ИСТИНА

The stored product pests Tenebrio molitor and Tribolium castaneum are model coleopterans from the family Tenebrionidae. The larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases (SP) in the middle and posterior midgut. We used high-throughput sequencing of T. molitor and T. castaneum larval gut mRNA, as well as T. castaneum genomic sequences, to identify, annotate and compare predicted SP data sets from both insects, and evaluate relative expression levels. In the gut transcriptome of T. molitor larvae, we found 183 sequences similar to SP of the S1 chymotrypsin family, but only 87 sequences contained conserved catalytic residues H57, D102, S195 representing active peptidases. The residual 96 sequences had substitutions in the catalytic triad and were classified as inactive homologs of serine peptidases (SPH). The genome of T. castaneum contains 178 SP sequences, of which 98 represent active peptidases, and 80 SPH. It is unclear why the genomes of both insects have retained such a high proportion of SPH, but we theorize that they may aid in protecting the insect from damaging cereal inhibitors. In the T. molitor gut transcriptome, 31 sequences were related to trypsins, two to chymotrypsins A/B, one to chymotrypsin C, 13 were chymotrypsin-like peptidases, 12 pancreatic elastases I, and the specificity of 28 peptidases was uncertain due to unusual residues in the substrate binding site. These data are reflective of a highly complex digestive organization in these larvae, presumably a result of high selection pressure of cereal inhibitors. The genome and gut transcriptome of T. castaneum contained many more trypsin sequences – 51, and fewer chymotrypsin sequences than those found in T. molitor: one chymotrypsin A/B, one chymotrypsin C, and only 5 chymotrypsin-like peptidases. Three sequences were annotated as pancreatic elastases I and 37 peptidases were with uncertain specificity. Most of the peptidases sequences were arranged in clusters containing up to 28 genes. Phylogenetic analysis suggested rapid evolution and often duplication of T. castaneum SP genes. Analysis of the relative expression levels of SP mRNA revealed that each group of SP usually contained one major SP with the expression level exceeding 50% of the total expression of the whole group, a number of peptidases with medium level of expression, and many SP with minor level of mRNA expression. Usually the major peptidase from each group of T. molitor and T. castaneum SP were not orthologous. Some SPH had rather high levels of mRNA expression, and this implies that they have important physiological functions. These data expand our understanding of digestion in Tenebrionidae and will help to identify the role of SPH in insects. This work was supported by RFBR grants 12-03-01057-a, 14-04-91167-NSFC_a