Anesthesia serves as an effective method to mitigate the stress response in aquatic animals during aquaculture and product transportation. In this study, we assessed the anesthetic efficacy of clove oil, tricaine methane-sulfonate (MS-222), ethanol, and magnesium chloride by anesthesia duration, recovery time, 24-hour survival rate, and the behavior of mud crabs (Scylla paramamosain). Additionally, the optimal anesthetic concentration for varying body weights of mud crabs was also investigated. The results revealed that clove oil emerged as the optimal anesthetic for mud crabs, with a 24-hour survival rate surpassing those observed in MS-222 and magnesium chloride treatments. Ethanol caused amputation and hyperactivity in mud crabs. Regression analyses between the optimal anesthetic concentration of clove oil and the weight categories of 0.03-27.50 g and 27.50-399.73 g for mud crabs yielded the following equations: y = 0.0036 x3 - 0.1629 x2 + 1.7314 x + 4.085 (R2 = 0.7115) and y = 0.0437 x + 2.9461 (R2 = 0.9549). Clove oil exhibited no significant impact on serum cortisol, glucose, lactate content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, or superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in mud crabs across different treatment groups. Anesthesia induced by clove oil in mud crabs resulted in an increase in inhibitory neurotransmitters such as glycine. However, the recovery from anesthesia was associated with elevated levels of the excitatory neurotransmitters L-aspartic acid and glutamate. In conclusion, clove oil proves to be a safe and optimal anesthetic agent for mud crabs, exerting no physiological stress on the species.

Dietary carbohydrates are anaerobically fermented by gut microbiota to short-chain fatty acids (SCFAs), conferring gut health benefits. Of all tested prebiotics, galactooligosaccharides (GOS) and resistant starch (RS) stimulated the SCFA production in mud crab (Scylla paramamosain), a crustacean model, to a greater extent than the other carbohydrates tested. Using in vitro anaerobic fermentation cultures, this study further explored the prebiotic potential of GOS and RS in mud crab by assessing their impacts on gut microbiota changes and SCFA production. Both GOS and RS significantly promoted SCFA production. Bacterial diversity in the GOS group was lower than in the RS or control group. GOS promoted the growth of Bacteroidetes, while RS promoted Tenericutes. A strong positive correlation was found between SCFA production and bacterial abundance; most bacteria per se correlated with each other. The findings demonstrated the prebiotic potential of GOS and RS in mud crab.

In this study, we investigated the hemocytes\' immune response to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection at the protein level. The differential proteomes from crab hemocytes infected with WSSV or V. alginolyticus were analyzed using the isobaric tags for relative and absolute quantitation approach immediately after infection. Using this approach, we identified 1,799 proteins by their by LC-MS/MS spectra and sequencing data. These included 157 upregulated proteins and 164 downregulated proteins after WSSV infection. Similarly, 243 proteins were determined to be differentially expressed during V. alginolyticus infection, of these, 121 were upregulated and 122 were downregulated after infection. Interestingly, among these differentially expressed proteins, 106 were up- or downregulated significantly in both WSSV and V. alginolyticus infection. Six genes, β-actin, myosin-9, anti-lipopolysaccharide factor isoform 4, anti-lipopolysaccharide factor 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were chosen for further study. The expression of these genes all showed a trend of upregulation at 24 h post-WSSV or V. alginolyticus infection except for myosin-9 in response to WSSV. To confirm the protective effects of the six genes, crabs were injected with specific dsRNAs before WSSV or V. alginolyticus challenge. The results showed that the knockdown of these genes led to an increase in the morbidity and mortality (P < 0.01) rate, and a decrease in infection time in WSSV-infected crabs. During the first 84 h, knockdown of these genes also led to an increase in the morbidity rates in V. alginolyticus -infected crabs, and results of four genes showed a higher mortality rate than that of the control after they were knocked down. This is the first report of the proteome response in crab hemocytes during WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crabs.

Histone H2A is known to participate in host immune defense through generating special antimicrobial peptides (AMPs), for which it has been an interesting research focus to characterize this kind of peptides in vertebrates and invertebrates. Although thousands of AMPs have been reported in variety of life species, only several AMPs are known in crabs and in particular no H2A-derived AMP has yet been reported. In the present study, a 38-amino acid peptide with antimicrobial activity was determined based on the sequence analysis of a histone H2A identified from the mud crab Scylla paramamosain. The histone H2A derived peptide was an AMP-like molecule and designated as Sphistin. Sphistin showed typical features of AMPs such as amphiphilic α-helical second structrue and positive charge net. The synthetic Sphistin exerted high antimicrobial activity against Gram-positive, Gram-negative bacteria and yeast, among which Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas stutzeri are important aquatic pathogens. Leakage of the cell content and disruption of the cell surface were observed in bacterial cells treated with Sphistin using scanning electron microscopy. It was proved that the increasing cytoplasmic membrane permeability of Escherichia coli was caused by Sphistin. Further observation under confocal microscopy showed that Sphistin could combine onto the membrane of Staphylococcus aureus, E. coli MC1061 and Pichia pastoris but not translocate into the cytoplasm. Moreover, the affinity of Sphistin with either LPS or LTA was also testified that there was an interaction between Sphistin and cell membrane. Thus, the antimicrobial mechanism of this peptide likely exerted via adsorption and subsequently permeabilization of the bacterial cell membranes other than penetrating cell membrane. In addition, synthetic Sphistin exhibited no cytotoxicity to primary cultured crab haemolymphs and mammalian cells even at a high concentration of 100 μg/mL for 24 h. This is the first report of a histone-derived Sphistin identified from S. paramamosain with a specific antimicrobial activity and mechanism, which could be a new candidate for future application in aquaculture and veterinary medicine.