BackgroundMosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.ResultsLab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.ConclusionMore than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn doublestranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins. The bacterium Bacillus thuringiensis (Bt)1 produces insecticidal crystal proteins, which upon ingestion by susceptible larvae get activated in the midgut, interact with specific receptor and form pores in the epithelium, resulting in the death of the larvae (1). Understanding the mechanism of action of Bt toxin and development of resistance in insects is fundamental in sustaining the use of Cry proteins in integrated pest management. One of the mechanisms of resistance development is an alteration in the binding ability and/or a decrease in the population of receptor molecules, which bind Bt toxin in the insect midgut (2). There have been intense efforts to characterize the nature of this receptor. As a result of several independent experiments employing ligand blot analysis and fluorescent labeling of insecticidal proteins, cadherin and aminopeptidase N (APN) have emerged as main putative receptor molecules (Ref. 3 and references there in). While the role of a receptor molecule in mediating the effect of Cry toxin is acknowledged, the identity of this receptor is still being worked out.Aminopeptidase N from Manduca sexta was the first molecule to be tentatively identified as a Cry toxin-binding protein (4, 5), and APN is the most extensively studied putative receptor, having been identified and isolated subsequently from other lepidopteran insect pests. Independently, a 210-kDa cadherin-like protein from M. sexta was shown to interact with Cry1Ab toxin (6) and later its presence and toxin interaction was also demonstrated from another insect, Bombyx mori (7). Relative abundance of APN in the posterior midgut (8) and lower binding constants of Cry toxin toward cadherin as compared with APN (9) raised apprehension about the role of APN as a receptor for Bt toxin in the insect midgut. Moreover,

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant.

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N-and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.The gram-positive bacterium Bacillus thuringiensis is known to produce parasporal crystalline inclusions during the late exponential phase of growth (8). These crystals consist of several polypeptides, some of which are insecticidal or nematocidal. Upon ingestion by insects, these toxins are proteolytically activated, and after interaction with specific receptors at the mid-gut, they cause larval death (5). Since these toxins are highly specific, they are extremely useful in controlling targeted agricultural pests. Over the past several years, more than 100 different polypeptides have been identified, and several of them have been employed in insect management programs (8). The diversity, specificity, and usefulness of these insecticidal polypeptides have encouraged searches among diverse niches for new strains displaying novel insecticidal polypeptides. In addition to the crystal-associated toxic polypeptides, some insecticidal proteins produced during vegetative growth of the bacteria have also been identified. These proteins, called vegetative insecticidal proteins (VIPs), were reported from about 15% of the B. thuringiensis strains analyzed (2). We have screened several strains of B. thuringiensis obtained from soil samples collected from different parts of India for the presence of homologues of the VIP. Based on the reported gene sequences, we designed PCR DNA primers for the detection of the vip gene in strains held in our collection. As a result of the screening program, we have cloned, sequenced, and expressed a vegetative insecticidal toxin-coding gene from one of the isolates in our collection. The toxicity spectrum of the Escherichia coli-expressed recombinant protein has been evaluated against five lepidopteran pests. By deletion analysis, we have characterized the minimal toxic polypeptide segment that retains insecticidal activity. The toxicity of deleted VIP against lepidopteran pests suggested a differential mode of action against different pests.Bacterial strains and plasmids. Different isolates of B. thuringiensis were enriched from soil samples collected from different geographical locations within India. For routine use in the laboratory, the isolates were maintained in nutrient medium (Difco), and for long-term storage, the isolates were stored as glycerol stocks at Ϫ70°C. E. coli strain M15 was obtained from Qiagen (Braunschweig, Germany) and, when required, was grown in Luria-Bertani (LB) medium at 37°C with shaking at 200 rpm.Oligonucleotide PCR primers. Primers to sc...

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