABI 3100, Applied Biosystems, Foster city, CA, USA) at Bangalore Genei, Bengaluru, India. The sequence obtained was analyzed for finding the closest homolog of the microbes using a combination of NCBI (National Centre for Biotechnology Information) GenBank and RDP (Ribosomal Data Base Project). The distance matrix was also based on nucleotide sequence homology (using Kimura-2 parameter). Finally, phylogenetic trees were constructed using Mega 3.1 software by the neighbor joining method with Boostarp analysis to obtain information on the molecular phylogeny.

2.5 Preparation of bioactive formulation

The strain of identified bacterium was inoculated in glucose peptone salt (GPS) medium (glucose 10.0 g/l, peptone 10.0 g/l, potassium dihydrogen phosphate 0.1 M, peanut oil 1.0 ml/l in water; pH=7). Laboratory bioassay of the formulation was followed by incubation on a rotary shaker at 37°C and 180 rev/ min for 48 hrs. The density was adjusted to 1.09 at OD540. The cells were grown to 6 × 107 CFU/ml. The CFU/ml of culture was calculated by using the following proportion:

CFU(×) = (Number of colonies × dilution of plate) × 1/ Volume of culture on plate

The cell mass was harvested by centrifugation at 10.000 rev/min for 15 min and formulation was prepared by mixing the cell mass with glycerine at 100g/l following the method of Pravakaran et al. (2003). This was taken as stock solution.

2.6 Bioassays

Entire study was conducted according to standard test methods (World Health Organization, 1975) with slight modifications.

The formulation was incorporated into 500 ml capacity beakers with different concentrations viz. 2.0, 1.5, 1.0, 0.5 and 0.3 mg/ ml for each instar larvae and the volume was made up to 250 ml. 25 larvae of each 4 instars were introduced separately in the beakers. Chlorine free water containing 25 percent glycerol served as control.

Percent mortalities were recorded after 24, 48 and 72 h of exposures which were corrected with Abbott’s formula (Abott, 1925). LC50 and LC90 values; were calculated following Finney (1971). For the larvicidal effect of formulation of the isolated bacterium, percent of mortality was subjected to completely randomised three-way factorial ANOVA, using different concentrations, different instars and days as variables to find the significance between the concentration of the bacterial formulation and mortality at different periods. Statistical analyses of the experimental data were performed using the software “STAT PLUS (2007)” and “SPSS ver. 11.0” to find out the LC50 and LC90 values, regression equations and regression coefficient values.

2.7 Sample preparation for Scanning Electron Microscopy (SEM)

Freshly died Cx. quinquefasciatus larvae were collected from the test solution of bacterial formulation. Then the larval gut was dissected out. The dissected gut was fixed in 2.5 % gluteraldehyde for 2 hrs. After the fixation, the gut was teared to open the innergut wall. It was then subjected to a dehydration procedure through upgraded alcohols: 50% alcolhol (5 mins), 70% alcohol (30 mins-with two changes), 90% alcohol (30 mins- with two changes) and absolute alcohol (30 mins-with two changes). The sample was then kept serially into 3 different ratios of mixtures of absolute alcohol: amyl acetate with 3 changes viz. 3:1 (30 mins), 2:2 (30 mins), 1:3 (30 mins). After that the gut was kept in amyl acetate for 30 mins. Then the sample was subjected to SEM.

2.8 Effect on non-target organisms

Four non-targeted organisms like Daphnia spp., Chironomus circumdatus larvae,Poecilia reticulata and tadpoles of Bufo sp. were selected. For acclimation to the laboratory each of them was kept in an environment similar to its natural habitat. As per the procedure used by Suwannee et al. (2006) the non-targets were exposed to the sublethal dose, LC50 (at 24 hrs for fourth instar larvae of Cx. quinquefasciatus) of the bacterial formulation. A set of control (without having the test solution) for each organism was run parallel. Number of dead non-targets was recorded after 24hrs, 48 hrs, 72 hrs of exposures, percent mortality was calculated and corrected using Abbott’s formula (Abott, 1925). Each experiment (including the control one) was replicated thrice and average mortality percentages were tabulated.

2.9 Field application

Trials were conducted in the outskirt of a village of Burdwan (24°28’ N and 86°56’ E) District, West Bengal, India. Necessary ethical approval has been taken (Approval No. VIHC/371 Dated: 29.09.2009). Applications were made in sewage drains with three different depth of water viz. 35-40 cm (drain-1), 15-35 cm (drain-2) and 5-25 cm (drain-3). The test plots contained different instars of Culex larvae. 1 ml of stock solution was liquefied with 100 ml of water and sprayed once over an area of 1 m2 (water surface) with back–pack sprayer (Killaspray,Model 4526, Hozelock, Birmingham, U.K., 16 litre capacity) in natural breeding habitats (sewage drains) of Cx. quinquefasciatus. The control drain (depth 35-40 cm) was kept untreated.

2.9.1 Sampling procedure

Afterwards all drains were examined daily and the average number of larvae calculated per standard 250 ml capacity mosquito dipper by taking 10 dips/ drain (three dips on each of two sides and four from the middle) from day 1 to day 27. Immatures of mosquitoes were classified in two categories: early instars (1st and 2nd), late instars (3rd and 4th) (World Health Organization, 2004) All larvae were counted, classified upto genus and instars and then returned to their respective sites.

The percentage reduction in larval mosquito densities was calculated using the formula of Mulla et al. (1971). 

Percentage reduction = 100 - [(C1/T1) × (T2/C2) × 100]

Where, C1= pre-treatment population density in control habitat;

C2= post-treatment population density in control habitat;

T1= pre-treatment population density in treatment habitat;  

T2= post-treatment population density in treatment habitat;  

3 Results

3.1 Characterization

The mosquito pathogenic bacterial stain was isolated and characterized based on the morphology, spore formation, nutritional features, physiological and biochemical characteristics. Isolate was creamy in colour. Enrichment and purified procedures were carried out which allowed the isolationof a species of spore forming bacterium. Isolated bacterium occurred singly, bacillus, with a length of approximately 2-4 μ. It was detected to be spore-forming, gram-negative, motile, bacilli. The optimal temperature and pH for growth lied around values of 37°C and 7.6, respectively. The determination of fermentation products showed that the isolate converts glucose to L (+) –lactic acid through a typical homo-fermentative pathway. The isolate showed its growth above 20°C and below 50°C within the pH range of 8.0 to 10.0, tolerated up to 4% NaCl. Isolate effectively utilized carbohydrates glucose but could not utilize others such as sucrose, maltose mannitol, fructose and galactose.

3.2 Antibiotic sensitivity test for the organisms

Sensitivity of isolated bacterial stain to some antibiotics in nutrient agar is presented in Table 1.

Table 1 Sensitivity of isolated bacterial stain to some antibiotics in nutrient agar

3.3 Bacterial characterization and identification by 16S rDNA

In order to gain more taxonomic information on the strain (Strain N), the 16S rDNA of the strain was partially sequenced. Based on the nucleotide homology and phylogenetic analysis, the bacterial strain was identified as Bacillus cereus (GenBank Accession Number: KM407516). The nearest homolog species was found to be Bacillus thuringiensis (GeneBank Accession Number: AM293345). Information regarding other close homologs of the strains has been provided in the alignment view of Table 2. The aligned sequence data have been presented in the Figure 1. The phylogenetic relations of the bacterial strain with other closely related species have been presented in the dendrogram (Figure 2).

Table 2 Alignment view using combination of NCBI GenBank and RDP database showing the close homologs of the sample ‘N’

Figure 1 Aligned sequence data (1550bp) of the isolated bacterium (N)

Figure 2 Dendrogram showing phylogenetic relations of the isolated bacterial strain (N) with other closely related bacteria

3.4 Larvicidal effect of bio active formulation

Results of instar specific larvicidal bioassay of Cx. quinquefasciatus with different concentrations of bioactive formulation of bacterium under study are presented in Table 3.

Table 3 Susceptibility of immature forms of Cx. quinquefasciatus to bioactive culture filtrate isolates of B. cereus

The results of three way factorial ANOVA (Table 4) computed on mortality of mosquito larvae by bacterial formulation using different concentrations and different instars as variables, revealed significant difference on mortality rates (P<0.05).

Table 4 Three way factorial ANOVA for comparison of mortality rates of Cx. quinquefasciatus larvae

First instar larvae were found to be most susceptible (LC50 =0.45 mg/ml) to the bioactive formulation in laboratory condition and corresponding LC50 values were 0.53 mg/ml, 0.65 mg/ml, 0.76 mg/ml for 2nd, 3rd and 4th instar larvae respectively, after 24 hours of exposure (Table 5). A clear dose dependent mortality was observed, as the rate of mortality (Y) was positively correlated with the concentration (X) of the formulation as evident from established regression equations. Regression coefficient R2 values were found near to 1 (Table 5). The ruptured larval mid gut wall along with protruding B. cereus obtained from a scanning electron microgram observation is presented in Figure 3.

Table 5 Larvicidal effect of the bioactive formulation of B. cereus against Cx. quinquefasciatus larvae

Figure 3 Infected gut wall of mosquito larva with bacteria (N) under SEM

3.5 Effect on non-targets

The LC50 of the bioactive formulation of B. cereus, for early fourth instar larvae of Cx. quinquefasciatus at 24h showed slight toxicity to the Chironomid- Chironomus circumdatus (Diptera: Chironomidae) larvae (10.00% and 14.33% after 48 hours and 72 hours respectively) (Table 6). It did not show any pathogenicity to the Daphnia spp. and P. reticulata upto 24 hours of exposure. But after exposure to bacterium it caused 6.30% and 13.66% death of Daphnia spp. and 7.66% and 14.00% death of P. reticulata after 48 hours and 72 h of exposures respectively. But it was non-toxic to the tadpoles even after 72 h of exposure.

Table 6 Effect of bioactive formulation of B. cereus on few non-target organisms at laboratory condition

3.6 Field application

When the single application of bioactive formulation of B. cereus was done to three different sewage drains, 100% mortality of all instars of Cx. quinquefasciatus was recorded on day 9 of application in drain 3 and on day 11 and 13 in drain 2 and 1 respectively. Recurrence of larvae (3.1%) was recorded on day 17 of application in drain 1 but in case of drain 2 and drain 3, 5.18 % and 1.72% larval recurrences were recorded respectively on day 19 after application. Residual activity of the formulation however was moderate as dips taken 17-19 days after treatment indicated quick and continuing re-colonization of all treated sites by early instars (Table 7).

Table 7 Effect of bioactive formulation (Percentage of reduction) of B. cereus on Cx. quinquefasciatus larvae in field trials in three different depths of sewage drains

4 Discussion

The effective control of larvae is a basic principle of Integrated Pest Management (IPM). Effective IPM involves understanding the local mosquito ecology and patterns of arbovirus transmission and then selecting the appropriate mosquito control tools. The most common methods of IPM include Environmental Management or Source Reduction, Larviciding and Adulticiding. Among these, larviciding is a general term for killing immature mosquitoes by applying agents, collectively called larvicides, to control mosquito larvae and/or pupae.

Mosquito control agencies have adopted the general view that larviciding is typically not as effective or as economical as permanent source reduction but is usually more effective than adulticiding. However, this view was derived long ago when wetlands were not considered to be as important as they are today. Many of the compounds used were different as were costs in terms of money, manpower, and equipment. The enlightened view of modern mosquito control professionals includes a strong commitment to minimizing environmental impacts.

Currently used mosquito larvicides, when applied properly, are efficacious and environmentally safe. Typically, there is less concern for the drift of mosquito larvicides than for the drift of adulticides, primarily due to the droplet size. Microbial larvicides are formulated to deliver a natural toxin to the intended target organisms. B. thuringiensis (Bt) is the most widely used agricultural microbial pesticide in the world, and the majority of microbial pesticides registered with the EPA are based on Bt. Bt products have been available since the 1950s. In the 1960s and 1970s, the World Health Organization (WHO) encouraged and subsidized scientific discovery and utilization of naturally occurring microbes.

As a result of those early studies and a whole body of subsequent work, new lines of mosquito control products have been developed: crystalline toxins of two closely related gram positive, aerobic bacteria – Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus (Bs). Mosquito control agents based on B.t. are the second most widely registered group of microbial pesticides. Highly successful Bti products have expanded the role of microbial agents into the public health arena (Barjac, 1990). Bacillus and Pseudomonas spp. are also potent pathogens of mosquitoes (Porter et al., 1993; Lacey, 1997; Krattiger, 1997; Cooping and Menn, 2001; Wirth et al., 2004; Teng et al., 2004; Chatterjee et al., 2008; Dangar, 2008). Mosquitocidal bacteria are environmentally friendly alternatives to chemical insecticides for controlling vector mosquitoes and therefore there have been tremendous world-wide efforts to isolate novel mosquitocidal bacteria with improved efficacy (Federici et al., 2006; Park et al., 2007; Chatterjee et al., 2010; Das et al., 2015). Despite these efforts, however, only B. thuringiensis, B. sphaericus, Brevibacillus laterosporus and Clostridium bifermentans are known to be mosquitocidal (Federici et al., 2006; Thiery et al., 1992; Orlova et al., 1998; Park et al., 2006). All of these bacteria produce parasporal endotoxin crystals during sporulation and these crystal proteins are believed to be responsible for their mosquitocidal activity.

Khyami-Horani et al. (1999) reported on B. cereus isolated from various habitats in Jordan showed activity against Culiseta longiareolata without providing the median lethal concentrations (LC50). Furthermore, it has been found to be the dominant bacterial species in the guts of Aedes aegypti and Cx. quinquefasciatus (Luxananil et al., 2001). There have been previous reports of colonization of B. cereus in insects, including various mosquito larval gut. B. cereus strains are also able to colonize in the guts of the mosquito larvae (Plearnpis et al., 2001). Insecticidal activity of spores of B. cereus against Ae.aegypti has already been determined (Dana et al., 1981). B.t. and B. cereus could also multiply in the insect haemocoel but they provoked septicaemia of the insects (Schnepf  et al., 1998; Stephens, 1952; Heimpel, 1955). So, the present study is significant and depicts the role of B. cereus (N) as mosquito larvicide in the natural environment.

In some places it is necessary to preserve diversity of organisms, especially those competitors to or predators upon targeted culicines. In such situations biological larvicides should be chosen because chemical larvicides are largely nonselective (Mohsen and Mulla, 1981; Frost and Sinniah, 1982) and affecting even vertebrates in some cases (Zinkl et al., 1981). Such larvicides should be used only in places where pollutions are too high to allow for biological diversity, provided that they are more cost-effective than biological examples.

In case of non-target organisms, tadpole of Bufo sp. showed excellent survival for consecutive 72 hrs after the treatment with LC50, whereas Chironomous circumdatus, Poecilia reticulata and Daphnia spp. showed survivility up to 24 hrs of treatment.

From the field trial, a potential importance of bioactive formulation @ 100 ml/m² is observed by having a significant efficacy to minimize the larval population in their habitats. It has also revealed its efficacy in a way where a single application of bioactive formulation lowers both the early and late instars larval population towards nil on day 13, which was also similarly effective in comparison to other larvicides. Another observation made during this study was that the recurrence of larval population was found in higher rate in the larger water bodies, which might be the cause that the residual effect of bioactive formulation was lower in the larger water body than that of smaller ones.

5 Conclusion

So it is concluded that isolated bacterial strain of B. cereus posses the ability to be acting as biocontrol agent, which appear promising for future utilization as new mosquito larvicide, since they offer an effective mosquito larvicidal activity along with long persistence in the natural habitats of mosquito larvae.

6 Acknowledgements

The authors are obliged to UGC-DRS for providing financial support.

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