BmCHSA-2b, a Lepidoptera specific alternative splicing variant of epidermal chitin synthase, is required for pupal wing development in Bombyx mori

- BmCHSA-2a and BmCHSA-2b were regulated by different promoters of BmCHSA, respectively.
- BmCHSA-2b was induced by high dose of 20E, while BmCHSA-2a expression was inhibited by 20E.
- BmCHSA-2b was up-regulated in mid-pupal wings; BmCHSA-2a was in the beginning and the end of pupa.
- RNAi of BmCHSA-2b resulted in abnormal wings and the decrease of wing area and chitin content.
- All data together indicate that BmCHSA-2b plays the role in the mid-wing development.

Insect chitin synthase A (CHSA) is an epidermis-specific enzyme that plays an essential role in insect development. In this study, the function and regulation of CHSA-2b, an alternative splicing variant of Bombxy mori CHSA that is discovered only in Lepidopteran insects, were investigated. Analysis of mRNA level showed that BmCHSA-2b was responsive to 20-hydroxyecdysone (20E) in pupal wing unlike BmCHSA-2a, which shares almost the identical sequence as BmCHSA-2b except the first 31 amino acids, suggesting that the expression of these two alternative splicing variants is driven by different promoters of CHSA gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that BmCHSA-2b was up-regulated in the wing of mid-pupa unlike BmCHSA-2a, which was up-regulated in epidermis and wing disc at the beginning and end of pupal stage. Further analysis reveals that the up-regulations of BmCHSA-2a and BmCHSA-2b in pupal wing were consistent with the increase of chitin content and wing area at the same stages, respectively. Furthermore, the higher transcription level of BmCHSA-2b in the mid-pupal wing of male than that in female was consistent with the chitin content of pupal wing between genders. Injection of double-stranded RNAs of BmCHSA-2b resulted in the decrease in the area and chitin content of the wing, and irregular and crimpled vein. All these results together suggest that B. mori evolves an extra promoter in CHSA gene to activate BmCHSA-2b expression in the wing of mid-pupal stage in response to 20E, and BmCHSA-2b is required for the wing development in the mid-pupa of B. mori.

When the pupa grows from the initiation to the end of pupa, 20E titer increases from basal level, reaches the peak in the mid-pupa and then drops down to basal line. The decline of 20E titer at the beginning and the end of pupa enhances the transcription of BmCHSA-2a, which expression is driven by the promoter P1 of BmCHSA, but high 20E titer in the mid-pupa inhibits the transcription of BmCHSA-2a. At the same time, the promoter P2 of BmCHSA in responding to high titer of 20E activates BmCHSA-2b expression. Up-regulation of BmCHSA corresponds with the pupal-wing enlargement. Therefore, the different responses of the promoters to 20E make the up-regulation of alterative splicing variants BmCHSA-2a and BmCHSA-2b at different development stages of pupa to ensure the growth of the pupal wing. Lepidoptera-specific BmCHSA-2b is responsible for mid-pupal wing growth.127