Expression Variations of Chloride Ion Channel Related Genes During Diapause Termination in Silkworm, Bombyx mori

Instant and refrigerated acid soaking are commonly used in cocoon production to prevent or break diapause, and provide developable silkworm eggs for sericulture, while their mechanisms have not been fully understood. This study aims to investigate the mechanisms by which hydrochloric acid (HCl) or dimethyl sulfoxide (DMSO) promotes embryonic development in silkworm Bombyx mori, focusing on the chloride ion (Cl^-) related gene expression profiles. Our results revealed that the HCl treatment of up to 6 min enhanced hatchability in freshly picked and cold-stored eggs, whereas a slight decrease in hatchability was observed in those treated with DMSO for 40 min. The genes encoding chloride ion channel proteins were cloned and transcriptionally analyzed by quantitative real-time PCR in B. mori 932 strain. Bioinformatics analysis showed that BmCLIC, BmGluCl, and Bmγ-Cl mainly clustered with Lepidopteran insect homologs, while there existed a relatively low conservation from B. mori to mammals. We further explored the mRNA levels of BmCLIC, BmGluCl, and Bmγ-Cl in different egg processing conditions and embryonic developmental stages following HCl or DMSO treatments. Our results revealed that the mRNA expression profiles of BmCLIC, BmGluCl, and Bmγ-Cl increased in 20 h post-egg oviposition and refrigerated eggs after acid soaking. Moreover, the relative expression level of BmCLIC and BmGluCl gene was the highest on the first day, and maximum relative expression level of Bmγ-Cl gene presented on the second day in diapause eggs after treatment, and then it showed diversity. The expression profiles of the candidate genes seem like the same trend of changes during the development of eggs treated with instant acid soaking or DMSO treatment. In general, in the production practice of soaking silkworm eggs with HCl or DMSO, Cl^- channel is likely to play an important subsidiary role to promot the relieving of diapause.