Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

Abstract

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p-nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

RNA interference and dietary inhibitors induce a similar compensation response in Tribolium castaneum larvae

“…T. castaneum has a midgut with a pH gradient of an acidic anterior to a neutral to basic posterior. In the acidic anterior midgut, proteolytic activity is mostly by cathepsin L and B cysteine peptidases (Vinokurov et al, 2009;Martynov et al, 2015). Reliance on cysteine peptidase activity in the anterior midgut of tenebrionid larvae is probably an adaptation to degrade inhibitors and protect serine peptidases in the posterior midgut (Prabhakar et al, 2007;Oppert et al, 2010).…”

“…Reliance on cysteine peptidase activity in the anterior midgut of tenebrionid larvae is probably an adaptation to degrade inhibitors and protect serine peptidases in the posterior midgut (Prabhakar et al, 2007;Oppert et al, 2010). Some cysteine peptidases also have become specialized for efficient digestion of cereal proteins (Vinokurov et al, 2009;Goptar et al, 2012;Martynov et al, 2015).…”

Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function

“…6, underlined) and three with a minor role when fed a normal diet. In a previous biochemical study, we described seven distinct cysteine peptidase activities from fractionation of gut enzymes in the T. castaneum gut, and all were anionic enzymes mostly in the anterior midgut (Vinokurov et al, 2009); we propose that the seven major cysteine cathepsin digestive genes listed here encode those seven anionic enzymes. Three cathepsin B or B-like genes (LOC657117, LOC657203, and LOC656957) and four cathepsin L genes (LOC659441, LOC659502, LOC660368, and LOC663145) were upregulated when T. castaneum larvae were fed digestive inhibitors ( Fig.…”

“…T. castaneum cysteine peptidase genes include cathepsin B and L peptidases, mostly arranged in clusters on chromosome 3, 7, 8 and 10, and also single genes encoding cathepsins F, K, and O on chromosome 7, 4 and 1(X), respectively. Empirical evidence of the biological function of each cysteine peptidase gene in T. castaneum is lacking, although our previous studies suggest that some function as major processors of food proteins in the larval gut (Oppert et al, 1993;Vinokurov et al, 2009;Oppert et al, 2010;Martynov et al, 2015). Gene expression studies indicated that cathepsin L genes LOC659441 and LOC659502 are the most highly expressed cysteine peptidases in the larval gut and are most likely encoding enzymes important in the early stages of cereal protein digestion, as they are located in the anterior midgut (Prabhakar et al, 2007;Morris et al, 2009;Vinokurov et al, 2009;Martynov et al, 2015).…”

“…The larval gut of T. castaneum is compartmentalized, with an acidic anterior portion that secretes a high concentration of C1 family cysteine peptidases, while the slightly alkaline posterior portion of the midgut has mostly serine peptidases (Prabhakar et al, 2007;Vinokurov et al, 2009). The presence of cysteine peptidases in the coleopteran gut has been proposed as an adaptation to avoid serine peptidase inhibitors in grain kernels (Terra & Ferreira, 1994;Terra & Cristofoletti, 1996;Vinokurov et al, 2009). Cysteine peptidases also provide efficient digestion of cereal grain proteins (Goptar et al, 2012).…”