Proteolytic Activity in the Midgut of Helicoverpa armigera (Noctuidae: Lepidoptera) Larvae Fed on Wild Relatives of Chickpea, Cicer arietinum.

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we evaluated 15 accessions of wild relatives of chickpea belonging to seven species and five genotypes of cultivated chickpea for their resistance to pod borer, Helicoverpa armigera, which is the most damaging pest of chickpea. The test genotypes were evaluated for resistance to H. armigera using detached pod assay. Data were also recorded on activity of the digestive enzymes in the midgut of the larvae fed on different wild relatives of chickpea. All the wild chickpea genotypes suffered lower pod damage and weight gained by the third-instar larvae of H. armigera was lower when fed on them compared with the cultivated chickpea. The accessions, IG 69979 (Cicer cuneatum), PI 599066, IG 70006, IG 70018, IG 70022 (Cicer bijugum), IG 599076 (Cicer chrossanicum), and IG 72933, IG 72953 (Cicer reticulatum), showed high levels of resistance to H. armigera. There were significant differences in protease activity in larval gut of H. armigera fed on different wild relatives of chickpea. Total protease, trypsin, and chymotrypsin activities were lowest in larva fed on PI 599066 (C. bijugum) compared with that in the larvae fed IG 69979 (C. cuneatum) and IG 70022 (C. bijugum). Aminopeptidase activity was highest in the larvae fed on IG 70022 (C. bijugum) and IG 599076 (C. chrossanicum), whereas lowest activity was recorded in the larvae fed on ICC 3137 and KAK 2 (susceptible checks). The variation in protease activities may be due to the presence of protease inhibitors in the wild relatives or hyperproduction of enzymes by the larvae as result of protease inhibitor activity of the wild relatives, resulting in low weight gain by larvae. The results suggested that wild relatives of chickpea with diverse mechanisms of resistance can be exploited to increase the levels and diversify the basis of resistance to H. armigera in cultivated chickpea.

Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 µg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

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

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.

Interaction of plant cell signaling molecules, salicylic acid and jasmonic acid, with the mitochondria of Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera is a polyphagous pest in Asia, Africa, and the Mediterranean Europe. Salicylic acid (SA) and jasmonic acid (JA) are the cell signaling molecules produced in response to insect attack in plants. The effect of these signaling molecules was investigated on the oxidative phosphorylation and oxidative stress of H. armigera. SA significantly inhibited the state III and state IV respiration, respiratory control index (RCI), respiratory complexes I and II, induced mitochondrial swelling, and cytochrome c release in vitro. Under in vivo conditions, SA induced state IV respiration as well as oxidative stress in time- and dose-dependent manner, and also inhibited the larval growth. In contrast, JA did not affect the mitochondrial respiration and oxidative stress. SA affected the growth and development of H. armigera, in addition to its function as signaling molecules involved in both local defense reactions at feeding sites and the induction of systemic acquired resistance in plants.