Insect Science (2013) 20, 485–496, DOI 10.1111/j.1744-7917.2012.01514.x ORIGINAL ARTICLE Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis Yunlong Yang 1 , Yu Cheng Zhu 2 , James Ottea 1 , Claudia Husseneder 1 , B. Rogers Leonard 1 , Craig Abel 3 , Randall Luttrell 2 and Fangneng Huang 1 1 Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, 2 Southern Insect Management Research Unit, Agricultural Research Service, United States Department of Agriculture, Stoneville, Mississippi, 3 Corn Insects and Crop Genetics Research Unit, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, USA Abstract Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is re- sponsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associ- ated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin- like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, includ- ing signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly as- sociated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes. Key words Bt, cDNA, chymotrypsin, enzymatic activity, resistance, trypsin Correspondence: Yu Cheng Zhu, Jamie Whitten Delta States Research Center, USDA-ARS, 141 Experiment Station Rd., P.O. Box 346, Stoneville, MS 38776, USA. Tel: +1 662 686 5360; fax: +1 662 686 5421; email: yc.zhu@ars.usda.gov Introduction Serine proteinases are common luminal midgut di- gestive enzymes of dietary proteins in many insect species including lepidopterans (Terra & Ferreira, 1994). Among the insect serine proteinases, midgut trypsins and Published 2012. This article is a U.S. Government work and is in the public domain in the USA. 485