1 Introduction

Majority of pathogens causing various deadly diseases all over the world are transmitted through different species of mosquitoes such as Anopheles, Culex, Aedes, Mansonia, Armigeres etc. (Porter et al., 1993; Cook et al., 2003; Poopathi et al., 2006). Filariasis is one of the most important vector-borne disease affecting Millions of people annually in many parts of the world. And in India, Culex quinquefasciatus is the vector of filariasis (Chandra et al., 2007). Indiscriminate use of chemical insecticides viz. DDT, gammexane, malathion, chlordane etc. caused resistance development and resulting in health and environment problems (Porter et al., 1993). Insecticide resistance among mosquitoes is a serious emerging problem in India. Therefore alternative control strategies need to be employed in mosquito management field. The insect gut is inhabited by a wide diversity of microorganisms as a result of its continuous exposure to the external environment. Diversification and evolutionary success of insects are dependent partially on their relationships with beneficial microorganisms, which are known to upgrade nutrient-poor diets, aid digestion of recalcitrant food components, protect from predators, parasites and pathogens, contribute to interspecific and intraspecific communication, affect efficiency in disease transmission, and regulate mating and reproductive systems (Maji et al., 2012). Gut bacteria play a major role in development of the insect vectors. In disease vectors, the microbes are thought to produce various growth factors or else bioactive compounds, which play a role in the growth, development, digestion, modulation of immune response, vector competence and survival of disease pathogens in the insect (Chernysh et al., 2002; Dillon and Dillon, 2004; Zhang and Brune, 2004; Rajagopal, 2009; Rani et al., 2009). The bacteria colonizing midguts of mosquito vectors have drawn special attention for their interaction with both the insect hosts and pathogenic organisms (Azambuja, 2005). Mosquitoesmaintain symbiotic association with a number of bacterial species which are harbored in different parts of the body and removal of micro-organisms often results into adverse effects on the host body (Roy et al., 2010). The understanding of the biology of mosquitoes would be incomplete without a comprehensive understanding of their gut microbes, as these have a significant impact on various life processes of the hosts. Little is known about the midgut microflora of Culex mosquitoes and very few studies have been conducted to study the midgut microbiota of Culex mosquitoes (Pidiyar et al., 2004; Demaio et al., 1996). The present study was aimed to characterize and control the midgut bacteria of Cx. quinquefasciatus that would be helpful in elucidating new mosquito management strategies in case of disease causing mosquito vectors.    

 

2 Materials and Methods

2.1 Collection of larvae

Larvae were collected from drains and organically rich polluted waters using the standard 350 ml dipper. The larvae were transferred to the Parasitological and Microbiology Research Laboratory, The Department of Zoology, The University of Burdwan. Some larvae were hatched to become adults and others were taken for dissection.

 

2.2 Isolation of mid guts bacteria

The body surface of larvae was sterilized with 70% ethanol (Pidiyar et al., 2004) in a sterile hood. Under sterile conditions, specimens were dissected individually and the midguts were mashed and suspended in sterile distilled water. A 100 µl aliquot of the contents was serially diluted up to 10⁻⁶ and plated onto the nutrient agar (NA) medium and incubated at 30° ± 1 °C for 24 h. The sterility of all reagents was checked during the entire procedure. The most prevalent colonies developed from the gut triturate of the Culex quinquefasciatus mosquito were isolated and purified colonies was maintained at 4 ± 0.1 °C on NA slants.

 

2.3 Characterization of bacterial isolate

Morphological, physiological and biochemical characters of the bacteria were studied following standard methods (Coolee and Miles, 1989; Logan and de Vos, 2009). Antibiotic sensitivity was tested using different antibiotic discs viz. kanamycin (30 µg/disc), nalidixic acid (30 µg/disc), rifampicin (5 μg/disc), doxycycline (30 µg/disc), gatifloxacin (10 µg/disc), vancomycin (30 µg/disc), gentamycin (10 µg/disc), ampicillin (10 µg/disc), ofloxacin (5 μg/disc), levofloxacin (5 μg/disc), streptomycin (10 µg/disc) following Brown (2004).The bacterium was phenotyped according to Logan and de Vos (2009).

 

2.4 Scanning electron microscopy of bacterial isolate

Bacteria were grown for 3 d on NA plates, smears were prepared on cover glasses, heat fixed over a flame for 1-2 sec followed by 2.5% glutaraldehyde (aqueous) for 45 min. The slides were then dehydrated passing through 50, 70, 90 and 100% alcohol for 5 min each. The specimens were gold coated and observed under a Scanning Electron Microscope (Hitachi-S-530) in The University of Burdwan.

 

2.5 Agar cup assay

Microbial susceptibility test of Neem (Azadirachta indica), citronella (Cymbopogon nardus) and Basak(Justicia adhatoda), plant leave extracts against the bacterial isolates CMG1 was done following Agar Cup Assay. Culture of bacterial isolates was spread out on the Muller Hinton agar plate and wells were made with corn borer and methanolic extract (0.5 gm crude powder/ 5 ml methanol) of each plant were added. The plates were then incubated at 37 ± 1˚C for 24 hrs. The sensitivity zones were measured.

 

3 Results

3.1 Characterization of the bacterial isolates

The bacterial isolate CMG1 was Gram positive; rod shaped and produced elliptical spores (Figure 1). CMG1 formed round, off-white, smooth, elevated colonies ranged between 2.5-3 mm in diameter. CMG1 was positive for catalase, methyl red, nitrate, oxidase, urease and gelatin hydrolysis test and negative for indole, Voges-Proskauer, Citrate, Starch hydrolysis and lipase test. CMG1 could tolerate upto pH 7.5 and 10% NaCl present in nutrient broth medium. Growth of CMG1 was observed in 50˚C temperature. The isolate could ferment glucose, fructose, mannitol and sucrose but unable to ferment lactose present in the nutrient broth (Table 1).

3.2 Sensitivity tests against different antibiotic and plant extracts

The isolate was sensitive against standard doses (μg/disc) of Doxycyclin hydrochloride, Tetracycline, Chloramphenicol, Gatifloxacin, Vancomycin, Ciprofloxacin and Nalidixic acid, but resistant to Ampicillin, Streptomycin and Gentamycin antibiotics. The isolate CMG1 showed sensitivity to the methanolic extract of neem [Azadirachta indica (25μl/well)] and citronella [Cymbopogon nardus (25 μl/well)]. CMG1 found to be resistant against the methanolic extract of Basak [Justicia adhatoda (25μl/well)] leaf (Table 2, Figure 2).

4 Discussions

Different Genera of Arthrobacter, Janibacter, Kytococcus, Leucobacter, Aerococcus, Sporosarcina, Vagococcus and Delftia were reported to be as midgut microflora of Culex mosquito (Tanada and Kaya, 1993). Comparative analysis of bacterial diversity from adult Culex mosquito revealed the high prevalence of genus Enterobacter, Bacillus, Staphylococcus, Enterococcus, Acinetobacter, Klebsiella, Pseudomonas and Aeromonas. It has been reported previously that species of Enterobacter are the most common bacteria isolated from insect gut (Tanada and Kaya, 1993). The midgut bacterial infection in wild mosquito populations may influence parasite transmission and could contribute to understand the variation in vectorial capacity observed by same species in different locations because naturally existing microorganisms in mosquito midgut have important roles to determine parasite survival and development. Several midgut bacteria like Acetobacter pomorum, Gluconobacter morbifer, Lactobacillus plantarum, Lactobacillus brevis and Commensalibacter intestine, have beneficial role on larval development (Rajagopal, 2009; Maji et al., 2012). Some of the immunological factors interacting with both pathogenic and commensal bacteria have also been identified (Lemaitre and Hoffmann, 2007; Ryu et al., 2008). During the present study, the morphological and biochemical analysis revealed that the mid gut bacterial isolate CMG1 belongs to Bacillus sp. Present observation was somewhat similar with the findings of Maji et al. (2013) where Paenibacillus nanensis and Bacillus cereus were established to have vital role in the fly development. Pseudomonas fluorescens has been proved to be an important gut bacteria playing important role in the larval development of Culex vishnui mosquitoes (Roy et al., 2010). Pidiyar et al. (2002; 2004) examined the mid-gut flora of Culex quinquifasciatus, responsible for the transmission of filaria and probably Japanese encephalitis. They used both the conventional, culture-based approach and culture-independent, 16S rRNA gene-based PCR–clone–sequencing. Many cosmopolitan bacteria were found in the Culex mid-gut, including a new species of the genus Aeromonas, named Aeromonas culicicola (Pidiyar et al., 2002). This bacterial species was 2000-fold more abundant in mosquitoes that were blood fed as compared to those which were not. The present isolated mid gut bacteria CMG1 was found to be sensitive against different antibiotics and plant extracts. Neem and Citronella extracts both showed higher sensitivity against CMG1. Whereas Mahesh et al. (2008) reported the antimicrobial effect of methanol leaf extracts of Acacia nilotica, Sida cordifolia, Tinospora cordifolia, Withania somnifer and Ziziphus mauritiana against Bacillus subtilis, Escherichia coli Pseudomonas fluorescens, Staphylococcus aureus and Xanthomonas axonopodis. Harraz et al. (2008) revealed thirteen wild plant extracts that exhibited a significant broad-spectrum antibacterial activity against Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Sarcina lutea, Bacillus subtilis, Mycobacterium phlei and Candida albicans.

 

5 Conclusion

The present investigation showed that the control of mosquito midgut bacteria Bacillus sp. CMG1 by plant extracts is not only cost effective but also environment friendly. As midgut bacteria play important role in the development of larva, it would be possible to control the mosquito larval population by controlling the midgut bacteria applying various plant extracts. The present study has shown a new approach towards economic and safer mosquito management.

 

Acknowledgement

The authors are thankful to University Grant Commission (UGC). Authors are grateful to The University of Burdwan, West Bengal, India for providing laboratory facility required for the work.

 

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