Changes in molting frequency and expression patterns of molting-related genes in Macrobrachium rosenbergii with exogenous calcium supplement in water

Exogenous calcium supplement in water is essential to crustacean aquaculture. To investigate the effects of exogenous calcium supplement on the molting of Macrobrachium rosenbergii, a culture experiment was carried out with different calcium concentrations in water (25 mg/L as the control group, 85 mg/L, 145 mg/L, and 205 mg/L). We found that the 145 mg/L group had the highest cumulative molting rate among the four groups. Notably, the 205 mg/L group exhibited the lower cumulative molting rate than the control group (P < 0.05), indicating a super high calcium concentration suppressed the molting of M. rosenbergii. Moreover, the 145 mg/L group exhibited higher activities of the molting-associated enzymes (glutamine synthetase, beta-N-Acetylglucosaminidase, and chitinase) at both pre-molt and post-molt stages. In the 145 mg/L group, the exoskeletal exocuticle was denser, the endocuticle was thickened, and the morphology of epithelial cell was columnar compared with the control group. RNA-seq analyses showed that some DEGs were significantly enriched in the lysosomal and calcium signaling pathways. The expression levels of genes, namely sphingomyelin phosphodiesterase (smpd), cathepsin D (cd), cathepsin L (cl), cathepsin B (cb), N-acetyl-beta-D-glucosaminidase (nase), legumain-like protein (llp), in the lysosomal pathway and genes, i.e., calmodulin-like (cam), phosphodiesterase (pde), eadenine nucleotide translocase (pka), calcium uniporter protein (mcu), adenine nucleotide translocase (ant), in the calcium signaling pathway were highest in the 145 mg/L group. Meanwhile, the expression levels of molting-related genes, including retinoid X receptor (rxr), ecdysone receptor (ecr), calcineurin (can), calreticulin (crt), ecdysis triggering hormone receptor (ethr), were significantly elevated in the 145 mg/L group, moreover, the expression of fushitarazu factor 1 (ftz-f1) and molt inhibitory hormone (mih) was inhibited. Notably, in 205 mg/L group, the expression of Na+/K+ ATPase alpha subunit (atpa) was suppressed, and the molting was impacted in M. rosenbergii. Overall, moderate calcium supplement in water could promote molting and exoskeleton mineralization of M. rosenbergii. This study provided a theoretical basis of exogenous calcium supplement in crustacean aquaculture.