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Female adults of the migratory locust, Locusta migratoria manilensis (Meyen), can sense seasonal photoperiod changes, which induces embryonic diapause as a key strategy to overwinter. Serine protease inhibitor genes (SPNs) were thought to play key roles during diapause, while few SPNs were functionally characterized. LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs; however, its biological function remained to be explored. So, we conducted RNAi knockdown of LmSPN2, resulting in a significant decrease of the egg diapause rate by 29.7%. Using yeast two-hybrid assays, co-immunoprecipitation, and pull-down methods, we found an interaction between LmSPN2 and LmSPN3, which was proved to be mediated by a glutamate (E331) binding site of LmSPN2. RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%, indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation. Double knockdown of two SPN genes resulted in a 26.4% reduction in diapause rate, indicating that LmSPN2 was the dominant regulatory signal. Moreover, we found four Toll pathway genes (easter, spätzle, pelle, and dorsal) upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3. Therefore, we speculate that two SPNs regulate diapause through the Toll pathway. Our results indicated that LmSPN2 positively regulates locust egg entry into diapause, while LmSPN3 is a negative regulator of embryonic commitment to diapause. Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway. This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

The efficacies of biological and conventional chemical insecticides against two major insect pests of alfalfa (aphids and thrips) were compared in three sites across China’s alfalfa belt. In addition, the persistence of the residues of chemical insecticides in alfalfa and their influence on the quality of alfalfa hay were examined. Efficacy varied among the different biological and chemical insecticides. The chemical insecticides were significantly more effective than biopesticides in a short time-frame. The efficacy period of biopesticides was significantly longer than that of chemical insecticides, and the corrected mortality rate of aphids in all regions was above 50% at 14 days after application. The analysis of pesticide residues showed that the residual doses of all the pesticides were within the allowed ranges after the safe period. The acid detergent fiber and neutral detergent fiber contents in alfalfa hay were higher and the protein content was lower in chemical insecticide treatments than in biopesticide treatments in Hebei. The relative feeding value of alfalfa hay treated with Metarhizium anisopliae IPP330189 was the highest among the treatments. Compared with chemical insecticides, the yield of alfalfa hay was higher in the biopesticides treatments. Biopesticides show a stronger control effect on insect populations and also a better improvement in the quality of alfalfa hay than chemical insecticides. This study provides a basis for exploring and developing a comprehensive control regime for alfalfa insect pests in the different alfalfa-growing regions in China, and for reducing chemical insecticide usage and improving forage quality.