Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis (Meyen) via the Toll pathway

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.