Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/22/2012
Publication Date: 7/26/2012
Citation: Oppert, B.S., Martynov, A.G., Elpidina, E.N. 2012. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts . Comparative Biochemistry and Physiology. 7(3): 233-242, doi: http://dx.doi.org/10.1016/j.cbd.2012.03.005. Interpretive Summary: Some insecticides are based on toxins produced by bacteria or other microbes, but these insecticides often don’t work well for beetle pests. In order to make these microbial toxins more effective for controlling beetles, we need to understand how they kill the insect. Most of the microbial toxins used for insect control are produced by the bacterium Bacillus thuringiensis, and we used one of these toxins in our study. We know that there are enzymes in the beetle gut that are involved in activating the toxin and making it soluble so that it can bind to the lining of the insect gut. Binding of the toxin starts a chain of events that we still don’t fully understand, but leads to killing the insect. To better understand this process, we determined how many enzymes are in the gut of mealworm larvae, and what changes are induced in these enzymes when mealworm larvae are fed toxin. Our results suggest that that mealworm larvae exposed to toxin are attempting to decrease toxicity of the toxin while maintaining efficient digestion. Knowledge of how the gut enzymes in mealworm larvae are affected by microbial toxins may help us to improve these important biopesticides for beetle pest control.
Technical Abstract: The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and posterior midgut, and the latter activate Bt Cry protoxins and are a determining factor in toxicity and resistance. Therefore, we evaluated the effect of Cry3Aa protoxin on the expression of cysteine and serine peptidase genes in the midgut of T. molitor larvae. High-throughput sequencing was used to obtain EST databases from midguts of one month old T. molitor larvae fed either a control diet or diet containing 0.1% Cry3Aa for 24 h. In addition, the temporal expression of peptidase genes was investigated by microarray analysis of transcripts from larvae fed Cry3Aa for 6, 12, or 24 h. Sequences encoding cysteine peptidases from the C1 family were similar to cathepsins B, L, and K. The expression of four cysteine cathepsin, including one encoding the major digestive cathepsin L, were decreased approximately 2-fold, and two were increased 2-fold in Cry3Aa-treated larvae. Transcripts of serine peptidases from the S1 family encoded active peptidases and homologs (SPH) lacking functional amino acids, and included trypsins, chymotrypsins, elastase 1, and unclassified serine peptidases. Transcripts encoding a highly-expressed digestive trypsin and chymotrypsin were 15- and 6-fold down-regulated, respectively, in Cry3Aa-treated larvae. Many serine peptidases and SPH expressed at relatively low levels in control larvae were not found in Cry3Aa-treated larvae, and overall serine peptidases were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. However, increased expression of several transcripts encoding a putative chymotrypsin and one transcript related to SPH was found in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases, and several serine peptidases transcripts with comparatively high expression were found only in Cry3Aa-treated larvae. We hypothesize that the insect may be attempting to decrease toxicity while maintaining efficient digestion. The data demonstrate that Cry3Aa intoxication in T. molitor induces widespread changes in peptidase gene expression.