Inhibitory effects of RNAi-mediated knockdown of EsDmrt-like gene on testicular development in the Chinese mitten crab Eriocheir sinensis

• The expression level of EsDmrt-like significantly decreased after RNAi.
• Gonadosomatic index of the testis was significantly decreased after RNAi.
• The seminiferous tubules were reduced and spermatogenesis was blocked.
• EsDmrt-like plays a critical role during spermatogenesis and testicular development.

EsDmrt-like is a novel testis-specific Dmrt gene identified from the Chinese mitten crab Eriocheir sinensis. To explore its biological functions, we performed RNA interference (RNAi) by injection of long double-stranded RNA (dsRNA) targeting the coding region of the EsDmrt-like gene. At 24 h post-injection with EsDmrt-like-dsRNA, the relative expression level of EsDmrt-like mRNA significantly decreased by 80% (P < 0.01) compared to negative control of PBS group, while no RNAi effect was detected using a dsRNA of the green fluorescent protein (GFP), indicating target sequence specificity of this interference effect. Although the EsDMRT-like protein could be obviously detected in the PBS group, its expression in the EsDmrt-like-dsRNA group disappeared in four of the five tested individuals. Taken together, the expressions of EsDmrt-like mRNA and its protein were successfully inhibited by RNAi. After extending RNAi treatment for one month, the crab testicular development exhibited inhibitory effects. The average testis size was reduced and the gonadosomatic index (GSI) was significantly decreased by 75% compared with the controls. The histological and immunohistochemical analysis showed that the spermatogenesis was blocked, with rare spermatozoa in the seminiferous tubules, and the immunohistochemical signal in the spermatogonia and spermatocytes became extremely weak in the EsDmrt-like-dsRNA group. These data demonstrated a critical role for EsDmrt-like in testicular development and spermatogenesis in E. sinensis, and in vivo repetitive injection of EsDmrt-like-dsRNA could work efficiently to inhibit male testicular development.Statement of relevanceThe Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture species and has high commercial value as a food source. EsDmrt-like is a novel testis-specific Dmrt gene identified from the E. sinensis. We performed RNAi by repetitive injection of long double-stranded RNA (dsRNA) targeting the coding region of the EsDmrt-like gene, and certified that EsDmrt-like plays a critical role during spermatogenesis and testicular development.