Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 25, 2010
Publication Date: February 1, 2011
Citation: Zhu, Y., Guo, Z., Chen, M., Zhu, K., Liu, X.F., Scheffler, B.E. 2011. Major Pesticide Receptors, Detoxification Enzymes, and Transcriptional Profile in Midgut of the Tobacco Budworm, Heliothis virescens (Lepidoptera: Noctuidae). Insect Molecular Biology. 106:296-307. doi: 10.1016/jip.2010.10.007. Interpretive Summary: Currently, control of the tobacco budworm (TBW) relies mainly on transgenic Bt plants expressing insecticidal Cry proteins (Bt), conventional chemical insecticides, and novel chemical and bio-pesticides. Most current insecticides reach target receptors in the midgut and hemolymph through the alimental cannel, with the exception of the contact insecticides. Long term and dominant use of a chemical may prompt resistance evolution in target populations. Insect midgut acts as a processing port of endo- and xeno-biotic substances. Besides food digestion and absorption, the insect midgut plays an important role in overcoming host plant defenses and in detoxifying insecticides. In this study, we sequenced 5,856 cDNAs which were assembled into 1,687 unique clusters. We identified at least 143 contigs and singletons that encoded 9 proteins, nearly a complete list of relevant genes associated with insecticide detoxification and resistance evolution in insects. Many of these 9 groups of proteins could be phylogenetically separated into additional sub-groups or isozymes that may be specialized for different functions or additively play the same function within the group. In addition to the 9 groups of proteins, we identified 48 different putative enzymes and numerous other proteins, many of which may be involved in detoxification which directly or indirectly affects resistance development. Profiling midgut gene transcripts in this study will certainly facilitate further study to examine and compare global gene expression levels between biological/physiological samples. More importantly, identification of multiple form gene transcripts (especially those for insecticide detoxification and resistance) may help identify the most important gene for future gene knockdown or protein inhibition with a plant-delivery system.
Technical Abstract: Insecticide resistance mechanisms, including those for Cry proteins (Bt) in the tobacco budworm, Heliothis virescens, are not well understood. Sequencing of midgut transcriptomes may facilitate the discovery of the genes responsible for resistance development which could then be targeted by new bio-pesticides or RNAi. A total of 5,856 Sanger sequences, obtained from this study, were assembled to 1,687 contigs (464) and singletons (1,233) with average length of 507 bp. Blast similarity search showed that 1,372 cDNAs from this study matched different genes or cDNAs in the GenBank and other insect sequence databases. Blast2go annotation identified 611 highly similar proteins with metabolic and cellular processes as major biological functions and catalytic activity and binding as major molecular functions. At least 143 contigs and singletons were associated with pesticide activation, detoxification, and resistance development. These cDNAs, with average length of 601 bp, matched 9 groups of pesticide resistance related genes. Major putative detoxification enzymes included 20 cytochrome P450 oxidases, 11 glutathione S-transferases, 9 esterases, 15 cytochrome oxidases, and 8 sodium channels. At least 80 cDNAs coded for Bt resistance related enzymes and potential receptors, including 58 serine proteinases, 5 alkaline phosphatases, 13 aminopeptidase, and 4 cadherins. Of the 143 contigs and singletons, 111 cDNA sequences seemed to be new resistance candidate gene transcripts in GenBank because they either priorly matched resistance candidate cDNAs of other species, or had low sequence identity with those previously sequenced from H. virescens. In addition to reporting a substantial increase of 111 new gut-specific candidate cDNAs associated with pesticide toxicity and resistance, this study provides a foundation for future research to develop a gut-specific DNA microarray for analysis of the global changes of gene expression in response to biological and chemical pesticides. Future development of RNAi and other resistance management strategies could benefit from this study and help continue research to identify key genes targetable by classic and novel approaches.