Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner)

Abstract

Helicoverpa armigera is one of the most important pests worldwide. Transgenic crops with toxin genes from Bacillus thuringiensis (Bt) have been deployed on a large scale to control this pest. The insecticidal activity of Bt is probably influenced by the insect midgut microbes, which vary across crop hosts and locations. Therefore, we examined the role of gut microbes in pathogenicity of Bt toxins in the H. armigera. Antibiotic cocktail was used for the complete elimination of the H. armigera gut microbes. Activated Cry1Ac, Bt formulation, and transgenic cotton resulted in larval weight loss and increase in mortality, but pretreatment of larvae with antibiotic cocktail significantly decreased larval mortality and increased the larval weight gain. Activated Cry1Ac and Bt formulation inhibited the activity of proteases in midgut of H. armigera larvae but showed no such effect in the larvae pretreated with antibiotic cocktail. Five protease bands in activated Cry1Ac and two in Bt formulation-treated larvae were inhibited but no such effect in the larvae pretreated with antibiotic cocktail. Cry1Ac protein was detected in Bt/Cry1Ac protoxin-fed larval gut extract in the absence of antibiotic cocktail, but fewer in larvae pretreated with antibiotic cocktail. The activity of antioxidant enzymes and aminopeptidases increased in larvae fed on Bt toxin, but there was no significant increase in antioxidant enzymes in larvae reared on toxin protein in combination with antibiotic cocktail. The results suggest that gut microbes exercise a significant influence on the toxicity of Cry1Ac and Bt formulation in H. armigera larvae. The implications of these results have been discussed in relation to development of insect resistance to Bt transgenic crops deployed for pest management.

Evaluation of flubendiamide‐induced mitochondrial dysfunction and metabolic changes in Helicoverpa armigera (Hubner)

“…One unit of enzyme activity was expressed as μM of p ‐nitroanilide (pNA) liberated min −1 ml −1 of enzyme (ε for pNA is 8.8 mM −1 cm −1 ). Total protease activity was measured using 1% azocasein as substrate (Visweshwar et al., ).…”

“…Trypsin and chymotrypsin were measured spectrophotometrically using N--benzoyl-DL-arginine-p-nitroanilide and N-glutaryl-L-phenylalanine p-nitroanilide as substrates, respectively (Lomate & Hivrale, 2013;Vinod, Sharma, & Kachole, 2010). Aminopeptidase and elastase were measured by incubating the gut extract with leucine p-nitroanilide and N-succinyl-alanine-alanine-alanine p-nitroanilide substrates, respectively (Li et al, 2004;Visweshwar, Sharma, Akbar, & Sreeramulu, 2015). Change in absorbance was measured at 410 nm.…”

Midgut microbiota and host immunocompetence underlieBacillus thuringiensiskilling mechanism

“…A heated debate on the B. thuringiensis killing mechanism started almost a decade ago, and a consensus has not yet been reached. Indeed, divergent opinions persist on the role played by the microbiota residing in the midgut of lepidopteran hosts and the septicemia it may cause after toxin-induced disruption of the lining epithelium (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Whether the gut paralysis and associated feeding cessation observed in larvae exposed to Cry pore-forming toxins or the septicemia induced by bacterial proliferation in the insect body cavity is the major factor contributing to eventual death remains to be resolved (5,32) Significance Bacillus thuringiensis and its toxins are widely used for insect control.…”

“…To circumvent the problems associated with the manipulation of the midgut microbiota with antibiotics (22)(23)(24)(25)(27)(28)(29)(30)(31) or of the immune response with immunosuppressive molecules (26,34), both of which have unpredictable multiple effects, we performed selective RNAi-mediated silencing of an immune gene (102 Sl, 102 Spodoptera littoralis), recently identified in Spodoptera littoralis (35). This gene is highly expressed in the immune cells circulating in the hemolymph (i.e., hemocytes) and modulates their incorporation in multilayered capsules formed around foreign intruders entering the body cavity (i.e., the hemocoel) (35).…”

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

“…Proteases from insects could be excellent candidates for industrial applications. Proteolysis is an essential part of food digestion in insects mediated by concerted action of several proteases (Anwar & Saleemuddin, 2002;Sanatan et al, 2013;Visweshwar et al, 2015). Serine protease from insect midguts have been characterized from Osphranteria coerulescens Redtenbacher, Periplaneta americana L., Glyphodes pyloalis Walker, and so on possess important features such as stability under high temperatures, and high activity under alkaline pH (Mahdavi et al, 2013;Sanatan et al, 2013;Sharifi et al, 2012).…”