1 Introduction

Among all the public health hazards known, being affected by a mosquito borne disease is one of the major concerns today. The ecology of the mosquito breeding, the reason behind their selection of a specific site for oviposition demands much attention. One such mosquito of vector status is the Asian tiger mosquito, Aedes albopictus, which is responsible for the dengue mayhem today. Popularly known as container breeders, these mosquitoes show “skip oviposition” behaviour (Mogi and Mokry, 1980), the activity of laying eggs in various small aquatic containers rather than showing a clutch pattern distribution (Fay and Perry, 1965; Chadee and Corbet, 1987; Apostol et al., 1994). These mosquitoes are shown to lay and hatch their eggs in small water holding containers, ranging from tree holes to artificial containers, discarded household products, earthen wares, coconut shells etc of varying shapes and sizes (Okogun et al., 2005), which are a pool of various micro and macro flora and fauna. Among these numerous bacterial species present are of a major importance, since bacteria are known to produce various by-products that act as strong ovipositional cues. Thus, identification of the bacterial species and thorough characterization of those species hold much relevance. Knowing the bacteria at the grass root level can act as mosquito controlling property.

2 Materials and Methods

2.1 Collection and processing of water sample and bacterial isolation

Water samples were collected from in and around various sites of Burdwan district throughout the time period of July 2014 to June 2015 in a seasonal basis of Monsoon, post monsoon, winter and summer. The collected water sample was brought to the Parasitology and Microbiology Research laboratory, Department of Zoology, The University of Burdwan, Burdwan for further processing. The water sample was then diluted to 10^-5 dilution and were then plated in Nutrient Agar (peptone: beef extract: NaCl: agar at 5:3:3:1 g/l) plates and kept in B.O.D. incubator at 30^̊C ± 1̊C for 24 hrs to obtain isolated colonies. The next day the isolated colonies were quadrant streaked following the methods of Pelczar et al. (1957). The pure cultures were then maintained in Nutrient Agar slants for further experimentations.

2.2 Phenotypic characterization of the bacterial isolates

2.2.1 Colony morphology

Colony characteristics (form, colour, elevation, margin, size, and consistency), morphology of the strains (shape and size of vegetative cells and spores, if any) were recorded following standard methods (Pelczar et al., 1957; Collee and Miles, 1989; Lacey, 1997).

2.2.2 Staining properties

Gram’s staining was done in order to study the morphology of the vegetative cells by standard protocol (Gram, 1884) and endospore staining was done in order to visualise spores, if any by standard protocol (Schaeffer and Fulton, 1933)

2.3 Biochemical characterization of the bacterial isolates

Indole test, Methyl red test (MR), Voges-Proskaeur's test (VP), Citrate test, nitrate reduction test, Urease test, Catalase test, carbohydrate fermentation test, Triple sugar iron test and hydrogen sulphide production test were done following standard protocol (Pelczar et al., 1957; Collee and Miles, 1989; Lacey, 1997). Total carbohydrate content was extracted by following the methodology of Reed et al. (1984) and estimated using anthrone and phenol sulphuric reagents (Dubois et al., 1951). Total DNA and total protein content was extracted following the protocol of Mahadevan and Sridhar (1986), and was estimated by diphenylamine method (Burton, 1986) and Folin-ciocalteau reagent (Lowry et al., 1951) respectively. Bacterial dry weight was also measured (Figure 1).

2.4 Extracellular enzymatic assay

Extracellular activity of the bacterial isolates was assayed by the following tests: Protease test (Gelatin hydrolysis, casein hydrolysis), Starch hydrolysis test, Lipase test (tween-20 hydrolysis, tween-80 hydrolysis), lecithinase test and chitin hydrolysis test.

2.5 Physiological assay

2.5.1 Effect of pH on isolates

Growth of each bacterial isolate was assayed on varying pH ranging from 4 to 12 in 100 ml nutrient broth culture, keeping the culture at 30̊C ± 1̊C for 24 hrs.

2.5.2 NaCl tolerance

Tolerance of bacterial isolates to varying degrees of NaCl concentration (2-12%) was recorded keeping the nutrient broth culture at 30̊C ± 1̊C for 24 hrs.

2.6 Antibiotic sensitivity assay

Antibiotic tests have been done with standard antibiotic discs following Brown (2004). Antibiotic discs like Ampicillin (10 µg/disc), Amoxycillin (10µg/disc), Polymixin B (50 µg/disc), Doxycyclin (30 µg/disc), Vancomycin (30 µg/disc), Levofloxacin (5 µg/disc), Kanamycin (30 µg/disc), Penicillin (2 µg/disc), Bacitracin (10 µg/disc), Nystatin (100 µg/disc), Ciprofloxacin (5 µg/disc), Erythromycin (15 µg/disc), Rifampicin (5 µg/disc), Tetracyclin (30 µg/disc), Nalidixic acid (30 µg/disc) were used to test the sensitivity or resistance of the isolates to the corresponding antibiotics. The zone of inhibition with respect to each of the antibiotics and respective isolates were recorded.

3 Results

A total of 18 bacterial isolates were screened from the breeding habitat water, among which KSG1, KSG2, KSG5 and BM3 were found to be prevalent throughout the year. The phenotypic characterization including the colony morphology and the staining properties are noted in Table 1. KSG1 is a Gram positive, spore forming, opaque, white, flat, partially smooth, dry colony having concentric alternate light and dark bands. KSG5 was also a Gram positive, spore former with slightly raised, circular, glossy, translucent colony. These two isolates were constantly found throughout the year in all the seasons. Isolates KSG2 and BM3 were found in all seasons but summer, also a fairly prevalent bacterial strain. KSG2 and BM3 both were Gram positive and non-spore former, with KSG2 being with round, pure white, flat and buttery colony and BM3 is being round circular, bright yellow and opaque, convex and buttery colony. Table 2 shows the biochemical tests performed. Catalase test showed positive results for all the isolates while indole production did not occur in any of them. KSG1, KSG2, BM7, BM8, BM9, BM12 and BW3 were found to be positive for MR test and BS3, KSG2, BM6 and BM13 were found to be positive for VP test. Nitrate reduction was found to be positive for all except for BS1 and BM13. KSG2, BM9 and BM12 showed the presence of urease enzyme and could also utilize citrate as their carbon source, whereas KSG1 was negative for citrate and positive for urease but BS3 showed the opposite results of KSG1 for both these tests. BS3, KSG1, KSG2, BM3, BM4, BM9 and BM10 fermented glucose only, whereas BS2, BS4, KSG5, BM6, BM8 and BM11 fermented glucose and lactose both. No fermentation was observed in TSI tests for the rest of the bacterial isolates. H2S production did not take place in any of the isolates. Dry weights of bacteria ranged from 0.000 2 g to 0.082 7 g, with BS2 being the lowest and BM4 being the highest. All the isolates were found to be negative for casein hydrolysis, tween 20, tween 80, lecithinase, chitin hydrolysis while KSG1, KSG2, BM3, BM4 and BM12 could hydrolyse starch. Gelatin was hydrolysed by BS3, BM8, and BM13 (Table 3). Both KSG1 and KSG5 showed no growth when antibiotics like doxycycline, vancomycin, levofloxacin, kanamycin, ciprofloxacin, erythromycin, tetracycline, nalidixic acid were applied, whereas KSG2 and BM3 were found to be sensitive for doxycycline, vancomycin, levofloxacin, bacitracin, ciprofloxacin, erythromycin, tetracycline (Table 4). All the bacterial isolates were seen to tolerate temperature range from 22̊C to 50 ̊C and 8% NaCl tolerance, except for BS1 and BM7 which could tolerate till 6%. Following the protocol of Logan and de Vos, 2009 the bacterial isolates were identified to be mainly Bacillus sp.

4 Discussions

Characterization of mosquito breeding water, whether it may be physicochemical or a cross sectional microbial study, is very important for the vector dynamics. Numerous bacterial isolates were identified in the breeding water, which suggests that there is an obvious role of the bacterial diversity in the mosquito oviposition. Previous studies conducted by Mondal et al. (2015), showed Bacillus sp to have role in the mosquito larval development and ovipositional responses. The biochemical studies show a primary investigation regarding the bacterial load of the breeding habitat. The bacteria present release several types of chemical cues which change the water composition in a variety of way, leading to be an ideal mosquito breeding ground (Mondal et al., 2015). In 2003, Trexler et al., showed that three bacterial species Psychrobacter immobilis, Sphingobacterium multivorum, and Bacillus sp from various sources yielded a great ovipositional response than control water without bacteria. The breeding water bacteria not only act as an oviposition attractant, but also play a major role in larval development by getting incorporated in the mosquito gut (Mondal et al., 2015). Reports of Thorsellia, a potential candidate for paratransgenesis, were shown to get incorporated in the mosquito midgut via their food type from the breeding ground, in Kenya (Lindh et al., 2005; Briones et al., 2008; Rani et al., 2009; Wang et al., 2011).

As important it is to determine the physicochemical parameters of the breeding habitat water, it is also equally important to study the microbial diversity and to find out that which bacteria is mainly responsible for mosquito breeding, and this can be done only by a preliminary biochemical, physiological and phenotypical characterization. Bacteria generally release semiochemicals and volatile substances which may act as an oviposition attractant for the mosquitoes. So in conclusion it can be said that identification of the bacteria and eliminating them may serve as an effective vector control strategy.

Acknowledgement

The authors are grateful to the The University of Burdwan, Burdwan, West Bengal, India for providing with the facilities to carry out their work.

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