Molecular characterization of TRPA1 and its function in temperature preference in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species, and its growth and development are regulated by temperature, but the molecular mechanisms of the responses to temperature remain unclear. Herein, we identified TRPA1 from E. sinensis, a member of the TRP family of heat receptor potential channels, performed RACE cloning and bioinformatics analysis, and investigated the effect of TRPA1 on temperature responses and molting by real-time PCR and RNA interference (RNAi). The open reading frame of Es-TRPA1 is 3660 bp, and the encoded protein has a molecular weight of 136.91 kDa, and is expressed in embryos and juveniles. RNAi-mediated silencing decreased Es-TRPA1 expression in juvenile crabs, molting rate was decreased, mortality was increased, and crabs avoided cold areas (4 °C) much less than control juvenile crabs. The results suggest that Es-TRPA1 is involved in regulating temperature adaptation and molting processes in E. sinensis. The findings lay a foundation for further exploration of temperature regulation mechanisms in E. sinensis and other crustaceans.

Microinjection-based CRISPR/Cas9 mutagenesis in the decapoda crustaceans Neocaridina heteropoda and Eriocheir sinensis.

CRISPR/Cas9 technology has been applied to many arthropods. However, application of this technology to crustaceans remains limited because of the unique characteristics of embryos. Our group has developed a microinjection system to introduce the CRISPR/Cas9 system into Neocaridina heteropoda embryos (one-cell stage). Using the developed method, we mutated the target gene Nh-scarlet (N. heteropoda scarlet), which functions in eye development and pigmentation. The results showed that both eye color and shape were altered in individuals in which Nh-scarlet was knocked out. Furthermore, this system was also successfully applied to another decapod crustacean, Eriocheir sinensis. DNA sequencing revealed that the zoeae with red eyes had an edited version of Es-scarlet. This study provides a stable microinjection method for freshwater crustaceans, and will contribute to functional genomics studies in various decapods.