A Novel Antimicrobial Peptide Sparanegtin Identified in Scylla paramamosain Showing Antimicrobial Activity and Immunoprotective Role In Vitro and Vivo

The abuse of antibiotics in aquaculture and livestock no doubt has exacerbated the increase in antibiotic-resistant bacteria, which imposes serious threats to animal and human health. The exploration of substitutes for antibiotics from marine animals has become a promising area of research, and antimicrobial peptides (AMPs) are worth investigating and considering as potential alternatives to antibiotics. In the study, we identified a novel AMP gene from the mud crab Scylla paramamosain and named it Sparanegtin. Sparanegtin transcripts were most abundant in the testis of male crabs and significantly expressed with the challenge of lipopolysaccharide (LPS) or Vibrio alginolyticus. The recombinant Sparanegtin (rSparanegtin) was expressed in Escherichia coli and purified. rSparanegtin exhibited activity against Gram-positive and Gram-negative bacteria and had potent binding affinity with several polysaccharides. In addition, rSparanegtin exerted damaging activity on the cell walls and surfaces of P. aeruginosa with rougher and fragmented appearance. Interestingly, although rSparanegtin did not show activity against V. alginolyticus in vitro, it played an immunoprotective role in S. paramamosain and exerted an immunomodulatory effect by modulating several immune-related genes against V. alginolyticus infection through significantly reducing the bacterial load in the gills and hepatopancreas and increasing the survival rate of crabs.

Effects of different temperatures on the growth and survival of mud crab (Scylla paramamosain) larvae

This study aimed to evaluate the effects of temperature on the growth and survival of mud crab (Scylla paramamosain) larvae in two stages including zoea-1 to megalopa (the first experiment) and megalopa to crablet-1 (the second experiment). Each experiment was conducted in a completely randomized design with four temperature levels (27, 30, 33, and 36℃) in triplicate. Stocking density of 200 ind./L for the first experiment and 4 ind./L for the second experiment. Fiberglass tanks of 500-L (containing 300-L and 250-L of 25‰ water for the first and second experiment, respectively) were used in the experiments. The results showed that S. paramamosain larvae at zoea-3 and zoea-4 died at 33℃ or above, while the highest survival rate of this stage was found at 27℃ (11.5%). The growth and larval stage index during the zoea to megalopa stage were significantly higher in 30℃ (p<0.05) compared to those in the 27℃ treatment. In the second experiment, the survival rates of crablet-1 were highest (72.9%) at 27℃ and lowest (34,7%) at 36℃. The growth rate of crablets at this stage increased with increased temperature. The highest carapace width and weight (3.41 mm and 0.030 g, respectively) were recorded at 36℃. The findings of this study suggested that mud crab larvae should be reared at the temperature range of 27-30℃ to maximize their production.

Comparative Genomic and Transcriptomic Analyses of CHHs and Their Putative Receptors in Scylla paramamosain, Portunus trituberculatus, and Eriocheir sinensis

Crustacean hyperglycemic hormone superfamily neuropeptides (CHHs) are typical crustacean eyestalk hormones that include the crustacean hyperglycemic hormone (CHH), moult-inhibiting hormone (MIH), vitellogenesis/gonad-inhibiting hormone (VIH/GIH) and mandibular organ-inhibiting hormone (MOIH), which are divided into two subfamilies: type I CHH (included CHH) and type II CHH (consisting of MIH, VIH/GIH, and MOIH). They are involved in various biological activities, such as metabolism, molting, reproduction, and osmotic regulation. Discovery of the ion transport peptide (ITP) in insects expanded the members of CHHs and revealed that CHHs are not restricted to crustaceans. In this study, we focused on three economically important crabs: the mud crab, Scylla paramamosain, the swimming crab, Portunus trituberculatus, and the Chinese mitten crab, Eriocheir sinensis. Their genomes, Pacbio full-length transcriptomic data as well as comparative RNA-seq data were obtained and used to analyze the genomic structures and expression patterns of CHHs and their putative receptors through bioinformatic methods. Two type I CHH members (CHH1 and CHH2) were identified, of which CHH1 had two splice variants, CHH1-v1 and CHH1-v2. One copy of type II CHH (MIH) was found in P. trituberculatus and E. sinensis. While most decapods, including S. paramamosain, have two copies of type II CHHs (MIH/VIH), these MIH/VIHs are adjacent to each other on the same chromosome. Besides type I and II CHH, ITP-like peptides have also been found in the three crabs, and they are mainly expressed in the eyestalk. Four, five, and three G protein-coupled receptors (GPCRs) were identified in S. paramamosain, P. trituberculatus, and E. sinensis, respectively, which might be putative CHH receptors. These GPCRs were divided into three groups. One group was composed of two contiguous genomic position GPCRs, and they were mainly expressed in the hepatopancreas. These findings provide a basis for further studies on CHHs receptor binding tests and on CHHs/GPCRs signaling pathways.