Molecular Entomology, 2025, Vol.16, No.1, 19-27 http://emtoscipublisher.com/index.php/me 25 3.4 Screening of larvae suitable for Bt toxin bioassay Through field plots and indoor observations, it was found that the larvae of the Ⅱ instar are most suitable for Bt toxin bioassay in terms of damage site, migration ability and body size: the larvae of the Ⅱ instar have a wide feeding range, feeding on both heart leaves and filaments and the surface of young ears, which is equivalent to a longer "window period" for field damage, which can represent the extensive exposure to Bt toxins; in addition, the adaptability of the larvae of the Ⅱ instar to the external environment and feed is significantly improved compared with the Ⅰ instar, and the individuals are larger and easier to handle, but they still maintain a high sensitivity to Bt toxins compared with older larvae (Wen et al., 2005). As the age increases, corn borer larvae show a gradually increasing tolerance to Bt toxins, which is the phenomenon of "age resistance". Studies have shown that the tolerance of older larvae to Bt toxins can be more than one order of magnitude higher than that of younger larvae (Huynh et al., 2022). For example, the tolerance of older larvae of some lepidopteran insects to Cry1Ab toxin is 12 to 23 times that of younger larvae (Hellmich et al., 2001). Therefore, if larvae of too old an age are used in bioassays, the actual lethal activity of the toxin may be underestimated, which is not conducive to timely detection of resistance signs (Dively et al., 2016). Considering the convenience of operation and test sensitivity, the selection of instar II larvae for Bt toxin bioassay is an ideal choice because it is easy to handle and can sensitively reflect the difference in toxicity. 4 Discussion This study successfully established a continuous generation artificial population of Asian corn borer in the laboratory by using corn cob feeding method. The results showed that under controlled conditions such as 26℃, high humidity, and suitable light, fresh corn cobs can effectively meet the growth and development needs of larvae, with a larval survival rate of over 91% and stable population reproduction, indicating that this method is easy to operate, has a healthy insect source, and is suitable for bioassays. This is consistent with the effect of using natural feed to feed corn borers in Wang et al. (2023). Compared with artificial feed, cob feeding depends on the corn planting cycle. If it is in the off-season, it is possible to consider introducing improved semi-artificial feed. Recent studies have shown that an optimized formula based on soybean meal, corn meal and vitamins can achieve efficient artificial feeding of corn borers (Rahayu and Trisyono, 2018; Alam et al., 2024). In addition, the agar-free formula reduces feed costs and avoids the problem of Bt toxin adsorption and inactivation (Wang et al., 2023). In terms of insect age identification, this study uses the width of the head shell as a judgment indicator to clearly divide the five instars, providing a standard for the unification of insect sources for bioassay. The larvae of the second instar are of moderate size, high sensitivity, and easy to operate, making them the ideal insect age for Bt toxin bioassay. Previous studies have shown that as the age increases, the tolerance of corn borers to Bt toxins significantly increases (Hellmich et al., 2001), so the second instar is more convenient to operate and has better sensitivity than the first instar. In terms of Bt toxin bioassay, the second instar larvae have been widely used in resistance monitoring research in my country. For example, Liu et al. (2022) used the second instar larvae to determine the sensitivity of multiple corn borer populations across the country to Cry1Ab, and no resistance mutations were found. Combined with the results of this study, it is recommended to use the second instar larvae as the standard test insect in the establishment of resistance baselines and field resistance monitoring, with a unified toxin concentration and feeding method to improve data consistency. In the future, it is possible to consider using transient gene editing without exogenous DNA to transform breeding strains to reduce insect source differences and further standardize the Bt toxin sensitivity assessment process (Dively et al., 2020). It is recommended that monitoring stations in various regions collect field egg masses and artificially raise them to instar II, use a unified bioassay procedure to establish a national sensitivity database, and combine metabolomics and transcriptomics to explore potential resistance mechanisms, providing scientific support for Bt corn resistance management.
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