Digestion of δ-Endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIc

The present study describes the correlation between gut protease activity of lepidopteran larvae of different instars, the inactivation of Bacillus thuringinesis δ-endotoxins in crystalline and noncrystalline forms, and the reduced susceptibility of advanced larval instars of Spodoptera littoralis to the toxin. The original assembly of δ-endotoxins in a crystal structure is essential for causing efficient larval mortality. Denaturation and renaturation (D/R) of δ-ednotoxin crystals increased the vulnerability of the toxin molecules to proteolysis, reduced their capability to kill neonate larvae of S. littoralis, but sustained most of their larval growth- inhibition activity. E. coli-produced CryIC δ-endotoxin applied as a fraction of inclusion bodies exerted a growth inhibition effect, similar to the molecules released from the crystals by denaturation and subsequent renaturation. Incubation of CryIC with gut juice of 1st or 2nd instar larvae, left part of the CryIC toxin intact, while the toxin was completely degraded when incubated with gut juice of 5th instar larvae. The degradation rate was consistent with the increase of protease specific activity of the gut juice during larval development. This increase in toxin degradation may account for the loss of sensitivity of 5th instar larvae to CryIC. Specific protease inhibitors such as PMSF and Leupeptin were shown to inhibit gut proteases activity in all instar larvae, while, 1,10 phenanthroline, TLCK and TPCK were effective only in young instar larvae. The differential effect of protease inhibitors on proteases obtained from different larval instars indicated that gut juice protease profiles change with larval age. The observed quantitative and qualitative differences in degradation of δ-endotoxin by larval gut proteases that occur during larval maturation may account for the difference in susceptibility to the δ-endotoxin. This finding should be taken into consideration when designing strategies for the development of transgenic crops expressing δ-endotoxins as potent insecticidal proteins.