Identification and expression pattern of three sex-related genes in the shrimp Neocaridina denticulata sinensis (Decapoda, Caridea)

Abstract

Abstract The sex determination and differentiation process of economically important crustaceans have been regarded as the focus of aquaculture for a long time, because of the sex-related weight differences. Neocaridina denticulata sinensis makes a suitable animal model for studying crustaceans because it can reproduce many times under artificial control and has a short reproductive cycle. Male and female sex characteristics of the adult rice shrimp Neocaridina d. sinensis are morphologically obvious, but not in embryos and juvenile stages. At present, sex-specific DNA markers have not yet been developed. To produce a reliable molecular marker for sex in Neocaridina and to investigate molecular sex differentiation, we therefore focused on identifying sex-specific transcriptomic differences. In this study, we found three sex-specific expression genes, NDM, Sushi, and NDF, after screening a large number of transcriptome data. NDM and Sushi are male-specific expression genes, and NDF is a female-specific expression gene. Semi-quantitative RT-PCR analysis showed that NDM and NDF can act as molecular markers for the sex identification of Neocaridina in different developmental stages, especially sex identification for embryos and juveniles with the same morphological characteristics. However, Sushi can only act as a molecular marker for the sex identification of Neocaridina in adult stages. Furthermore, in situ hybridization showed that a strong positive signal of NDM was detected in the male testis. At the same time, we explored the relationship between these three genes and sex differentiation. The results of RNA interference treatment show that knockdown of nd-IAG (Neocaridina denticulata sinensis insulin-like androgenic gland hormone) can change the expression of NDM and NDF. On the basis of the expression of the male-specific gene NDM and the female-specific gene NDF, we developed a molecular test that for the first time allows the unambiguous sex determination of Neocaridina samples lacking external sex-specific features from juvenile stages onward.