# Lack of Tolerance Development Following Oral Exposure Tosublethal Cry1 and Vip3Aa Proteins in Spodoptera exigua (Hübner, 1808)

## TL;DR

This study finds that exposing beet armyworm larvae to low doses of Bt insecticidal proteins does not lead to increased tolerance in the insects or their offspring.

## Contribution

The study demonstrates that sublethal exposure to purified Cry1 and Vip3Aa proteins does not induce tolerance in Spodoptera exigua.

## Key findings

- Feeding neonate larvae with sublethal doses of Cry1 and Vip3Aa proteins did not increase their tolerance to lethal doses.
- Offspring of exposed larvae showed no increased tolerance and were sometimes more susceptible.
- Sublethal exposure to purified Bt proteins is unlikely to compromise the long-term effectiveness of Bt-based pest management.

## Abstract

Farmers often rely on natural insecticidal proteins produced by the soil bacterium Bacillus thuringiensis (Bt) to protect crops from damaging caterpillars. These proteins have been incorporated into transgenic plants and have been highly effective. However, there are growing concerns because insect populations are developing tolerances and resistances to these toxins. Previous studies have suggested that exposure to low doses of Bt-derived products could give an advantage to insects, increasing their tolerance. In this study, we tested whether feeding neonate larvae of the beet armyworm (Spodoptera exigua) with Cry1 and Vip3Aa proteins would increase their tolerance and whether this effect would be transmitted to their offspring. Our results showed that feeding on neonates with these proteins did not lead to a biologically relevant increase in tolerance when larvae were later exposed to lethal doses. Moreover, the offspring of the exposed larvae did not show increased tolerance; in fact, they were in some cases slightly more susceptible. In conclusion, our findings indicate that it is highly unlikely that tolerance will develop in this species through this mechanism with the tested proteins. Therefore, sublethal exposure is not expected to compromise the long-term effectiveness of Bt-based pest management strategies, thus supporting the continued use of these proteins in agriculture.

Insecticidal proteins derived from Bacillus thuringiensis (Bt) have been effectively employed in controlling lepidopteran pests, notably in transgenic crops targeting Spodoptera species. However, concerns have arisen regarding the long-term efficacy due to the emergence of tolerant and resistant insect populations. Prior research suggested that repeated exposures to Bt, which contains a mixture of spores and crystals, may contribute to the development of tolerance; however, the specific effects of sequential exposure to purified Cry1 and Vip3Aa proteins remain unclear. This study aimed to assess whether prior exposure of Spodoptera exigua neonate larvae to sublethal concentrations of Cry1Ab, Cry1Ca or Vip3Aa proteins would heighten their tolerance upon subsequent exposure, and whether such effects would extend to their offspring. Pre-exposure to Cry1Ab or Vip3Aa did not affect larval responses to the toxin. For Cry1Ca, a slight increase was observed under one treatment condition, but the effect was not considered biologically relevant. Transgenerational analysis revealed no enhancement of tolerance; rather, there was a negative impact on the offspring’s response in some cases. These findings indicate that although previous studies have documented that sublethal contact with bacterial preparations may significantly affect insect tolerance, exposure to sublethal doses of purified Cry1 and Vip3Aa proteins is unlikely to lead to the development of tolerance in S. exigua.