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

<em>Arsenophonus</em> GroEL Interacts with CLCuV and Is Localized in Midgut and Salivary Gland of Whitefly <em>B. tabaci</em>

<div><p>Cotton leaf curl virus (CLCuV) (<em>Gemininiviridae: Begomovirus</em>) is the causative agent of leaf curl disease in cotton plants (<em>Gossypium hirsutum</em>). CLCuV is exclusively transmitted by the whitefly species <em>B. tabaci</em> (Gennadius) (Hemiptera: Alerodidae). <em>B. tabaci</em> 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 <em>Hamiltonella</em> has been shown to interact with Tomato yellow leaf curl virus (TYLCV). The present study was initiated to find out whether endosymbionts of <em>B. tabaci</em> are similarly involved in CLCuV transmission in Sriganganagar (Rajasthan), an area endemic with cotton leaf curl disease. Biotype and endosymbiont diversity of <em>B. tabaci</em> were identified using MtCO1 and 16S rDNA genes respectively. Analysis of our results indicated that the collected <em>B. tabaci</em> population belong to AsiaII genetic group and harbor the primary endosymbiont <em>Portiera</em> and the secondary endosymbiont <em>Arsenophonus</em>. The GroEL proteins of <em>Portiera</em> and <em>Arsenophonus</em> were purified and <em>in-vitro</em> interaction studies were carried out using pull down and co-immunoprecipitation assays. <em>In-vivo</em> interaction was confirmed using yeast two hybrid system. In both <em>in-vitro</em> and <em>in-vivo</em> studies, the GroEL protein of <em>Arsenophonus</em> was found to be interacting with the CLCuV coat protein. Further, we also localized the presence of <em>Arsenophonus</em> in the salivary glands and the midgut of <em>B. tabaci</em> besides the already reported bacteriocytes. These results suggest the involvement of <em>Arsenophonus</em> in the transmission of CLCuV in AsiaII genetic group of <em>B. tabaci</em>.</p> </div

Rooted phylogenetic tree constructed for partial 16S rRNA gene of isolates cultured from <i>Helicoverpa armigera</i> gut samples.

<p>A neighbor-joining analysis with Jukes–Cantor correction and bootstrap support was performed on the gene sequences. Bootstrap values are given at nodes. Entries against (j) represent generic names and accession numbers (in parentheses) are from public databases. Entries from this work are represented as: clone number and accession number (in parentheses).</p

Phylogenetic affiliation of bacteria isolated by culturing method from 5^th instar larval midguts of <i>H. armigera</i> reared on cotton leaves based on complete 16S rRNA sequence.rRNA sequence.

<p>Phylogenetic affiliation of bacteria isolated by culturing method from 5^th instar larval midguts of <i>H. armigera</i> reared on cotton leaves based on complete 16S rRNA sequence.rRNA sequence.</p

Phylogenetic affliation of bacteria identified by culture independent analysis from 5^th instar larvae of midguts of <i>H. armigera</i> reared on cotton leaves based on complete 16S r RNA.

<p>Phylogenetic affliation of bacteria identified by culture independent analysis from 5^th instar larvae of midguts of <i>H. armigera</i> reared on cotton leaves based on complete 16S r RNA.</p

Pairwise comparison for similarity of T-RFLPs from the midgut of <i>Helicoverpa armigera</i> larvae collected on different host plants from Pachora location and artificial diet.

<p>Pairwise comparison for similarity of T-RFLPs from the midgut of <i>Helicoverpa armigera</i> larvae collected on different host plants from Pachora location and artificial diet.</p

Correspondence analysis of T-RFLP data sets derived from <i>BfaI</i> digestion of 16S rDNA from gut bacterial communities of <i>H. armigera</i> of different crops collected from various locations in India.

<p>Axis 1 explains 27.93% of significance and shows the difference between tomato, ladyfinger, castor and sorghum crop plants when compared to the other crops. Axis 2 with 18.56% of significance, illustrates the difference of tomato from other crop plants and artificial diet from rest of the samples. Cluster one includes Chickpea from Pachora (P), Delhi (D), Bangalore (B) and Coimbatore (C), Cotton from P, D, B and C; Sunflower, and Redgram from Pachora. Second cluster has lady finger, sorghum and castor from Pachora. Third cluster consists of tomato crop from P, D, B and C. Artificial diet group forms the 4^th cluster.</p