Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

Salinity is a pivotal environmental factor that governs crustacean survival and development through its regulatory effects on key physiological processes, including osmoregulation and metabolic homeostasis. In the mud crab Scylla paramamosain, salinity tolerance of the megalopa plays an important role in larval survival rates and aquaculture yield. Here, we employed a combined transcriptomic and proteomic strategy to comprehensively dissect the molecular adaptive mechanisms of S. paramamosain megalopa exposed to acute and prolonged low-salinity stress (8‰) compared to control condition (17‰). Illumina-based transcriptome sequencing generated 81.71 Gb of high-quality clean data, which were assembled into 42,210 unigenes. LC-MS/MS-based proteomic profiling identified 51,390 unique peptides, corresponding to 5909 confidently quantified proteins. Transcriptomic profiling identified 2627 differentially expressed genes (DEGs) under acute low-salinity stress, comprising 1332 upregulated and 1295 downregulated genes compared to the control group. In contrast, a total of 733 DEGs were identified under prolonged low-salinity exposure, including 390 upregulated and 343 downregulated genes. Parallel proteomic analysis identified 199 differentially expressed proteins (DEPs) in the acute stress group, with 105 upregulated and 94 downregulated relative to the control group. Under prolonged stress, 206 DEPs were detected, including 124 upregulated and 82 downregulated proteins compared to the control group. Significant GO term and KEGG pathway enrichments contained metal ion binding, oxidoreductase activity, nucleus, apoptotic process, innate immune response, and amino acid metabolism, suggesting that megalopa employ coordinated regulatory mechanisms involving metabolic reprogramming, immunity system modulation, ion homeostasis maintenance and cell cycle regulation to adapt to hypoosmotic stress. Integrated multi-omics analysis identified 17 genes displaying significant concordant differential expression at both mRNA and protein levels during acute hypoosmotic stress, versus only 5 gene-protein pairs during prolonged stress exposure, indicating extensive post-transcriptional regulation and protein turnover mechanisms in sustained hypoosmotic condition. To the best of our knowledge, this study established the first integrative transcriptome-proteome framework elucidating hypoosmotic adaptation (8‰) mechanisms in S. paramamosain megalopa. The identified molecular signatures offer actionable targets for selective breeding of salinity-tolerant strains and precision management of megalopa culture under suboptimal salinity condition, while fundamentally advancing our mechanistic understanding of osmoregulatory plasticity across decapod crustaceans.

The feeding rhythm is one of the key factors determining the success of artificial breeding of S. paramamosain. To understand the feeding rhythm of the different zoea larva developmental stages of S. paramamosain, the feeding rate, digestive enzyme activity, and expression of metabolism-related genes were investigated in the present study. The results showed that the S. paramamosain feeding rate has strong diurnal feeding rhythm, being significantly higher at 10:00–14:00 from stages ZI to ZIV. While the feeding rate peaked at 14:00 on Days 10 and 11, the peak shifted to 18:00 on Day 12. The activity of digestive enzymes amylase, pepsin and lipase decreased at night but increased in the daytime, showing a single-phase rhythm similar to that of the feeding rate, suggesting that the digestive enzyme activity was closely associated with the feeding rate during the larval development. Compared to pepsin and lipase, the activity of amylase was the most consistent with feeding rate. In particular, amylase activity peaked at 18:00 on Day 12. Due to its synchronicity with feeding activity, the activity of amylase could provide a potential reference for determining the best feeding time during zoea stages in S. paramamosain breeding. Moreover, the relative mRNA expression of metabolism-related genes SpCHH and SpFAS at most tested points was lower from 10:00 to 14:00, but higher at 18:00 to 6:00 of the next day. On the other hand, the expression patterns of SpHSL and SpTryp were converse to those of SpCHH and SpFAS. Our findings revealed that the S. paramamosain zoea has an obvious feeding rhythm, and the most suitable feeding time was 10:00–18:00 depending on different stages. The feeding rhythm is a critical aspect in aquaculture, influencing a series of physiological functions in aquatic animals. This study provides insights into the feeding rhythm during the zoea development of S. paramamosain, making a significant contribution to optimizing feeding strategy, improving aquafeed utilization, and reducing the impact of residual feed on water environment.

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, methoprene-tolerant (Met) of Scylla paramamosain was obtained and characterized, which we named Sp-Met. Sp-Met contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of Sp-Met was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in Sp-Met and Sp-Kr-h1 was consistent with the immersion effect of MF. The most interesting finding is that knockdown of Sp-Met blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of Sp-Met significantly reduced the expression of Sp-Kr-h1 and two ecdysone signaling genes, Sp-EcR and Sp-E93. However, only the reduction in Sp-Kr-h1 could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→Kr-h1 signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects.

Bait feeding plays a pivotal role in the artificial nursery process, as different baits can significantly influence water quality and microbial community structure, thereby directly affecting the growth and development of cultured organisms. In this study, four types of baits, including live Artemia nauplii (AN), frozen copepods (FC), frozen Artemia nauplii (FA), and formulated diet (FD) were used to feed megalopa-stage mud crabs (Scylla paramamosain) until their metamorphosis into the first juveniles. The effects on water quality, microbial community structure, metamorphosis survival rate, and molting duration were statistically analyzed. Results indicated that the AN group had 88 unique bacteria OTUs, followed by 80, 64 and 27 unique OTUs in the FA, FD and FC groups, respectively. Proteobacteria predominated in both AN and FA groups, while Bacteroidetes demonstrated the highest percentage in the FC and FD groups. Actinobacteria maintained consistently low relative abundance in all treatments. The FA group exhibited the highest Chao1, ACE and observed richness indices (P > 0.05), suggesting the greatest bacterial community richness. The AN group displayed the highest Pielou’s evenness and Shannon index, along with the lowest Simpson′ index (D) (P < 0.05), indicating a more uniform species distribution and higher community diversity. In addition, the AN group exhibited the lowest concentrations of ammonia nitrogen and nitrite among the four treatments. Moreover, the AN group showed the highest abundance of the beneficial genus Marivita, whereas the FD, FC, and FA groups had a significant increase in the harmful genus Tenacibaculum. Compared with those in the FA, FC and FD groups, the megalopae in the AN group also demonstrated the highest metamorphosis survival rate and the shorter molting duration, underscoring the critical role of live Artemia nauplii in improving water quality, and stabilizing microbial communities. These findings suggested that live Artemia nauplii were the optimal feed choice for enhancing the success of S. paramamosain megalopa breeding. Future studies could explore the integration of appending probiotics and periodic removal of harmful bacteria in the aquaculture water to further optimize microbial structure, thereby improving the metamorphosis survival rate of S. paramamosain megalopa. Moreover, strategies such as partial substitution of live Artemia nauplii with frozen Artemia could be investigated to reduce seedling cultivation costs and advance S. paramamosain aquaculture technology in the further research.