Different digestive protease complements in Colorado potato beetles fed functional variants of the tomato cystatin SlCYS8

Colorado potato beetles ingesting plant cystatins compensate for the loss of digestive Cys protease activities by a multicomponent defensive strategy involving the overexpression of cystatin-sensitive Cys proteases to outnumber the inhibitory proteins, the expression of Cys proteases insensitive to the cystatins, and the expression of proteases from alternative functional classes. We previously engineered single variants of the tomato cystatin, SlCYS8, exhibiting differential inhibitory spectra against digestive Cys protease populations of the potato beetle. Our goal, in the present study, was to determine whether such functional variants of an original, wild-type cystatin would induce differential compensatory responses in the insect, as inferred from a kinetic analysis of midgut Cys protease patterns upon recombinant cystatin ingestion. In brief, potato beetle larvae fed potato leaves painted with either SlCYS8 variants showed comparable growth rates after one or three days, but variable leaf consumption rates indicating an effect on the efficiency of dietary protein digestion. Midgut protease profiles also differed depending on the cystatin variant, with more, or on the contrary less, cathepsin L-like and cathepsin B-like activities compared to control larvae ingesting no recombinant cystatin or control larvae ingesting the original, wild-type version of SlCYS8. Our data point, overall, to the presence of different digestive protease complements in potato beetle larvae ingesting plant cystatins diverging in their basic protease inhibitory spectrum towards Cys proteases.