Temporal transcriptome analysis provides molecular insights into night-time molting of juvenile mud crab (Scylla paramamosain)

Abstract

Molting is an important physiological trait in crustaceans, through which they can realize metamorphosis, growth, and reproduction. In this study, the diurnal differences in molting of Scylla paramamosain were investigated with a 9-week experiment, and the temporal transcriptome was introduced to investigate the potential roles of circadian clock in entraining the molting rhythm. The results conducted from 6 molting cycles showed that crabs exposed to a simulated natural photoperiod (SNP, 12 L:12D) molted primarily during the nocturnal phase (61.54 % - 82.50 %), which is significantly higher than daytime (26.82 ± 7.20 % vs. 73.18 ± 7.20 %) (P < 0.05). The melatonin and ecdysone content in the haemolymph of crabs reared under SNP conditions exhibited obvious daily rhythms (P < 0.05); phase analysis showed that the melatonin content reached its peak at ZT0/ZT24, and the ecdysone content peaked during the photophase. Temporal transcriptome analysis from crabs exposed to natural photoperiod revealed that 4296 expressed genes (38.34 %) displayed significant daily rhythmicity. GO and KEGG enrichment analysis showed that rhythmic expressed genes were mainly involved in catalytic activity on nucleic acid, ribosome biogenesis, mitochondrial protein-containing complex, protein acetylation, and pathways such as mRNA biogenesis, ribosome biogenesis, spliceosome, DNA repair, and cell cycle. In addition, diurnal expressed genes (peak at ZT0/ZT24-ZT8) mainly included methyltransferase activity, ATP hydrolysis activity, chromatin binding, and protein methyltransferase activity terms. Nocturnal expressed genes (peak at ZT12-ZT20) were primarily involved in spliceosome, arginine biosynthesis, circadian rhythm, amino sugar and nucleotide sugar metabolism, and nitrogen metabolism, which providing insight into the circadian entrainment of crustacean molting. Moreover, the expression of circadian clock-related genes (cry1, cry2, period, clock, and cycle), 20E signaling pathway-related genes (rxr, hr4, ftzf1), and chitin metabolism-related genes (g6pi, gna, pagm, pgm, chit, and nag) displayed significant diurnal oscillations (P < 0.05). Overall, the present research confirmed that the molting of S. paramamosain under natural photoperiod display a daily rhythm, that may be entrained by the rhythmic expression of genes involved in circadian clock, arginine biosynthesis and chitin metabolism pathways.