Molecular Entomology, 2025, Vol.16, No.1, 19-27 http://emtoscipublisher.com/index.php/me 22 2.4.2 Larval age identification method In order to establish a standard for judging larval age by morphological indicators, samples of corn borer larvae of various ages were collected during the feeding process (especially after the larvae molted). The head of the larvae was observed under a dissecting microscope, and the width of the head shell was measured as the main basis for age determination. At the same time, the body length of the larvae was measured (the straight length from the head to the tail end). Multiple larvae were measured to obtain the range of head shell width and body length for each age. Since corn borer larvae stop feeding and the head shell color darkens at the moment of molting, behavioral and morphological characteristics can be combined to assist in judging the age. The age boundaries were divided by statistically analyzing the head shell width data, and compared with the standards reported in the literature for verification (Ng et al., 1993; Gardner et al., 2001; Pintilie et al., 2023). 2.4.3 Field feeding site survey Plots were set up in maize fields in Sanya to observe and record the damaged sites of larvae of different ages under natural field conditions. Field plots without pesticide application were selected to regularly collect several different parts of corn heart leaves, tassels, female ears and stems, and record the age of corn borer larvae found in them. By dissecting the damaged parts of the plants, the feeding location preferences of larvae of different ages were determined, such as heart leaves, leaf sheaths, filaments, grains or stem pith, etc. (Guo et al., 2018; Zastempowski et al., 2024). This field survey is used to assist the laboratory in analyzing the relationship between larval age and feeding behavior, and to provide a basis for screening suitable insect ages for bioassay. 2.4.4 Bt toxin bioassay method The focus of this study is to screen insect ages suitable for Bt toxin sensitivity determination, so the Bt toxin dose-effect bioassay was not directly implemented in the experiment. In existing studies, the commonly used Bt toxin bioassay uses the artificial feed poisoning method: the purified Bt crystal toxin is added to the semi-artificial feed without agar solidification in a gradient concentration series, and the first-instar larvae of corn borer that have been newly hatched for less than 12 hours are inoculated on the surface of the poisoned feed. After 7 days of feeding under appropriate conditions, the mortality rate is counted to calculate the toxin concentration that mediates 50% mortality (LC50) (Farhan et al., 2023; Smith and Farhan, 2023). This method has been used to monitor the sensitivity baseline of corn borer field populations in different regions of China to toxic proteins such as Cry1Ab and Cry1F (Rahayu and Trisyono, 2018). This study will analyze the selection of appropriate insect age for bioassay in the discussion combined with the experimental results. 3 Results and Analysis 3.1 Laboratory rearing generation record of Asian corn borer Under constant temperature of 26 ℃, it takes about 34~37 days for Asian corn borer to complete a generation cycle (egg-larva-pupa-adult-egg) (Figure 4). There are slight differences in the development period of male and female individuals: females take about 36.2±0.72 days to complete a generation on average, and males take 34.8 ±0.72 days on average. There is little difference between the two sexes in the length of the larval and pupal stages, but male pupae emerge slightly earlier than female pupae. This data is basically consistent with the results of 27~34 days obtained by Rahayu et al. (2018) under artificial rearing conditions. The width of the head shell of corn borer larvae at different ages is significantly different and there is no overlap, which can be used as a reliable basis for age judgment. The entire feeding period of the larvae is generally about 17~19 days, during which they undergo 4 molting. Molting usually occurs during feeding intervals and is accompanied by behavioral changes (such as stopping feeding, spinning silk to fix the body, etc.). 3.2 Division of larval age by morphological characteristics By measuring the length of the abdominal tube and the width of the head shell, the experiment divided the larvae of the Asian corn borer into five age stages (Figure 5). The head shell width and body length range of the larvae of each age were measured in this experiment as follows: Instar larvae: body length is about 3.0~8.2 mm, head shell width is about 0.50~0.82 mm. The body color is milky white, the head is light yellow-brown, and there are scattered small black spots.
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