Molecular Entomology, 2025, Vol.16, No.1, 11-18 http://emtoscipublisher.com/index.php/me 13 leaves required by larvae of all ages. When feeding natural feed, care should be taken to keep the leaves fresh. The base of the harvested branches and leaves can be inserted into a bottle with water or fresh leaves can be replaced frequently. Young larvae usually gather on the back of the leaves to feed on the surface mesophyll. It is necessary to ensure that the leaves do not dry up to increase their feeding rate. Scientific formulation, high-frequency feeding, and frequent replacement and cleaning are the key to ensuring the high survival rate and normal development of Spodoptera litura larvae. 2.4 Experimental methods 2.4.1 Soybean seed pre-treatment and planting Seed pre-treatment: Soybean seeds stored in refrigeration were activated in a 25°C light incubation box for 2 hours, then transferred to a cool place to cool down (Didorenko et al., 2023). Sandy loam was filled to 2/3 of the height in plastic planting containers, and approximately 15 soybean seeds were evenly sown in each container, with a covering soil thickness approximately equal to the diameter of the seeds, followed by thorough watering. Considering the large food intake of Spodoptera litura larvae, additional soybean plants were planted outdoors for backup: a plot of land was selected, surface weeds were removed, soil was deeply turned, and shallow furrows (5 cm wide, 2 cm deep) were dug at 10 cm intervals. After watering, seeds were sown, and soil was leveled. The seeds sprouted in approximately 4 days. 2.4.2 Larvae rearing and emergence The entire experiment was conducted in an insect rearing room. The temperature was maintained around 25.5°C with a relative humidity of 23%, using natural light plus daylight lamps with a 12-hour light and 12-hour dark cycle. Thirteen pupae were placed in white emergence containers and covered with nylon mesh, with soybean plants placed nearby. Daily observations were made on the color changes of the pupae (from light green to black to dark brown), and the plants were watered every three days to keep them fresh, maintaining a soybean plant fresh weight moisture content of ≥80%. After the adults emerged, 10% honey water (1 mL honey + 9 mL tap water) was prepared and placed in open petri dishes with soaked cotton for nutrition. The honey solution was replaced daily to enhance adult reproductive vitality. The body length of the adults was measured using image analysis software (ImageJ v1.53), with n=20 samples. 2.3.3 Adult reproduction and egg hatching Two days (48 hours) after emergence, the adults began mating and laying eggs. The eggs were mostly laid on the nylon mesh, pot walls, and the surfaces of soybean leaves in clusters, and they hatched in situ without human intervention, allowing natural hatching (Islam et al., 2020). Egg diameter was measured using a micrometer (n=50). 2.4.4 Larvae rearing management The newly hatched larvae (1.02±0.15 mm in body length) are black in color and were transferred to the undersides of soybean leaves using a soft brush or paintbrush. The larvae were continuously observed to ensure they did not fall off or move, and they were promptly returned to the leaf surface if necessary. Rearing density was kept ≤5 larvae per plant (during the 3-4 leaf stage), and the food was replaced daily to ensure sufficient food supply. Large larvae (≥4th instar) require more food, and thus potted or field-grown soybeans were prepared in advance. The larvae were then reared in individual containers (1 larva per container) to reduce density and prevent cannibalism. The body length growth curve was observed and recorded daily. 2.4.5 Pupal treatment After defecating, the mature larvae enter the prepupal stage and should be separated for individual rearing to prevent cannibalism or disturbance during pupation. The larvae were observed for molting, deformation, and color
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