Function of sarco/endoplasmic reticulum Ca²⁺-ATPase in calcium regulation and its effect on molting of Chinese mitten crab (Eriocheir sinensis)

The Chinese mitten crab (Eriocheir sinensis) is a vital freshwater economic crab in China. Its growth is limited by exoskeleton. In the process of growth and development, the exoskeleton must be shed periodically in order to complete its phased growth. A highly complex calcium regulation mechanism is needed in the molting cycle of E. sinensis. Sarco/endoplasmic reticulum Ca²⁺ATPase SERCA (SERCA) transports Ca²⁺ from the cytosol to the sarco/endoplasmic reticulum (SR/ER), where Ca²⁺ is mainly stored in cells. In order to improve the molecular mechanism of molting of E. sinensis, a series of functional analyses of the SERCA gene, the key factor of Ca²⁺ signal transduction, was carried out in this study. The experimental group prepared four kinds of culture water with different Ca²⁺ concentrations by adding analytical pure CaCl₂ (40 mg/L, 160 mg/L, 320 mg/L) to the fresh water, and the control group without CaCl₂. The relative expression of genes were tested using qRT-PCR method. The result showed that the relative expression of genes related to Ca²⁺ signal pathway SERCA, PMCA, RyR and NCX in Y-organs and gill tissues increased, the relative expression of MIH in eyestalks decreased. The concentration of Ca²⁺ and ecdysone in hemolymph increased tested using ELISA method. The results showed that high concentration of Ca²⁺ could promote the absorption of Ca²⁺ from the water environment and transport across cells to hemolymph through the joint regulation of SERCA, PMCA, RyR and NCX in the gills of E. sinensis, thus increasing the concentration of Ca²⁺ in the hemolymph. In Y-organs, the relative expression of genes related to the Ca²⁺ signal pathway was increased, and the Ca²⁺ signal was up-regulated, which was involved in the regulation of intracellular calcium homeostasis. Meanwhile, the MIH gene was down-regulated in the eyestalks. This series of reactions promoted the transformation of E. sinensis to the premolt stage, accompanied by an increase in the concentration of ecdysone in hemolymph. After injecting the ds RNA of SERCA gene to E. sinensis for 24 h, 48 h, 72 h and 96 h, the knock down efficiency of SERCA gene were 65.67 %, 55.5 %, 32.10 % and 15.43 %, respectively, which showed it could effectively knock down the expression of SERCA gene, meaning it can be used in the next experiment. After knocked down of SERCA gene by RNAi and no Ca²⁺ was added to the culture water, and knocked down SERCA gene in high calcium environment (320 mg/L of Ca²⁺ was added to the culture water), the relative expression of PMCA and NCX genes in Y-organs increased, while the relative expression of RyR gene decreased. The concentration of intracellular Ca²⁺ was tested using Fluo-3 AM probe and it was increased. It suggested that the SERCA gene played an important role in maintaining intracellular Ca²⁺ concentration and intracellular calcium homeostasis in E. sinensis. Continuous knock down of SERCA gene expression could significantly prolong (P < 0.05) the molting interval and decrease the weight gain rate and survival rate of E. sinensis. SERCA is the critial factor of Ca²⁺ signal transduction, which not only participates in the regulation of molting, but also plays an important role in many cellular functions, knock down of SERCA gene expression may cause intracellular calcium homeostasis imbalance, interfere with Ca²⁺ signal, and cause adverse effects on other physiological activities, thus reducing the survival rate of E. sinensis. Continuous knock down of the SERCA gene in a high calcium environment resulted in shorter molting interval but aggravated intracellular calcium disorder and decreased survival rate compared with continuous knock down of the SERCA gene group. The results of this experiment will provide an essential reference for the further study of the molting mechanism of E. sinensis. It suggests that SERCA gene plays an important role in molting of E. sinensis. The study will improve the molecular mechanism of molting of E. sinensis, provide basic reference for further study of the molting mechanism and genetic improvement of E. sinensis, and provide theoretical support in the exploration of scientific culture and improving culture efficiency.