Influence of Ephestia kuehniella stage larvae on the potency of Bacillus thuringiensis Cry1Aa delta-endotoxin

- -Toxicity of Cry1Aa decreased during development of Ephestia kuehniella larvae stage.
- -Persistence of activated 58 kDa toxin inside L1 midgut caused an interesting toxicity.
- 4 caseinolytic activities appeared in L5 midgut, while 1 activity in the L1 one.
- Putative receptors APN1, APN2, APN4 were detected in both larvae instars.
- Same binding profile of Cry1Aa was found in both larvae instars.

The economically important crop pest Ephestia kuehniella was tested at two stages of larval development for susceptibility to Bacillus thuringiensis Cry1Aa toxin. Bioassays showed that toxicity decreased during the development of larvae stage. In fact, Cry1Aa toxins from BNS3-Cry− (pHT-cry1Aa) showed low toxicity against the first-instar larvae (L1) with a LC50 value of about 421.02 μg/g of diet and was not toxic against the fifth-instar (L5), comparing to the BLB1 toxins used as positive control which represent a LC50 value of about 56.96 and 84.21 μg/g of diet against L1 and L5 instars larvae, respectively. Effects of Cry1Aa toxins were reflected in histopathological observations by the weak destruction of midgut epithelium, slight hypertrophy of epithelial cells, and minor alteration of brush border membrane (BBM) detected mainly in L1 larvae stage comparing to the more extensive damage caused by BLB1 toxins. Interestingly, in vitro proteolysis of Cry1Aa toxins was found to correlate with the difference of toxicity during larval stage development. In fact, the weak proteinase activity detected inside the L1 midgut has led to the persistence of the Cry1Aa active forms (65 and 58 kDa) during prolonged incubations, causing the alterations described previously. Three subfamilies of aminopeptidase (APN) receptors were detected in both larvae instars with different intensities and molecular weights (150 kDa and 55 kDa for APN1, and 90 kDa for APN2 and APN4). Remarkably, binding assay using Cry1Aa toxin seems to have no direct correlation with larval stages toxicity differences, since same putative receptors were detected. Understanding the reasons for the clear differences in the effectiveness of Cry1Aa toxins during larval development stages of E. kuehniella is very important for the design of future improvement insecticidal approaches and for the accomplishment of resistance prevention strategies.

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