Structural and Functional Analysis of Groundnut bud necrosis virus (GBNV) Using Computational and Biochemical Approaches.

Groundnut bud necrosis virus (GBNV) belonging to the genus Orthotospovirus is transmitted by its vector Thrips palmi. It is a tri-segmented RNA virus that consists of L, M, and S RNA segments. We analysed the secondary structure features of GBNV proteins through various software and predicted the transmembrane helix, glycosylation, and signal peptidase sites within the GBNV protein sequences (GN, GC, N, NSm, and NSs). In glycoprotein sequence, extended strands are predominant (52.87%) whereas the N protein sequence mostly contains alpha helices (47.46%). The random coils are present in movement protein (43.97%) and structural protein (39.41%). We generated the 3D structure of GN and N protein using SWISS MODEL software and quality is validated through PROCHECK and PDBsum software. We also expressed the GBNV proteins (GN, GC, N, NSm, and NSs) in bacterial expression system. The recombinant proteins were used to raise polyclonal antibodies in mice. Our study will be useful in understanding GBNV protein structures in further detail by analysing the important domains that interact with the thrips proteins. This will further aid us in understanding virus-vector relationship through the application of protein-protein interaction and other immunodiagnostic techniques.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Mucin Protein of Aedes aegypti Interacts with Dengue Virus 2 and Influences Viral Infection.

Dengue, caused by dengue virus (DENV), is the most prevalent vector-borne viral disease, posing a serious health concern to 2.5 billion people worldwide. DENV is primarily transmitted among humans by its mosquito vector Aedes aegypti; hence, the identification of a novel dengue virus receptor in mosquitoes is critical for the development of new anti-mosquito measures. In the current study, we have identified peptides which potentially interact with the surface of the virion particles and facilitate virus infection and movement during their life cycle in the mosquito vector. To identify these candidate proteins, we performed phage-display library screening against domain III of the envelope protein (EDIII), which plays an essential role during host cell receptor binding for viral entry. The mucin protein, which shared sequence similarity with the peptide identified in the screening, was cloned, expressed, and purified for in vitro interaction studies. Using in vitro pulldown and virus overlay protein-binding assay (VOPBA), we confirmed the positive interaction of mucin with purified EDIII and whole virion particles. Finally, blocking of mucin protein with anti-mucin antibodies partially reduced DENV titers in infected mosquitos. Moreover, mucin protein was found to be localized in the midgut of Ae. aegypti. IMPORTANCE Identification of interacting protein partners of DENV in the insect vector Aedes aegypti is crucial for designing vector control-based strategies and for understanding the molecular mechanism DENV uses to modulate the host, gain entry, and survive successfully. Similar proteins can be used in generating transmission-blocking vaccines.

Understanding the role of insects in the acquisition and transmission of antibiotic resistance.

Antibiotic resistance (AR) is a global healthcare threat that requires a comprehensive assessment. Poorly regulated antibiotic stewardship in clinical and non-clinical settings has led to a horizontal dissemination of AR. A variety of often neglected elements facilitate the circulation of AR from antibiotic sinks like concentrated animal feeding operations and healthcare settings to other environments that include healthy human communities. Insects are one of those elements that have received underwhelming attention as vectors of AR, despite their well-known role in transmitting clinically relevant pathogens. We here make an exhaustive attempt to highlight the role of insects as zoonotic reservoirs of AR by discussing the available literature and deriving realistic inferences. We review the AR associated with insects housing various human-relevant environments, namely, animal farm industry, edible-insects enterprise, healthcare institutes, human settlements, agriculture settings and the wild. We also provide evidence-based accounts of the events of the transmission of AR from insects to humans. We evaluate the clinical threats associated with insect-derived AR and propose the adoption of more sophisticated strategies to understand and mitigate future AR concerns facilitated by insects. Future works include a pan-region assessment of insects for AR in the form of AR bacteria (ARB) and AR determinants (ARDs) and the introduction of modern techniques like whole-genome sequencing, metagenomics, and in-silico modelling.

Detection of unprecedented level of antibiotic resistance and identification of antibiotic resistance factors, including QRDR mutations in Escherichia coli isolated from commercial chickens from North India.

AIM: This study aimed to investigate the occurrence of antibiotic resistance phenotype and simultaneously understand its genetic basis in Escherichia coli isolated from the cloacal swabs of commercial chickens from north India. METHODS AND RESULTS: Escherichia coli isolates were assessed for susceptibility to 14 different antibiotics using the disc-diffusion technique and were screened for the presence of 22 antibiotic resistance genes (ARGs) by employing PCR. Isolates were found to be highly resistant to fluoroquinolones (nalidixic acid 91%, norfloxacin 73% and ciprofloxacin 66%), tetracycline (71%), beta-lactams (ampicillin 49% and amoxicillin/clavulanic acid 37%), co-trimoxazole (48%), streptomycin (31%) and chloramphenicol (28%); and comparatively less resistant to cefazolin (13%), amikacin (10%), aztreonam (4%), gentamicin (4%) and ceftriaxone (3%). Sixty-three percent of isolates were resistant to more than four different drugs. Abundance of plasmid-borne ARGs like tetA (83%), sul3 (44%), aadA1 (44%), strA (43%), strB (41%), qnrS (38%), sul2 (28%) and aac(6)-Ib-cr (15%) was observed among the isolates. Forty-five percent of isolates possessed more than five different ARGs. Quinolone resistance-determining region (QRDR) mutations within gyrA and parC genes were found to be the major determiners of quinolone resistance. QRDR mutations included leu83, asn87 and gly87 within gyrase-A polypeptide and ile80 and lys84 within topoisomerase IV (encoded by parC). CONCLUSIONS: Our findings suggest the abuse of antibiotics as feed additives and prophylactic drugs in Indian poultry sector. It also projects this industry as an active hotspot for the replication and selection of ARGs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings would provide evidence to the authorities for formulating effective strategies for restricting antibiotic usage as non-therapeutic agents in food animals. Occurrence of both plasmid-borne and chromosome-borne resistance towards quinolones can drive movement of resistance phenotype across bacterial species and vertical movement of resistance along the bacterial generations, respectively, which can pose mitigation challenges.

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus-2 and inhibits viral replication.

Dengue virus (DENV) comprises of four serotypes (DENV-1 to -4) and is medically one of the most important arboviruses (arthropod-borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV-2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti-lachesin antibody resulted in a significant reduction in DENV replication.

Implication of the Whitefly, Bemisia tabaci, Collagen Protein in Begomoviruses Acquisition and Transmission.

Begomoviruses are the largest group of plant viruses transmitted exclusively by the whitefly, Bemisia tabaci (Gennadius), in a persistent, circulative, and nonpropagative manner. Begomoviruses in association with B. tabaci cause enormous loss to world agricultural crops. Transmission, retention, and circulation of begomovirus in B. tabaci are facilitated by its interaction with several proteins of the insect and its endosymbionts. However, very few such proteins have been identified from B. tabaci that are involved in this specific interaction. Here, we have performed yeast two-hybrid assay between B. tabaci complementary DNA expression library and the coat protein (CP) of tomato leaf curl New Delhi virus (ToLCNDV) and cotton leaf curl Rajasthan virus (CLCuV). Collagen was the common protein found to be interacting with both of the viruses. The collagen protein was found to be localized in gut layers of B. tabaci. Additionally, pull-down and dot-blot assays confirmed the association of endogenous collagen with ToLCNDV CP. Immunolocalization analysis also showed colocalization of ToLCNDV particles and collagen within insect gut. Finally, B. tabaci fed on anticollagen antibody and exhibited ∼46% reduction in ToLCNDV transmission, suggesting a supportive role for collagen in virus transmission.

A thioredoxin-like protein of Bemisia tabaci interacts with coat protein of begomoviruses.

Bemisia tabaci (whitefly) is the sole vector of begomoviruses, which transmits them in a persistent and circulative manner from infected to healthy plants. During this process, begomoviruses interact with various proteins in the insect vector B. tabaci that would play a specific role in the virus transmission. Identification and characterization of such proteins are important to understand the complete process of virus transmission. Coat protein (CP) of begomoviruses is the only protein which is reported to interact with proteins of the insect vector B. tabaci. In this study, we performed yeast two-hybrid assay using CP of cotton leaf curl Rajasthan virus (CLCuV) and Tomato leaf curl New Delhi virus (ToLCNDV) as bait in separate experiments and cDNA prepared from total RNA of B. tabaci was used as prey. Yeast two-hybrid assay resulted in identification of a thioredoxin-like protein (TLP) from CLCuV yeast two-hybrid library. Later TLP was also found to interact with CP of ToLCNDV. In vitro pull-down assay showed TLP interaction with CP of both CLCuV and ToLCNDV. TLP was found to interact with ToLCNDV virus particles isolated from tomato leaves.

Tissue damage induced midgut stem cell proliferation and microbial dysbiosis in Spodoptera litura.

In the past decade, gut microbiota has come to the fore in search for the cause of disregulation in intestinal homeostasis. Here, we report a possible link between gut microbial dynamics and stress-inducing factors using the leaf worm moth Spodoptera litura as a model organism. Investigation reveals that S. litura exhibits dysbiosis i.e. alteration in the gut microbiota composition that might induce or suppress inflammation upon exposure to dextran sulfate sodium salt, a tissue damaging agent (DSS, 40 kD). It primarily corresponds to an expansion of the bacterial phylotypes Enterobacter sp., Pseudomonas sp., Escherichia sp. and Acinetobacter sp. belonging to subclass Gammaproteobacteria. To assess the role played by gut residents in midgut inflammation, we re-colonized the axenic insects with Pseudomonas, Enterobacter and Acinetobacter individually. We observed that Pseudomonas and Enterobacter monoassociated insects exhibit inflammatory effects like damage to gut epithelium and hyperproliferation of stem cells under stress conditions. Conversely, Acinetobacter promotes fitness in larvae and reduces inflammatory effects of DSS. However, we failed to detect phenotypic inflammatory changes like midgut epithelium damage and stem cell proliferation in axenic insects reared on DSS-supplemented diet. Our results highlight that gut commensals that apparently remain low in abundance and benign under typical conditions can exert modulatory (positive or negative) effects on host fitness in the presence of stimulator.

Influence of Groundnut bud necrosis virus on the Life History Traits and Feeding Preference of Its Vector, Thrips palmi.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

Insecticide resistance status in the whitefly, Bemisia tabaci genetic groups Asia-I, Asia-II-1 and Asia-II-7 on the Indian subcontinent.

The present study is a summary of the current level of the insecticide resistance to selected organophosphates, pyrethroids, and neonicotinoids in seven Indian field populations of Bemisia tabaci genetic groups Asia-I, Asia-II-1, and Asia-II-7. Susceptibility of these populations was varied with Asia-II-7 being the most susceptible, while Asia-I and Asia-II-1 populations were showing significant resistance to these insecticides. The variability of the LC50 values was 7x for imidacloprid and thiamethoxam, 5x for monocrotophos and 3x for cypermethrin among the Asia-I, while, they were 7x for cypermethrin, 6x for deltamethrin and 5x for imidacloprid within the Asia-II-1 populations. When compared with the most susceptible, PUSA population (Asia-II-7), a substantial increase in resistant ratios was observed in both the populations of Asia-I and Asia-II-1. Comparative analysis during 2010-13 revealed a decline in susceptibility in Asia-I and Asia-II-1 populations of B. tabaci to the tested organophosphate, pyrethroid, and neonicotinoid insecticides. Evidence of potential control failure was detected using probit analysis estimates for cypermethrin, deltamethrin, monocrotophos and imidacloprid. Our results update resistance status of B. tabaci in India. The implications of insecticide resistance management of B. tabaci on Indian subcontinent are discussed.

Detection and Localization of Wolbachia in Thrips palmi Karny (Thysanoptera: Thripidae).

Thrips palmi Karny is a globally distributed polyphagous agricultural pest. It causes huge economic loss by its biological behaviors like feeding, reproduction and transmission of tospoviruses. Since T. palmi shows close morphological similarities with other thrips species, we employed mitochondrial cytochrome oxidase 1 (mtCO1) gene as a molecular marker. BLAST analysis of this sequence helped us to identify the collected specimen as T. palmi. We observed the female to male ratio of about 3:1 from collected samples and suspected the presence of Wolbachia. The presence of Wolbachia was detected by PCR using genus specific primers of 16S rRNA gene. Further confirmation of Wolbachia strain was achieved by conducting PCR amplification of three ubiquitous genes ftsZ, gatB and groEL. A phylogenetic tree was constructed with concatenated sequences of ftsZ and gatB gene to assign supergroup to Wolbachia. Finally, we localized Wolbachia in abdominal region of the insect using fluorescent in situ hybridization with the help of confocal microscope. Our result confirmed the presence of Wolbachia supergroup B strain for the first time in T. palmi.

Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis (Hemiptera: Aleyrodidae) adults using 16S rDNA amplicon pyrosequencing and FISH.

Dialeurolonga malleswaramensis Sundararaj (Hemiptera: Aleyrodidae) is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene (mtCO1) helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswaramensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization (FISH) in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.

Infection of Bacterial Endosymbionts in Insects: A Comparative Study of Two Techniques viz PCR and FISH for Detection and Localization of Symbionts in Whitefly, Bemisia tabaci.

Bacterial endosymbionts have been associated with arthropods and large number of the insect species show interaction with such bacteria. Different approaches have been used to understand such symbiont- host interactions. The whitefly, Bemisia tabaci, a highly invasive agricultural pest, harbors as many as seven different bacterial endosymbionts. These bacterial endosymbionts are known to provide various nutritional, physiological, environmental and evolutionary benefits to its insect host. In this study, we have tried to compare two techniques, Polymerase chain reaction (PCR) and Flourescence in situ Hybridisation (FISH) commonly used for identification and localization of bacterial endosymbionts in B. tabaci as it harbors one of the highest numbers of endosymbionts which have helped it in becoming a successful global invasive agricultural pest. The amplified PCR products were observed as bands on agarose gel by electrophoresis while the FISH samples were mounted on slides and observed under confocal microscope. Analysis of results obtained by these two techniques revealed the advantages of FISH over PCR. On a short note, performing FISH, using LNA probes proved to be more sensitive and informative for identification as well as localization of bacterial endosymbionts in B. tabaci than relying on PCR. This study would help in designing more efficient experiments based on much reliable detection procedure and studying the role of endosymbionts in insects.

Elimination of Arsenophonus and decrease in the bacterial symbionts diversity by antibiotic treatment leads to increase in fitness of whitefly, Bemisia tabaci.

Bemisia tabaci is an invasive agricultural pest with more than 24 genetic groups harboring different bacterial endosymbionts categorized into obligatory and facultative endosymbionts. Arsenophonus is one of the facultative endosymbionts prevalent in B. tabaci of Indian sub-continent. Not much is known about the functional role of this endosymbiont in its host. Some studies have revealed its involvement in virus transmission by B. tabaci, but how it effects the biology of B. tabaci is unknown. In this study, tetracycline was used to eliminate Arsenophonus from B. tabaci to study its effects with regard to development and other fitness parameters. Bacteria specific 16S Polymerase chain reaction (PCR) was used to ascertain Arsenophonus absence with differential effects on other secondary endosymbionts present in B. tabaci. Our results revealed that Arsenophonus negative (A(-)) whiteflies had more fecundity, increased juvenile developmental time, increased nymphal survival and increased adult life span as compared to control (A(+)) whiteflies. Thus, our results demonstrate that A(+) whiteflies have lesser fitness as compared to A(-) whiteflies. These observations give a new insight about the probable role of Arsenophonus in B. tabaci, that need to be explored further.

Draft Genome Sequence of the Paenibacillus sp. Strain ICGEB2008 (MTCC 5639) Isolated from the Gut of Helicoverpa armigera.

Paenibacillus sp. strain ICGEB2008 (MTCC 5639) is a Gram-positive cellulolytic bacterium, isolated from the gut of Helicoverpa armigera. Here, we report the draft genome sequence of Paenibacillus sp. ICGEB2008. The annotation of the ~5.7-Mb sequence indicated a cluster of genes related to the glycosyl hydrolase family and the butanediol biosynthesis pathway.

Genome sequence of Acinetobacter sp. strain HA, isolated from the gut of the polyphagous insect pest Helicoverpa armigera.

In this study, Acinetobacter sp. strain HA was isolated from the midgut of a fifth-instar larva of Helicoverpa armigera. Here, we report the draft genome sequence (3,125,085 bp) of this strain that consists of 102 contigs, 2,911 predicted coding sequences, and a G+C content of 41%.

Arsenophonus GroEL interacts with CLCuV and is localized in midgut and salivary gland of whitefly B. tabaci.

Cotton leaf curl virus (CLCuV) (Gemininiviridae: Begomovirus) is the causative agent of leaf curl disease in cotton plants (Gossypium hirsutum). CLCuV is exclusively transmitted by the whitefly species B. tabaci (Gennadius) (Hemiptera: Alerodidae). B. tabaci contains several biotypes which harbor dissimilar bacterial endo-symbiotic community. It is reported that these bacterial endosymbionts produce a 63 kDa chaperon GroEL protein which binds to geminivirus particles and protects them from rapid degradation in gut and haemolymph. In biotype B, GroEL protein of Hamiltonella has been shown to interact with Tomato yellow leaf curl virus (TYLCV). The present study was initiated to find out whether endosymbionts of B. tabaci are similarly involved in CLCuV transmission in Sriganganagar (Rajasthan), an area endemic with cotton leaf curl disease. Biotype and endosymbiont diversity of B. tabaci were identified using MtCO1 and 16S rDNA genes respectively. Analysis of our results indicated that the collected B. tabaci population belong to AsiaII genetic group and harbor the primary endosymbiont Portiera and the secondary endosymbiont Arsenophonus. The GroEL proteins of Portiera and Arsenophonus were purified and in-vitro interaction studies were carried out using pull down and co-immunoprecipitation assays. In-vivo interaction was confirmed using yeast two hybrid system. In both in-vitro and in-vivo studies, the GroEL protein of Arsenophonus was found to be interacting with the CLCuV coat protein. Further, we also localized the presence of Arsenophonus in the salivary glands and the midgut of B. tabaci besides the already reported bacteriocytes. These results suggest the involvement of Arsenophonus in the transmission of CLCuV in AsiaII genetic group of B. tabaci.

LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization.

BACKGROUND: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. RESULTS: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. CONCLUSION: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Host plant induced variation in gut bacteria of Helicoverpa armigera.

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.