ABSTRACT
In this study, the results showed that the population of midgut bacteria and larval mortality due to Cry1Ac are significantly reduced in antibiotic-treated larvae from Bt-susceptible, -resistant and field-collected strains (96S, BtR, FS respectively) of Helicoverpa armigera. The percentage reduction of larval mortality with increasing concentrations of antibiotics was significantly different among strains with the smallest effect observed in FS. It has been suggested that antibiotics could influence the toxicity of Cry1Ac, possibly by eliminating gut bacteria, hence gut bacteria might be playing essential roles in Bt-induced killing of H. armigera. But elimination of midgut microflora with antibiotics had no effect on resistance level.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Endotoxins/metabolism , Gastrointestinal Microbiome/drug effects , Hemolysin Proteins/metabolism , Insecticide Resistance/drug effects , Moths/microbiology , Pest Control, Biological/methods , Animals , Bacillus thuringiensis Toxins , Moths/drug effectsABSTRACT
A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.
Subject(s)
Alkaline Phosphatase/metabolism , Bacillus thuringiensis , Bacterial Proteins/pharmacology , CD13 Antigens/metabolism , Cadherins/metabolism , Endotoxins/pharmacology , Hemolysin Proteins/pharmacology , Insect Proteins/metabolism , Insecticides/pharmacology , Intestines , Moths/metabolism , Animals , Bacillus thuringiensis Toxins , Larva/metabolismABSTRACT
Cry1A and Cry2A toxins, which are widely used in Bt transgenic crops, can specifically bind to insect midguts and exert their insecticidal effects. There are interactions between insect midgut-binding proteins and Cry1A toxins; however, little is known about the insect protein that specifically binds to Cry2A. Midgut membrane-bound alkaline phosphatases (ALPs), which are important for the binding of proteins to Cry1A, play dominant roles in Cry1A-mediated toxicity in some lepidopteran larvae. In this study, we cloned and expressed one partial ALP2 peptide from susceptible Spodoptera exigua larvae and studied the binding characteristics of SeALP2 with Cry2Aa. The ALPs proteins was expressed at all larval stages and highly expressed in the first and second instar larvae. The heterologously expressed SeALP2 peptide bound specifically to Cry2Aa with a high affinity. Knocking down ALP2 in vivo revealed that it plays an important role in the susceptibility of S. exigua to Cry2Aa. Based on these findings, we propose that ALP2 in S. exigua serves as a functional receptor for Cry2Aa.
