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Background: Evolutionary adaptation drives organismal adjustments to environmental pressures, exemplified in the diverse morphological and ecological adaptations seen in Decapoda crustaceans, particularly brachyuran crabs. Crabs thrive in diverse ecosystems, from coral reefs to hydrothermal vents and terrestrial habitats. Despite their ecological importance, the genetic mechanisms underpinning their developmental processes, reproductive strategies, and nutrient acquisition remain poorly understood. Results: Here, we report a comprehensive genomic analysis of the green mud crab Scylla paramamosain using ultralong sequencing technologies, achieving a high-quality chromosome-level assembly. The refined 1.21 Gb genome, with an impressive contig N50 of 11.45 Mb, offers a valuable genomic resource. The genome exhibits 33,662 protein-coding genes, enriched in various pathways related to development and environmental adaptation. Gene family analysis shows expansion in development-related pathways and contraction in metabolic pathways, indicating niche adaptations. Notably, investigation into Hox gene regulation sheds light on their role in pleopod development, with the Abd-A gene identified as a linchpin. Post-transcriptional regulation involving novel-miR1317 negatively regulates Abd-A levels. Furthermore, the potential role of fru gene in ovarian development and the identification of novel-miR35 as a regulator of Spfru2 add complexity to gene regulatory networks. Comparative functional analysis across Decapoda species reveals neo-functionalization of the elovl6 gene in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFA), suggesting its importance in environmental adaptation. Conclusions: Our findings shed light on various aspects of crab biology, including genome sequencing, assembly, and annotation, as well as gene family expansion, contraction, and regulatory mechanisms governing crucial developmental processes such as metamorphosis, reproductive strategies, and fatty acid metabolism. [ABSTRACT FROM AUTHOR]

Understanding the nutrition of crabs has a key role in ensuring the success and sustainability of their culture, as providing a well‐balanced, cost‐effective and sustainable diet that ensures the survival, growth and health of crabs is crucial. The present review is the first to focus primarily on the current state of knowledge of the nutrient requirements and related nutritional aspects in farmed crab species. The most common farmed and studied crabs are the Chinese mitten crab (Eriocheir sinensis Milne‐Edwards, 1853), giant mud crab (Scylla serrata Forsskal, 1775), green mud crab (Scylla paramamosain Estampador, 1949) and swimming crab (Portunus trituberculatus Miers, 1876). The article reviews how levels of dietary protein and lipid, the two most important and expensive macronutrient ingredients for most marine animals, directly affect reproduction, growth performance and survival of crabs, and the important impacts they have on immune response and antioxidant capacity. Furthermore, essential amino acids, and essential fatty acids, especially the long‐chain polyunsaturated fatty acids, as well as key lipid classes such as cholesterol and phospholipid will be discussed in terms of dietary requirements. Nutrient digestibility is a crucial method to determine protein quality, and studies on this topic in crabs were covered. The replacement of fishmeal and fish oil, as the predominant ingredients traditionally used in aquafeeds for marine animals, with more sustainable alternatives in diet formulations for crabs are also discussed. Modern 'omics' studies and high‐throughput technologies as fast‐growing approaches in protein and lipid research are also covered. Crabs generally require approximately 35%–50% protein, 5%–10% lipid, ~2.5% arginine, ~2.5% lysine, 1.5%–2.5% phenylalanine, 2.2% leucine, 0.7% tryptophan, 0.7% taurine, 1%–2% each of eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid, 1% cholesterol and ~2% phospholipid in their diets. [ABSTRACT FROM AUTHOR]

Soft-shell crabs have attracted consumers' attention due to their unique taste and nutritional value. To evaluate the feasibility of harvest optimisation of soft-shell mud crabs, the proximate composition, mineral composition, and total carotenoid, amino acid, and fatty acid contents of edible parts of male and female soft-shell mud crabs at different moulting stages were determined and compared from a nutritional value perspective. The results showed that the sex and moulting stages could significantly affect the nutritional values of the edible portions of soft-shell crabs. The female or male soft-shell crabs in the postmoult Ⅰ stage had a much richer mineral element content than that in other moulting stages. The total carotenoid content in female soft-shell crabs was significantly higher than that in male crabs in all moulting stages, while male soft-shell crabs had better performance in amino acid nutrition than female soft-shell crabs. Moreover, it was found that soft-shell crabs in the postmoult Ⅱ stage had significantly higher contents of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), while significantly lower contents of saturated fatty acids (SFA) than those in other stages. The present study will provide a reference basis for the diversified cultivation of soft-shell crabs, and further promote the development of the mud crab industry. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is an importantly economical mariculture crab. To figure out the optimized conditions in the culture of juvenile crabs, three experiments were conducted to examine the effects of diet, salinity and light condition on the growth performance (body weight gain, BWG; carapace width gain, CWG; carapace length gain, CLG) and moulting cycle (MC) of crablets from V to VII stages. The growth performances of the crablets from stages V–VII have no significant differences after feeding different diets. However, the MC of crablet V fed Artemia or mixed fish were significantly shorter than that fed artificial diet. The CLG of crablet VI in salinity 25‰ was significantly higher than that in salinity 15‰, while the CWG and CLG of crablet VII in salinity 15‰ were significantly higher than that in salinity 5‰. The MC of the crablets V–VI reared in salinity 25‰ and 15‰ were significantly shorter than that in salinity 5‰, while it was significantly shorter in salinity 25‰ than that in salinity 15‰ from crablets VI–VII. For light conditions, the BWG of crablet VI with full cover was significantly higher than that with half cover. Whereas, the MC of the crablets V and VI with full cover was significantly shorter than that without cover or half cover. These findings indicated that crablet V and VI may prefer to exposing to 25‰ salinity and dark environment and feeding on Artemia for faster growth. Thus, the current findings may serve as a guide for establishing appropriate conditions in the aquaculture of juvenile mud crabs. [ABSTRACT FROM AUTHOR]

This study focused on the effect of common natural diets, scadfish and squid on the growth performance, morphology, histology and fatty acids of ovary and hepatopancreas of female orange mud crabs, Scylla olivacea. Twenty individuals of newly mature crabs (body weight = 69.21 ± 2.19 g, carapace width = 7.18 ± 0.07 cm) with 10 individuals per treatment were cultured for 60 days with ambient water parameters. Twenty individuals of control crabs were also collected from the wild. The highest body weight gain (BWG), carapace width gain (CWG), gonadosomatic index (GSI) as well as oocyte and tubule diameter were recorded during the captivity, with the mud crabs fed with scadfish leading (BWG = 36.58%, CWG = 14.90%, GSI = 0.47%, oocyte diameter = 47.19 μm, tubule diameter = 167.82 μm). Likewise, high concentrations of ∑PUFA, ∑EPA, ∑DHA and ∑n3 were reported in scadfish‐fed crabs compared to both squid‐fed and wild‐caught crabs (p < 0.05). In contrast, lower fatty acids were noted in the hepatopancreas of crabs from all treatments, suggesting an occurrence of nutrient transfer from the hepatopancreas to the ovaries. Overall results concluded that scadfish is preferred as the main diet for crabs in captivity. [ABSTRACT FROM AUTHOR]

The influence of temperature on sex determination has been reported previously on many crustaceans. The interpretability of temperature capability to regulate the steroidogenic pathway, which in turn governs the offspring sex of mud crab, still remains unclear. Thus, the aim was to determine the effects of temperature on sex percentage and steroidal pathway in purple mud crab, Scylla tranquebarica. In sex percentage analysis, the eggs (28°C and 30°C), larvae (28°C, 30°C and 32°C) and juveniles (26°C, 28°C, 30°C) were exposed to different temperatures. The results revealed that exposure to a higher temperature during larvae (32°C) and juvenile (30°C) stages probably initiated the maleness (58.7%) and femaleness (60.8%) mechanism respectively. In steroid hormone analysis, the eggs (26°C, 28°C, 30°C and 32°C), larvae (29°C, 30°C, 31°C and 32°C) and juveniles (26°C, 28°C, 30°C and 32°C) were exposed to different temperatures. The steroidal assay corroborated the results of the sex percentages, which found higher dihydrotestosterone (172.4 pg/g) and testosterone (580.2 pg/g) levels when the larvae were incubated at a higher temperature (32°C) while higher estrone (136.9 pg/g) and estradiol (540.7 pg/g) levels when the juveniles were reared at high temperature (30°C). In brief, the temperature plays a significant role to redirect the mud crab sex during the larval and juvenile stages. [ABSTRACT FROM AUTHOR]

Simple Summary: The eyestalk is a key organ in crustaceans that produces neurohormones and regulates a range of physiological functions. Eyestalk displacement was discovered in some first-generation (F1) offspring of the novel interspecific hybrid crab (Scylla serrata ♀ × S. paramamosain ♂). To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, high-quality transcriptome was reconstructed using single-molecule real-time (SMRT) sequencing. A total of 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in hybrid crabs with displaced eyestalks (DH). The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant gene ontology (GO) terms were related to the cuticle or chitin. Overall, this study highlights the underlying genetic mechanisms of eyestalk displacement and provide useful knowledge for mud crab (Scylla spp.) crossbreeding. The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring's eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding. [ABSTRACT FROM AUTHOR]

The main focus of the study was to assess the effect of lipid levels on the reproduction of the female orange mud crab, Scylla olivacea. Four isonitrogenous feed (~420 g/kg) were formulated with different lipid percentages: T 1 = 60 g / kg , T 2 = 80 g / kg , T 3 = 100 g / kg , and T 4 = 120 g / kg. A feeding trial was carried out on 120 matured female mud crabs for 90 days with 30 replicates in each treatment. Based on the morphological, histological, and hormonal analysis of each crab, the performances of each experimental diet were tested. The results of the experiment disclosed positive body weight gain (BWG) and specific growth rate (SGR), with the highest value recorded in crabs that were fed T4 (BWG: 14.43 ± 1.14 g ; SGR: 0.10 ± 0.01 % da y − 1 ). The highest gonado-somatic index (GSI) and hepato-somatic index (HSI) were noted in mud crabs that were fed T4, each with a value of 14.51 ± 1.13 % and 5.23 ± 0.55 % , respectively (P < 0.05). The histological assessment revealed that the highest oocyte diameter was recorded in mud crabs fed T4 (192.80 ± 21.93 μm), where the oocyte diameter was correlated with the progesterone (Pg) concentrations in the ovary with r = 0.73. Evidence also indicated that Pg and estradiol (E2) are important in the ovarian development in S. olivacea and their concentrations were affected by the lipid levels. An overall analysis revealed that 120 g/kg of lipid in the diet contributes to optimal reproductive performance in Scylla olivacea. Yet, a further experiment is expected to be carried out in the future to determine the nutrient-dose response as 120 g/kg lipid may not be the optimum lipid level for reproductive and growth performance in S. olivacea. Moreover, the extrapolation of data from this study can be used as guidelines in feed formulation for developments in future research in adult crustaceans. [ABSTRACT FROM AUTHOR]

Interest in the production of crustacean aquaculture has focussed primarily on mud crab species; however, mud crab production still relies on wild‐caught juveniles due to the low production of viable seeds of mud crab. Thus, the aim of this study was to enhance mass propagation of purple mud crab seed by studying the relation of temperature on growth performance of the early life stage of purple mud crab. Several data such as survival, incubation duration and morphometric measurements were evaluated after temperature was exposed on the egg, larval and crablet stages. For the egg stage, high hatching success (78.95% and 75.00%) was noted within a limited temperature range (28°C and 30°C respectively). Also, significant increment of final egg diameter (0.33 ± 0.05 mm) after the berried crab incubated at 30℃. For larval stage, the highest survival rate was found when the larvae were reared at 30℃ and 31°C with 68.79 ± 2.69 and 66.98 ± 2.63% respectively. The largest morphometric measurements for megalopa (CW: 2.80 ± 0.15 mm) and C1 (CW: 2.86 ± 0.12 mm) were observed at 31℃. The yielded total juvenile at the end of the larval experiment indicated the highest production was obtained for temperature 31°C (1,095 ± 47.3 individuals) followed by 30°C (1,002 ± 84.7 individuals). For the crablet stage, the crablets were able to survive in a wide range of temperatures (26°C–32°C) with >95% survival; however, the temperature 26℃–29°C indicated larger morphometric measurements (CW: 6.24–6.39 mm). In brief, the optimal temperature for egg, larval and crablet stages was recommended within 28℃–30℃, 30℃–31℃ and 26℃–29℃ respectively. [ABSTRACT FROM AUTHOR]

Keywords: egg; juvenile; larva; mud crab; temperature EN egg juvenile larva mud crab temperature 707 713 7 01/07/22 20220201 NES 220201 INTRODUCTION A series of morphological variations started in early juvenile as an adaptability process to ensure the capability of mating and reproducing during adulthood (Parvizi et al., 2017). If sexual dimorphism occurred, the line between male and female is widening with the course of juvenile growth gl Copulatory organs, chelae, and abdomen can be used to differentiate between male and female juveniles. The traits of large chela in male crabs are considered to be evolved due to intra-sexual selection, in which the male crabs fight aggressively to access the female crabs (Yoshino et al., 2011). Most findings of brachyuran crabs applied the abdominal width as secondary characters to define the sexual maturity of female crabs (Waiho et al., 2016a; 2016b). [Extracted from the article]

This experiment aimed to evaluate the efficiency of formulated feeds with different lipid levels (T1 = 60 g/kg, T2 = 80 g/kg, T3 = 100 g/kg and T4 = 120 g/kg) in the biochemical composition of female orange mud crab, Scylla olivacea, during the fattening period. Experiments were conducted on 120 crabs with BW and CW of 186.42 ± 37.90 g and 10.24 ± 0.66 cm, respectively, for 90 days. The crabs were reared in tanks, and dissections were performed in 10 individuals per treatment every 30 days. Overall assessments disclosed that mud crabs fed the T4 showed the highest BWG (1.80 ± 0.78 g, 13.78 ± 1.81 g and 14.51 ± 3.81 g), SGR (0.03 ± 0.02%day−1, 0.19 ± 0.01%day−1 and 0.28 ± 0.02%day−1), GSI (9.90 ± 1.05%, 11.31 ± 1.68% and 17.30 ± 2.13%) and HSI (4.17 ± 0.52%, 5.22 ± 0.40% and 5.06 ± 0.45%) during fattening periods. Meanwhile, EPA and DHA were the most important fatty acids required for maturing ovaries in mud crabs. During 60‐ and 90‐day culture period, higher EPA and DHA were noted in the ovary of mud crabs fed with T4 (EPA: 4.79 mg/g and 6.69 mg/g; DHA: 4.55 mg/g and 6.85 mg/g) compared with hepatopancreas (EPA: 1.84mg/g and 2.47 mg/g; DHA: 3.28 mg/g and 3.71 mg/g) (p <.05). The low fatty acid composition in the hepatopancreas indicates a nutrient transfer from the hepatopancreas to the ovaries for maturation. Two‐way ANOVA suggested that 120 g/kg of feed with 60–90 days of duration would be the best for crab rearing. [ABSTRACT FROM AUTHOR]

Ovarian development in animals is a complicated biological process, requiring the simultaneous coordination among various genes and pathways. To understand the dynamic changes and molecular regulatory mechanisms of ovarian development in mud crab (Scylla paramamosain), both histological observation and whole transcriptome sequencing of ovarian tissues at different mating stages were implemented in this study. The histological results revealed that ovarian development was delayed in unmated females (60 days after courtship behavior but not mating), who exhibited an oocyte diameter of 56.38 ± 15.17 μm. Conversely, mated females exhibited accelerated the ovarian maturation process, with females reaching ovarian stage III (proliferative stage) 23 days after mating and attained an average oocyte diameter of 132.19 ± 15.07 μm. Thus, mating process is essential in promoting the rapid ovarian development in mud crab. Based on the whole transcriptome sequencing analysis, a total of 518 mRNAs, 1502 lncRNAs, 18 circRNAs and 151 miRNAs were identified to be differentially expressed between ovarian tissues at different mating stages. Notably, six differentially expressed genes (DEGs) associated with ovarian development were identified, including ovary development-related protein , red pigment concentrating hormone receptor , G2/mitotic-specific cyclin-B3-like , lutropin-chorio gonadotropic hormone receptor , renin receptor , and SoxB2. More importantly, both DEGs and targets of differentially expressed non-coding RNAs (DEncRNAs) were enriched in renin-angiotensin system, TGF-β signaling, cell adhesion molecules, MAPK signaling pathway, and ECM-receptor interaction, suggesting that these pathways may play significant roles in the ovarian development of mud crabs. Moreover, competition endogenous RNA (ceRNA) networks were constructed while mRNAs were differentially expressed between mating stages were involved in Gene Ontology (GO) biological processes such as developmental process, reproduction, and growth. These findings could provide solid foundations for the future development of female mud crab maturation enhancement strategy, and improve the understanding of the ovarian maturation process in crustaceans. [Display omitted] • Mating process promotes rapid ovarian development in S. paramamosain. • Six genes are identified to be connected with ovarian development. • Pathways related to cell proliferation, differentiation, and migration are highlighted. • Competition endogenous RNA networks at different mating stages are constructed. [ABSTRACT FROM AUTHOR]

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans. Author summary: Exosomes are small membrane vesicles of endocytic origin which are widely involved in the regulation of a variety of pathological processes in mammals. Yet, although the antibacterial function of exosomes has been discovered for many years, the relationship between exosomes and hemolymph microbiota homeostasis remains unknown. In the present study, we identified the miRNAs packaged by exosomes that were possibly involved in Vibrio parahaemolyticus infection by modulating hemolymph microbiota homeostasis in crustacean mud crab Scylla paramamosain. Moreover, it was found that miR-224 was densely packaged in exosomes after Vibrio parahaemolyticus challenge, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex in recipient hemocytes, then the released TRAF6 was further interacted with Ecsit to regulate ROS and ALFs levels, which eventually affected hemolymph microbiota homeostasis to cope with pathogenic bacteria infection. Our finding is the first to reveal the relationship between exosomes and hemolymph microbiota homeostasis in animals, which shows a novel molecular mechanism of invertebrate resistance to pathogenic microbial infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying sex differentiation and gonad development remain elusive in crustaceans. Here, we report the cloning and characterization of a Sry-related high-mobility group family B2 gene (SpSoxB2) from an economic crustacean species, the mud crab Scylla paramamosain. The SpSoxB2 encodes a putative HMG-box protein (317 amino acids) clustered with invertebrate Sox14 homologues in the SoxB2 subgroup, but shared low similarity with SpSox14. SpSoxB2 was expressed in the hepatopancreas, thoracic ganglion, and gonads (relatively higher expression in testis than ovary). In hepatopancreas, SpSoxB2 expression was not significantly affected by sex or gonadal development stages. In testis, it was demonstrated that the SpSoxB2 expression level increased from Stage I to Stage III and then decreased afterwards. During ovarian development, the SpSoxB2 expression level continually increased from Stage I to Stage V. Furthermore, fluorescence in-situ hybridization showed that SpSoxB2 transcripts were present in both somatic and developing germ cells in the gonads. In testis, SpSoxB2 transcripts were strongly localized in spermatocytes and spermatids. In ovary, SpSoxB2 transcripts were detected in follicle cells, oogonia and oocytes, with the strongest signal in follicle cells. Our results demonstrated that the SpSoxB2 gene may play an important role during gonadal development in mud crab. [ABSTRACT FROM AUTHOR]

Mud crab, Scylla paramamosain is one of the most important crustacean species in global aquaculture. To determine the genetic basis of sex and growth-related traits in S. paramamosain , a high-density genetic linkage map with 16,701 single nucleotide polymorphisms (SNPs) was constructed using SLAF-seq and a full-sib family. The consensus map has 49 linkage groups, spanning 5,996.66 cM with an average marker-interval of 0.81 cM. A total of 516 SNP markers, including 8 female-specific SNPs segregated in two quantitative trait loci (QTLs) for phenotypic sex were located on LG32. The presence of female-specific SNP markers only on female linkage map, their segregation patterns and lower female: male recombination rate strongly suggest the conformation of a ZW/ZZ sex determination system in S. paramamosain. The QTLs of most (90%) growth-related traits were found within a small interval (25.18–33.74 cM) on LG46, highlighting the potential involvement of LG46 in growth. Four markers on LG46 were significantly associated with 10–16 growth-related traits. BW was only associated with marker 3846. Based on the annotation of transcriptome data, 11 and 2 candidate genes were identified within the QTL regions of sex and growth-related traits, respectively. The newly constructed high-density genetic linkage map with sex-specific SNPs, and the identified QTLs of sex- and growth-related traits serve as a valuable genetic resource and solid foundation for marker-assisted selection and genetic improvement of crustaceans. [ABSTRACT FROM AUTHOR]

Background: Mud crabs, Scylla spp., are commercially important large-size marine crustaceans in the Indo-West Pacific region. As females have the higher growth rate and economic value, the production of all female stocks is extremely essential in aquaculture. However, the sex determination mechanism is still unclear. Development of sex-specific genetic markers based on next-generation sequencing proved to be an effective tool for discovering sex determination system in various animals. Results: Restriction-site associated DNA sequencing (RAD-seq) was employed to isolate sex-specific SNP markers for S. paramamosain. A total of 335.6 million raw reads were obtained from 20 individuals, of which 204.7 million were from 10 females and 130.9 million from 10 males. After sequence assembly and female-male comparison, 20 SNP markers were identified to be sex-specific. Furthermore, ten SNPs in a short sequence (285 bp) were confirmed heterozygous in females and homozygous in males in a large population by PCR amplification and sequencing. Subsequently, a female-specific primer was successfully designed according to the female-specific nucleotide which could amplify an expected band from females but not from males. Thus, a rapid and effective method for molecular sexing in S. paramamosain was developed, meanwhile, this method could successfully identify the sex of S. tranquebarica and S. serrata. Finally, nine and four female-specific SNP markers were detected in S. tranquebarica and S. serrata, respectively. Conclusions: Sex-specific SNP markers were firstly identified in crab species and showed female heterogamety and male homogamety, which provided strong genetic evidence for a WZ/ZZ sex determination system in mud crabs S. paramamosain, S. tranquebarica and S. serrata. These findings will lay a solid foundation for the study of sex determination mechanism, sex chromosome evolution, and the development of mono-sex population in crustaceans. [ABSTRACT FROM AUTHOR]

In this study, we reported the characterization of the first transcriptome of the mud crab (Scylla paramamosain). Pooled cDNAs of four tissue types from twelve wild individuals were sequenced using the Roche 454 FLX platform. Analysis performed included de novo assembly of transcriptome sequences, functional annotation, and molecular marker discovery. A total of 1,314,101 high quality reads with an average length of 411 bp were generated by 454 sequencing on a mixed cDNA library. De novo assembly of these 1,314,101 reads produced 76,778 contigs (consisting of 818,154 reads) with 5.4-fold average sequencing coverage. The remaining 495,947 reads were singletons. A total of 78,268 unigenes were identified based on sequence similarity with known proteins (E≤0.00001) in UniProt and non-redundant protein databases. Meanwhile, 44,433 sequences were identified (E≤0.00001) using a BLASTN search against the NCBI nucleotide database. Gene Ontology (GO) analysis indicated that biosynthetic process, cell part, and ion binding were the most abundant terms in biological process, cellular component, and molecular function categories, respectively. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed that 4,878 unigenes distributed in 281 different pathways. In addition, 19,011 microsatellites and 37,063 potential single nucleotide polymorphisms were detected from the transcriptome of S. paramamosain. Finally, thirty polymorphic microsatellite markers were developed and used to assess genetic diversity of a wild population of S. paramamosain. So far, existing sequence resources for S. paramamosain are extremely limited. The present study provides a characterization of transcriptome from multiple tissues and individuals, as well as an assessment of genetic diversity of a wild population. These sequence resources will facilitate the investigation of population genetic diversity, the development of genetic maps, and the conduct of molecular marker-assisted breeding in S. paramamosain and related crab species. [ABSTRACT FROM AUTHOR]

Microsatellite markers from a transcriptome sequence library were initially isolated, and their genetic variation was characterized in a wild population of the mud crab (Scylla paramamosain). We then tested the association between these microsatellite markers and the growth performance of S. paramamosain. A total of 129 polymorphic microsatellite markers were identified, with an observed heterozygosity ranging from 0.19 to 1.00 per locus, an expected heterozygosity ranging from 0.23 to 0.96 per locus, and a polymorphism information content (PIC) ranging from 0.21 to 0.95 per locus. Of these microsatellite markers, 30 showed polymorphism in 96 full-sib individuals of a first generation family. Statistical analysis indicated that three microsatellite markers were significantly associated with 12 growth traits of S. paramamosain. Of these three markers, locus Scpa36 was significantly associated with eight growth traits, namely, carapace length, abdomen width (AW), body height (BH), fixed finger length of the claw, fixed finger width of the claw, fixed finger height of the claw, meropodite length of pereiopod 2, and meropodite length of pereiopod 3 (MLP3) (P<0.05). Locus Scpa75 was significantly associated with five growth traits, namely, internal carapace width, AW, carapace width at spine 8, distance between lateral spine 2 (DLS2), and MLP3 (P<0.05). Locus Spm30 was significantly associated with BH, DLS2, and body weight (P<0.05). Further analysis suggested a set of genotypes (BC at Scpa36, BC and BD at Scpa75, and AC at Spm30) that have great potential in the selection of S. paramamosain for growth traits. These findings will facilitate the development of population conservation genetics and molecular marker-assisted selective breeding of S. paramamosain and other closely related species. [ABSTRACT FROM AUTHOR]

Microsatellite markers from a transcriptome sequence library were initially isolated, and their genetic variation was characterized in a wild population of the mud crab (Scylla paramamosain). We then tested the association between these microsatellite markers and the growth performance of S. paramamosain. A total of 129 polymorphic microsatellite markers were identified, with an observed heterozygosity ranging from 0.19 to 1.00 per locus, an expected heterozygosity ranging from 0.23 to 0.96 per locus, and a polymorphism information content (PIC) ranging from 0.21 to 0.95 per locus. Of these microsatellite markers, 30 showed polymorphism in 96 full-sib individuals of a first generation family. Statistical analysis indicated that three microsatellite markers were significantly associated with 12 growth traits of S. paramamosain. Of these three markers, locus Scpa36 was significantly associated with eight growth traits, namely, carapace length, abdomen width (AW), body height (BH), fixed finger length of the claw, fixed finger width of the claw, fixed finger height of the claw, meropodite length of pereiopod 2, and meropodite length of pereiopod 3 (MLP3) (P<0.05). Locus Scpa75 was significantly associated with five growth traits, namely, internal carapace width, AW, carapace width at spine 8, distance between lateral spine 2 (DLS2), and MLP3 (P<0.05). Locus Spm30 was significantly associated with BH, DLS2, and body weight (P<0.05). Further analysis suggested a set of genotypes (BC at Scpa36, BC and BD at Scpa75, and AC at Spm30) that have great potential in the selection of S. paramamosain for growth traits. These findings will facilitate the development of population conservation genetics and molecular marker-assisted selective breeding of S. paramamosain and other closely related species. [ABSTRACT FROM AUTHOR]

Abstract: In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species. [Copyright &y& Elsevier]

Abstract: The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNAHis differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy. [Copyright &y& Elsevier]

Abstract: In this study, we first examined the utility of COI sequences as DNA barcoding for identification of genus Scylla, which includes four species: Scylla paramamosain, Scylla serrata, Scylla tranquebarica and Scylla olivacea. The mean intraspecific Kimura 2-parameter distances were 0.003 for S. paramamosain, 0.014 for S. serrata, 0.017 for S. olivacea and 0.006 for S. tranquebarica. The interspecific K2P distances were higher than the intraspecific distances: the minimum interspecific distance (0.092) was between S. paramamosain and S. tranquebarica while the maximum interspecific distance (0.196) was between S. paramamosain and S. olivacea. No overlap was found between intraspecific and interspecific distances, suggesting the existence of a distinct barcoding gap. The maximum-likelihood (ML) phylogenetic tree consisted of four distinct clusters, each containing individuals from one species only. Furthermore, a molecular species identification method was developed successfully, by which these four species could be identified rapidly and effectively from each other though a PCR reaction. [Copyright &y& Elsevier]

The article discusses a study which isolated and characterized 54 polymorphic microsatellite markers in Scylla paramamosain. The study included 29 Scylla paramamosain gathered from Ningde, China, which were analyzed using amplified fragment length polymorphisms (AFLP) of sequences containing repeats (FIASCO) technique. Study authors said that FIASCO is an effective method for isolating microsatellite markers, while gene heterozygosity is effective for assessing the genetic structure.

Simple Summary: Nursery culture knowledge is important for successful commercial seed production especially for mud crab, genus Scylla, a highly valued delicacy. The aim of this review paper is to summarise the status and information on the current nursery culture stage of the mud crab. Various aspects of mud crab hatchery described in this paper are expected to facilitate practitioners and lay people to easily understand a mud crab nursery. This review also provides guidelines for researchers in conducting future research and development on a mud crab nursery in order to increase the production of mud crab crablets for the farming industry. The nursery stages of mud crab, genus Scylla, proceed from the megalopa stage to crablet instar stages. We review the definition and several of the key stages in mud crab nursery activities. The practice of the direct stocking of megalopa into ponds is not recommended due to their sensitivity. Instead, nursery rearing is needed to grow-out mud crabs of a larger size before pond stocking. Individual nursery rearing results in a higher survival rate at the expense of growth and a more complicated maintenance process compared with communal rearing. The nursery of mud crabs can be done both indoors or outdoors with adequate shelter and feed required to obtain a good survival percentage and growth performance. Artemia nauplii are still irreplaceable as nursery feed, particularly at the megalopa stage, while the survival rate may be improved if live feed is combined with artificial feed such as microbound diet formulations. Water quality parameters, identical to those proposed in tiger shrimp cultures, can be implemented in mud crab rearing. The transportation of crablets between different locations can be done with or without water. The provision of monosex seeds from mud crab hatcheries is expected to become commonplace, increasing seed price and thus improving the income of farmers. Numerous aspects of a mud crab nursery including nutrition; feeding strategies; understanding their behaviour, i.e., cannibalism; control of environmental factors and practical rearing techniques still need further improvement. [ABSTRACT FROM AUTHOR]

Simple Summary: The nursery phase of mud crab (Scylla spp.)—from megalopa to several, further crablet stagesneeds to be carried out in optimal environmental conditions, until they reach a larger size suitable to being stocked to a pond. This study observed the behavioral thermoregulation of crablet instar phases, and determined the effects of several levels of water temperature on growth, survival, molting cycle, gill condition, and sex ratio in the nursery phase of S. paramamosain. In this study, optimal temperature for the long-term nursery phase of S. paramamosain, beginning with megalopa stage, was found to be 28 to 30 °C, with the possibility that water temperature could affect the sex ratio of mud crabs. Findings of this study could result in increased crablet production from hatcheries, and assist in realizing potential of monosex seed production of S. paramamosain, through temperature treatment. This study was carried out to determine the physiological changes (survival, growth, molting cycle, sex differentiation, and gill condition) of mud crab, Scylla paramamosain crablet at different water temperatures of 24, 28 and 32 °C, and ambient temperature of 27 to 30 °C. Thermoregulatory behavior, represented by preferred temperature (29.83 ± SD 2.47 °C), critical thermal minimum (17.33 ± SD 0.58 °C), critical thermal maximum (40 ± SD 0.00 °C), and thermal tolerance interval (22.67 ± SD 0.58 °C), were checked for Crablet 1 stage only (with ambient temperature as acclimation temperature).Both low (24 °C) and high (32 °C) temperatures were associated with lower growth performance, and survival rate (p < 0.05), in comparison with both 28 °C and ambient temperature treatments.Male ratio at low temperaturetreatment (24 °C) was higher (80.09 ± SD 18.86%) than for other treatments (p < 0.05), observed as 44.81 ± D 10.50%, 41.94 ± SD 19.44%, and 76.30 ± SD 5.13% for 28 °C, 32 °C and ambient temperature treatments, respectively. However, there was no significant difference observed between 24 °C, 28 °C, and ambient temperature treatments. Anatomical alterations of gill lamellae of S. paramamosain crablet for both 32 °C, and 24 °C treatments, appeared thinner and paler than at both 28 °C, and ambient temperature treatments. Based on this study, temperature of 28 to 30 °C was recommended as the optimal temperature for the long-term nursery phase of S. paramamosain. [ABSTRACT FROM AUTHOR]

In modern aquaculture, inter-specific hybridization enhances traits, but its effects on embryonic development and growth in mud crabs (genus Scylla) remain underexplored. This study investigated the embryonic development and growth performance of Scylla paramamosain , Scylla serrata , and their hybrid offsprings from reciprocal crosses. We found that S. paramamosain and the hybrid (S. paramamosain ♀ × S. serrata ♂) produced smaller eggs with shorter development periods than S. serrata and the hybrid (S. serrata ♀ × S. paramamosain ♂). Both hybrids exhibited higher growth rates in the crablet stage than their purebred parents. Notably, the hybrid (S. serrata ♀ × S. paramamosain ♂) demonstrated significant heterosis in body weight and height, while the hybrid (S. paramamosain ♀ × S. serrata ♂) showed single-parent heterosis. While the shape of the frontal lobe spines in hybrids depended on the male parent. Development durations varied, with the longest observed in the hybrid (S. paramamosain ♂ × S. serrata ♀). Discriminant analysis of growth traits showed high accuracy across developmental stages, supporting the potential of inter-specific hybridization for enhancing mud crab aquaculture. This research provides valuable insights and germplasm resources for the aquaculture industry. [Display omitted] • Interspecific hybridization is feasible in Scylla species. • The hybrid crab (S. serrata ♀ × S. paramamosain ♂) have enhanced growth traits. • The hybrid crab (S. serrata ♀ × S. paramamosain ♂) exhibit both mid-parent and single parent heterosis. • There is a high degree of genetic compatibility among Scylla species. [ABSTRACT FROM AUTHOR]

Genomic selection (GS) and genome-wide association studies (GWAS) have been widely implemented to accelerate the genetic improvement of important economic traits. Developing an efficient and reliable high-throughput genotyping tool is the premise and basis of implementing GS and GWAS on the mud crab (Scylla paramamosain). In this study, leveraging the whole-genome resequencing data of 146 mud crabs from the southeast coast of China, a total of 16,714,943 high-quality SNPs were discovered. Based on polymorphic information content and evenly distribution on 49 chromosomes, a total of 40,352 SNPs was selected to develop a 40 K liquid SNP array (named "Xiexin No. 1") using the genotyping by target sequencing (GBTS) technology. The genotyping performance test showed that this SNP array had a high site polymorphism (over 92.70%) and individual calling rate (97.02%). Furthermore, the mean of SNP concordance, genotype concordance, non-reference sensitivity, and non-reference discrepancy between "Xiexin No. 1" SNP array and resequencing were identified to be 0.986, 0.954, 0.942, and 0.097, respectively. Using this SNP array data, the genomic heritabilities were evaluated as 0.887 ± 0.065, 0.643 ± 0.108, 0.599 ± 0.111, and 0.624 ± 0.111 for body weight (BW), carapace length (CL), carapace length (CW), and body height (BH) in a cultured population (233 crabs). Furthermore, five significant SNPs associated with growth traits were identified by GWAS. After annotation, one candidate gene (XP_027221997.1) associated with BW and one candidate gene (XP_027210446.1) associated with CW and BH were identified. Most importantly, the prediction accuracy of GBLUP for BW, BH, CL, and CW were 0.535 ± 0.064, 0.544 ± 0.101, 0.574 ± 0.087, and 0.585 ± 0.083, with the unbiasedness values of 0.988 ± 0.22, 1.015 ± 0.274, 1.011 ± 0.196 and 1.037 ± 0.235. In conclusion, the "Xiexin No. 1" SNP array provides an efficient and reliable platform for high-quality genotyping, which would greatly promote the genetic studies and thus accelerate the genetic improvement of mud crabs. • The first 40-K liquid SNP array was developed in mud crab (S. paramamosain). • This SNP array contained 40,352 SNPs that uniformly covered on 49 chromosomes. • This SNP array had a high polymorphism (over 92.70%) and calling rate (97.02%). • This SNP array had a good performance in GS, GWAS, and heritability estimations. • This SNP array provides an efficient platform for genetic studies in mud crab. [ABSTRACT FROM AUTHOR]

Soft-shell crabs are highly valued for their commercial significance and culinary appeal. To enhance production of soft-shell crabs, careful attention should be paid to the water calcium concentration in their culture. This study investigated the influences of different levels of calcium concentrations on the molting and growth of mud crab (Scylla paramamosain) within a meticulously controlled recirculating aquaculture system (RAS). We established three distinct water calcium concentration gradients (200, 300, and 450 mg/L) to comprehensively assess feeding habits, molting patterns, growth, and tissue calcium levels of mud crab. Initially, we observed a gradual reduction in water calcium concentrations with increments of crab molting events in RAS. Throughout one molting cycle, the feeding incidence of the crabs remained consistently high from 4 days to 37 days post-molt, irrespective of different calcium concentrations. Nonetheless, high water calcium levels (300 and 450 mg/L) significantly shortened molt intervals and accelerated exoskeletal calcification. Specifically, the molt intervals were 52.30 ± 2.72 d, 41.65 ± 5.24 d, and 37.70 ± 3.02 d, while the carapace hardening durations were 21.51 ± 4.33 h, 17.23 ± 3.38 h, and 13.46 ± 2.76 h at water calcium concentrations of 200, 300, and 450 mg/L, respectively. Moreover, elevated calcium concentrations also significantly enhanced weight gain of the mud crab during molting. Noteworthy, regardless of fluctuations in water calcium concentration, the condition factor of the molting crabs was approximately 63%, which could be an indicator of crab selection in soft-shell crab cultivation. Furthermore, the dynamic changes in calcium concentrations were disparate among different tissues in the post-molt stage. Gill and exoskeleton tissues exhibited an upward trend in calcium concentrations, while hemolymph and hepatopancreas tissues displayed distinct patterns of decrease followed by increase. Gill and exoskeleton tissues were identified as primary sites for calcium absorption and utilization, evidenced by a positive correlation between high calcium concentrations and increased calcium content in these tissues at 6 h and 3 d post-molt. Overall, our findings provide practical evidence for optimizing aquaculture management strategies through adjustments in water calcium concentration, thus to enhance production efficiency and profitability in soft-shell mud crab aquaculture. • Higher water calcium significantly shortened molt intervals, accelerated carapace hardnessduring molting cycles. • The condition factor of crabs at around 63% could be an indicator for crab selection in soft-shell crab cultivation. • Dynamic changes in calcium concentrations varied among different tissues in the post-molt stage. • Gill and exoskeleton tissues were identified as primary sites for calcium absorption and utilization. [ABSTRACT FROM AUTHOR]

Chitin is a kind of insoluble structural polysaccharide and plays different roles in different species. In crustaceans, it forms the structural components in the exoskeleton. In our previous studies, novel mud crab hybrids have been produced from the interspecific hybridization of Scylla serrata ♀ × S. paramamosain ♂. Some of the hybrid crabs have been found to be morphologically (eyestalk) abnormal, but the genetic mechanism remains unknown. To address this question, we performed whole-transcriptome RNA sequencing on the control group (normal hybrids), abnormal hybrids, and S. paramamosain to uncover the genetic basis underlying this morphological abnormality. A total of 695 mRNAs, 10 miRNAs, 44 circRNAs, and 1957 lncRNAs were differentially expressed between normal and abnormal hybrids. Several differentially expressed genes (DEGs) associated with chitin and cuticle metabolism were identified, including chitin synthase , chitinase , chitin deacetylase , β-N-acetylglucosaminidase , β-1,4-endoglucanase , N-alpha-acetyltransferase, cuticle proprotein , early cuticle protein , and arthrodial cuticle protein. Functional analysis showed that DE miRNAs, DE circRNAs, DE lncRNAs, and lncRNA/circRNA-miRNA-mRNA network were enriched in pathways related to the amino acid, carbohydrate, and glycogen metabolism. Considering the importance of the chitin and cuticle in exoskeleton formation, it can be concluded that the changes in the chitin and cuticle biosynthesis might have caused the eyestalk abnormality in hybrid crabs. These findings can lay the solid foundation for a better understanding of the important roles of chitin and cuticle related genes and the development of hybridization techniques in crustaceans. [ABSTRACT FROM AUTHOR]

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5′-UTR and a 672 bp 3′-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N -glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene. [ABSTRACT FROM AUTHOR]

Infection by the rhizocephalan parasite Sacculina beauforti can have detrimental effects on mud crab Scylla olivacea. However, the molecular changes that occur during rhizocephalan infection are poorly understood. Due to the disruption in the reproductive system after infection, the gonadal transcriptomic profiles of non-infected and infected Scylla olivacea were compared. A total of 686 and 843 unigenes were differentially expressed between non-infected and infected males, and females, respectively. The number of DEGs increased after infection. By comparing shared DEGs of non-infected and infected individuals, potential immune- and reproduction-related of host, and immune- and metabolism-related genes of parasite are highlighted. The only shared KEGG pathway between non-infected and infected individuals was the ribosome pathway. In summary, findings in this study provide new insights into the host-parasite relationship of rhizocephalan parasites and their crustacean hosts. • Sacculina beauforti 's infection resulted in increased genes being differentially expressed. • Infection induced up-regulation of immune-related genes and down-regulation of reproduction-related genes in Scylla olivacea. • Infection induced up-regulation of immune- and metabolism-related genes in Sacculina beauforti. • Significant up-regulation of genes in ribosome pathway after infection. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain , the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans. • The miRNA profiling during WSSV infection was obtained. • 152 miRNAs were differentially expressed in WSSV-infected hemocytes. • Differentially expressed miRNAs were correlated with immune responses of hemocytes. [ABSTRACT FROM AUTHOR]

Lysin motif (LysM)-containing proteins function as pattern-recognition receptors in plants to recognize different N -acetylglucosamine-containing ligands, thereby triggering specific defense responses against pathogens. However, the biological functions of these proteins in animals remain unclear. In this study, we characterized a novel LysM protein, designated as Sp LysMD3, in mud crab Scylla paramamosain. The cDNA sequence of SpLysMD3 had 1058 bp with an open reading frame of 840 bp encoding a protein with 279 amino acid residues. The deduced protein contained a LysM domain and a transmembrane region. SpLysMD3 was highly expressed in gills, intestine, muscle, and hemocytes and upregulated after challenges with bacteria, suggesting that it may be involved in antibacterial defense. Binding assay showed that Sp LysMD3 possessed specific binding activities to all tested microorganisms as well as bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN), indicating that Sp LysMD3 was an important LPS- and PGN-binding protein in mud crab. Bacterial clearance assay revealed that coating bacteria with Sp LysMD3 accelerated bacterial clearance in vivo. The promotion of bacterial clearance by Sp LysMD3 was further determined by using SpLysMD3 -silenced crabs injected with S. aureus or V. parahemolyticus. Silencing SpLysMD3 dramatically suppressed the bacterial clearance. Meanwhile, knockdown of SpLysMD3 also severely impaired the expression of a specific set of antimicrobial peptides (AMPs); moreover, Sp LysMD3 overexpression can enhance the promoter activity of SpALF2. These results suggested that SpLysMD3 affected bacterial clearance by regulating AMPs. Collectively, all the results demonstrated that Sp LysMD3 may function as a potential receptor involved in innate immunity by binding to LPS and PGN and by regulating AMPs to eliminate invading pathogen. This study provided new insights into the biological functions of LysM proteins in animals and the mechanisms underlying the antibacterial activity of crustaceans. • Sp LysMD3 was membrane-bound protein with a lysin motif. • Sp LysMD3 displayed strong binding activity to PGN and LPS. • Sp LysMD3 knockdown suppressed bacterial clearance in vivo and some AMPs' expression. • Sp LysMD3 may act as a potential PRR involved in antibacterial immunity of mud crab. [ABSTRACT FROM AUTHOR]

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC , a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development. • 596 out of 115,491 novel piRNAs were gonadal differentially expressed. • Predicted potential target genes of piRNAs were involved in gametogenesis- and reproduction-related pathways. • piRNAs potentially regulate gametogenesis and gonadal development in crustaceans. [ABSTRACT FROM AUTHOR]

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (Sp BAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. Sp BAG1 was found to be significantly up-regulated at 6 h–24 h, but down-regulated from 48 h–72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of Sp BAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that Sp BAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of Sp BAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab. •Sp BAG1 was up-regulated in mud crab in the early stage of WSSV infection. •RNAi of Sp BAG1 can increase the apoptotic rate in mud crab. •Sp- BAG1 acts as an apoptosis inhibitor to promote virus infection in mud crab [ABSTRACT FROM AUTHOR]

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (Sp TGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). Sp TGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of Sp TGase. Phylogenetic analysis revealed a close relation between Sp TGase to its homolog Es TGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of Sp TGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus , lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant Sp TGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. Sp TGase was found to directly interact with another important blood coagulation component clip domain serine protease (Sp cSP). Moreover, knockdown of Sp TGase resulted in a decreased expression of both clotting protein precursor (Sp preCP) and Sp cSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest Sp TGase play an important role in the hemolymph clotting in mud crab S. paramamosain. • Sp TGase with a 2304 bp ORF encoding 767 amino acids was characterized in mud crab. • Recombinant Sp TGase agglutinated towards both Gram (-) bacteria and yeast. • Sp TGase directly interacted with a blood coagulation component Sp cSP. • Si Sp TGase induced a downregulation of Sp preCP and Sp cSP and elongated clotting time. [ABSTRACT FROM AUTHOR]

Abstract Mud crab (Scylla paramamosain) is an economically important crustacean species for fisheries and aquaculture industry in China and other Southeast Asian countries. In this study, the sex ratio, morphological dimorphism, and the characteristics of morphological traits between female and male S. paramamosain juveniles at three different pond-culture stages were determined. The sex ratio skewed towards female crablets as growth and development proceeded. Three growth traits exhibited significant differences between sexes when the crablets were at two months of pond-culture stage, however, the number of growth traits with significant differences increased to seven when the crablets were at three and four months of pond-culture stages. Further, significant positive correlations were detected among all the 190 trait-pairs, irrespective of the pond-culture stages of the crablets. Apart from this, the path coefficients and determination coefficients varied between sexes among the three pond-culture stages, suggesting that the growth traits have different effects on body weight (BW) of female and male crablets during different pond-culture stages. The effect of growth traits on BW in both sexes at different pond-culture stages was determined by constructing six multiple regression equations. Analysis of the width-weight relationship revealed that male crablets had positive growth allometry whereas the females had isometric growth. In addition, an obvious sexual dimorphism in cheliped size and abdomen width was observed with growth and development. A discriminant function equation was successfully constructed for sex identification based on morphological indexes, with a high accuracy of 90.4%. Highlights • The sex ratio and sexual dimorphism of Scylla paramamosain crablets were evaluated. • The morphological traits affecting body weight varied during the different developmental stages. • A sex discriminant equation based on morphological indexes was constructed, with a high accuracy of 90.4%. • The findings serve as the baseline information for prospective artificial selective breeding and farming of S. paramamosain. [ABSTRACT FROM AUTHOR]

Abstract Vibrio parahaemolyticus is one of the major pathogens caused diseases in cultured mud crab (Scylla paramamosain). Mud crabs lack an adaptive immune system, their defenses depend almost on innate immunity. Evaluation of the molecular responses of mud crabs to pathogens is essential for control of disease occurrence in farmed animals. In this study, the impacts of V. parahaemolyticus on immunity-related genes and metabolites in mud crabs of different groups (PG, SG and MG refer to controlled, survival and moribund groups, respectively) were investigated. Our results revealed that V. parahaemolyticus infection stimulated significant expressions of immune-related genes (prophenoloxidase, alpha 2-macroglobulin, lysosomal-associated membrane protein, Rab5, C-type lectin B and anti-lipopolysaccharide factor 5) in the MG within 72 h post-infection. The ATP content was significantly reduced in all tissues except muscle of moribund mud crabs. A total of 668 metabolites (including 190 down-regulated and 145 up-regulated) were identified and assigned to 77 pathways in both SG and MG. Metabolites involved in the saturated fatty acid are up-regulated, whereas unsaturated fatty acid and amino acid metabolisms are down-regulated in the immune system of mud crabs during the bacterial infection in MG. Furthermore, a reduction of hemocyte number and an increase of microbial abundance was found in MG. Our results demonstrated that V. parahaemolyticus induced death of mud crabs through reducing the metabolites associate with energy biosynthesis and innate immune system (i.e. proliferation of hemocyte and melanization), resulting in decrease of ATP in different tissues and failed to clearance of pathogens, respectively. The findings of this study provide a basic information of the responses of mud crab on bacterial infection, which is essential for prevention and control of diseases in mud crab aquaculture. Highlights • The immune-related genes are up-regulated upon pathogen infection in MG. • Metabolites involved in saturated fatty acid are induced, unsaturated fatty acid and amino acid metabolisms repressed. • The metabolites associate with energy biosynthesis and innate immune system are down-regulated. • ATP in different tissues and hemocyte are decreased, whereas microbial abundant is increased. [ABSTRACT FROM AUTHOR]

Abstract Serine proteases (SPs) are important in various immune responses, including prophenoloxidase (proPO) activation, antimicrobial peptides (AMPs) synthesis, and hemolymph coagulation in invertebrates. In this study, SP3 and SP5 of mud crab (Scylla paramamosain) were studied. SP3 and SP5 were expressed in all examined tissues (mainly in hemocytes), and are associated with the immune responses of mud crab to Vibrio parahemolyticus and Staphylococcus aureus , as well as interacted with TRAF6, and are involved in the activation of anti-lipopolysaccharide factors (ALFs) probably through the TLR/NF-κB pathway. Depletion of SP3 inhibited the expression of ALF1, ALF2, ALF3, and ALF6, while knockdown of SP5 significantly decreased ALF5, and ALF6. Furthermore, both SP5 and TRAF6 regulated the PO activity in the hemolymph of mud crab. Overexpression assay showed that both SP3 and SP5 could enhance the promoter activities of ALFs in mud crab. Taken together, the results of this study indicate that SP3 and SP5 might play important roles in the immune system of mud crab against pathogen invasion. Highlights • Full-length of SP3 with 1366 bp, and SP5 with 1655bp were isolated from mud crab. • SP3 and SP5 interacted with TRAF6 and were associated with the activation of ALFs. • Both SP5 and TRAF6 regulate PO activity in the hemolymph of mud crab. [ABSTRACT FROM AUTHOR]

Abstract Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be associated with the reactive oxygen species (ROS) hydrogen peroxide (H 2 O 2 ) production at the plasma membrane and crucial in host biological processes. In this study, Sp DUOX1 and Sp DUOX2 of mud crab ( Scylla paramamosain ) were identified and studied. Both Sp DUOX1 and Sp DUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase homology domain in the extracellular region, transmembrane regions, and three EF (calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the intracellular region. The Sp DUOXs were expressed in all tissues examined, but mainly in hepatopancreas, heart, and mid-intestine. The expression of the Sp DUOXs in the hemolymph of mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA interference (RNAi) of the Sp DUOXs resulted in reduced ROS production in hemolymph. The bacterial count increased in the hemolymph of mud crabs injected with Sp DUOX1 or Sp DUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while Proteobacteria were significantly reduced following Sp DUOX2 knockdown. There was a significant increase in the relative abundance of the genera Marinomonas , Pseudoalteromonas , Shewanella , and Hydrogenoph in Sp DUOX2 depleted mud crabs compared with the controls. Our current findings therefore indicated that Sp DUOXs might play important roles in maintaining the homeostasis in the hemolymph microbiota of mud crab. Highlights • Full-length of Sp DUOX1 with 4786 bp and Sp DUOX2 with 5093 bp was isolated from mud crab. • Sp DUOX1 and Sp DUOX2 might participate in maintaining bacterial homeostasis in the hemolymph of mud crabs. • The crabs with reduced Sp DUOX2 gene expression showed a profound difference in bacterial community composition. [ABSTRACT FROM AUTHOR]

Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain , although a Toll-like receptor ( Sp Toll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 ( Sp Toll2) from S. paramamosain . The full-length cDNA of Sp Toll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that Sp Toll2 clustered with Drosophila Toll1, and shared high homology with Pt Toll4. Real-time qPCR analysis showed that Sp Toll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both Sp Toll1 and Sp Toll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus , Staphylococcus aureus , Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant Sp Toll1-LRR and Sp Toll2-LRR proteins could bind to V. parahaemolyticus , S. aureus , Escherichia coli , and Beta Streptococcus . In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of Sp AMPs after the challenges with V. parahaemolyticus or S. aureus . The data suggested that Sp Toll1and Sp Toll2 could regulate the transcripts of several AMPs and four immune related mediators ( Sp MyD88, Sp Tube, Sp Pelle and Sp TRAF6) at different scale. While silencing of Sp Toll1 post pathogens challenge attenuated the expression of Sp Histin, Sp ALF1 and Sp ALF5 in mud crab's hemocytes, depletion of Sp Toll2 post pathogens challenge inhibited the expression of Sp ALF1-6, Sp GRP, Sp Arasin and Sp Hyastastin. Furthermore, the results of overexpression assay also showed Sp Toll1 and Sp Toll2 could enhance the promoter activities of Sp ALFs in mud crab. Taken together, these results indicated that Sp Toll1 and Sp Toll2 might play important roles in host defense against pathogen invasions in S. paramamosain. [ABSTRACT FROM AUTHOR]

The increasing global demand for mud crabs (genus Scylla ) and threats to the wild populations highlight the urgency of fully rearing them in captivity. Despite considerable progress in mud crab production, most crab farms still rely heavily on wild-caught crablets and juveniles while the low and inconsistent success rates of larviculture remain as the main bottleneck impeding the development of mud crab aquaculture. Over the years, numerous studies have been conducted to determine the optimum larval rearing parameters, the ontogenic changes in digestive function and feeding behaviour, and the diets for different larval stages. These data, however, are dispersed and not summarised to inform culture practices. This review provides an update on the current progresses and to pinpoint the gaps in knowledge regarding mud crab larval rearing. We include all four mud crab species under the genus Scylla , i.e. Scylla serrata, Scylla olivacea, Scylla tranquebarica and Scylla paramamosain . Knowledge compiled in this review serves as an important guideline for prospective mud crab larviculture. Future research should gear towards filling in the gaps in our knowledge to advance mud crab larval rearing, thus fully incorporating mud crab into the aquaculture sector. [ABSTRACT FROM AUTHOR]

Abstract Phagocytosis and apoptosis are key cellular innate immune responses against bacteria and virus in invertebrates. Class B scavenger receptors (SRBs), which contain a CD36 domain, are critical pattern recognition receptors (PRRs) of phagocytosis for bacteria and apoptotic cells. In the present study, we identified a member of SRB subfamily in mud crab Scylla paramamosain , named Sp -SRB. The full-length cDNA of Sp -SRB is 2593 bp with a 1629 bp open reading frame (ORF) encoding a putative protein of 542 amino acids, and predicted to contain a CD36 domain with two transmembrane regions at the C- and N-terminals. Real-time qPCR analysis revealed that Sp -SRB was widely expressed in all tissues tested, and the expression of Sp -SRB was up-regulated upon challenge with Vibrio parahaemolyticus, white spot syndrome virus (WSSV), lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (PolyI:C). Moreover, in vitro experiments indicated that recombinant Sp -SRB protein ( rSp -SRB) could bind to fungi, Gram-positive and Gram-negative bacteria. RNA interference of Sp -SRB resulted in significant reduction in the expression level of phagocytosis related genes, antimicrobial peptides (AMPs) and Toll-like receptors (TLRs), which consequently led to impairment in both bacterial clearance and the phagocytotic activity of hemocytes. In addition, we found that Sp -SRB had the ability to attenuate the replication of WSSV proliferation in mud crab S. paramamosain. Collectively, this study has shown that Sp -SRB contributed to bacteria clearance by enhancing phagocytosis and up-regulating the expression of AMPs possibly in a TRLs ( Sp Toll 1 and Sp Toll 2)-dependent manner. Besides, Sp -SRB inhibited the replication of WSSV in S. paramamosian probably through enhancement of hemocytes phagocytosis of apoptotic cells. Highlights • Full-length of Sp -SRB with 2593 bp was isolated from mud crab. • Sp -SRB promoted bacteria clearance by enhancing phagocytosis and up-regulating the expression of AMPs. • Sp -SRB inhibited the replication of WSSV in S. paramamosian. [ABSTRACT FROM AUTHOR]

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, Sp CTL-B, was identified from the hemocytes of mud crab Scylla paramamosain . The full-length of Sp CTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted Sp CTL-B protein contains a single carbohydrate-recognition domain (CRD). Sp CTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of Sp CTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant Sp CTL-B (r Sp CTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β- hemolytic Streptococcus, Escherichia coli , Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae . r Sp CTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria ( S. aureus, β- hemolytic Streptococcus ) and Gram-negative bacteria ( E. coli, V. parahaemolyticus , A. hydrophila, V. alginolyticus ) in a Ca 2+ -dependent manner. The agglutination activities of r Sp CTL-B could be inhibited by D-mannose and LPS, but not by d -fructose and galactose. The antimicrobial assay showed that r Sp CTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When Sp CTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) ( Sp Crustin, Sp Histin, Sp ALF4 (anti-lipopolysaccharide factor), Sp ALF5 and Sp ALF6) were significantly decreased in hemocytes. In our study, knockdown of Sp CTL-B could down-regulate the expression of Sp STAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of Sp CTL-B in hemocytes in mud crab. Collectively, our results suggest that Sp CTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain . [ABSTRACT FROM AUTHOR]

Type I crustins are crucial effectors of crustacean immune system. Various type I crustins with high sequence diversity possess different antimicrobial activities. To date, the mechanism on how the sequence diversity of type I crustins affects their antimicrobial activities is largely unclear, and how different crustins function together against bacterial invasion still remains unknown. In this study, we identified two novel type I crustins, namely, Sp Crus3 and Sp Crus4, from an economically important crab, Scylla paramamosain . Either SpCrus3 or SpCrus4 was highly expressed in gill. After challenges with Vibrio parahemolyticus or Staphylococcus aureus , SpCrus4 was up-regulated, whereas SpCrus3 was down-regulated. No significant expression change of SpCrus3 and SpCrus4 was observed after white spot syndrome virus injection, suggesting that these two genes may not participate in the antiviral immune responses. SpCrus3 and SpCrus4 had the common 5′ terminus and high similarity of 66.06%, but Sp Crus4 exhibited stronger antimicrobial activity than that of Sp Crus3. Microorganism-binding assay results revealed that both Sp Crus3 and Sp Crus4 exhibited binding ability to all tested microorganisms. Furthermore, the polysaccharide-binding assay showed that these two proteins exhibited strong binding activity to bacterial polysaccharides, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN). Sp Crus3 and Sp Crus4 exhibited stronger binding activity to LPS or LTA than to PGN. Moreover, Sp Crus4 showed stronger binding activity to LTA than that of Sp Crus3, which may be responsible for the significantly distinct antimicrobial activity between these two proteins. In addition, Sp Crus4 displayed stronger agglutination activity against several kinds of microorganisms than that of Sp Crus3. This increased agglutination activity may also contribute to the strong antibacterial activity of Sp Crus4. On the basis of all these results, a possible antibacterial mode exerted by Sp Crus3 and Sp Crus4 was proposed as follows. SpCrus3 was highly expressed in normal crabs to maintain low-level antibacterial activity without bacterial challenges. When crabs were challenged with bacteria, large amount of Sp Crus4 was generated to exhibit strong antibacterial activity against bacterial invasion. This study provides new insights to understand the antibacterial functions and mechanisms of type I crustins. [ABSTRACT FROM AUTHOR]

Crustins, the main AMP family in Crustacea, are generated as isoforms in many species and implicated in innate immune responses, but their detailed molecular mechanisms on susceptible bacteria remain largely unclear. Type II and type I crustins are distinguished by glycine-rich region (GRR), which is a major marker motif, and some type II crustins exhibit stronger antibacterial activities than their GRR deletion mutants. In the present study, a novel crustin, namely, SpCrus5 , was functionally characterized from a commercially valuable crab Scylla paramamosain . Sp Crus5 contained a typical cysteine-rich domain at the N-terminus, a conserved WAP domain in the center, and a special GRR at the C-terminus, which is located in a site that differs from that of GRRs in typical type II crustins found between signal peptides and cysteine-rich domains. Sp Crus5 shared high similarities with most type II crustins, and it was more closely related to type II crustins than to other retrieved crustins. SpCrus5 was predominantly expressed in gills and remarkably upregulated after the crabs were challenged with Vibrio parahemolyticus or Staphylococcus aureus , suggesting that SpCrus5 might participate in antibacterial immune responses. To further elucidate how this C-terminal GRR affects the function of Sp Crus5, we harvested a GRR deletion mutant ( Sp Crus5-ΔGRR) by deleting the GRR. Liquid growth inhibition assays demonstrated that the antimicrobial activity of Sp Crus5 was stronger than that of Sp Crus5-ΔGRR, and the antibacterial spectrum of the former toward Gram-negative bacteria was broader than that of the latter. Binding assays revealed that the microorganism-binding ability and polysaccharide-binding activity of Sp Crus5 were stronger than those of Sp Crus5-ΔGRR. Sp Crus5 or Sp Crus5-ΔGRR agglutinated all tested Gram-positive bacteria. Therefore, the antibacterial activities of Sp Crus5 were stronger and broader than those of Sp Crus5-ΔGRR, and the binding ability and agglutination activity might contribute to the antimicrobial activity of Sp Crus5. These results revealed that the C-terminal GRR was necessary to produce an efficient antibacterial activity of Sp Crus5. Sp Crus5 was highly identical with most type II crustins and it functioned as many type II crustins did, indicating that Sp Crus5 was more likely an atypical type II crustin than a type I crustin. This study revealed that Sp Crus5 participated as an essential antimicrobial effector in immune responses and provided new insights into the underlying mechanisms of the sequence and function diversity of crustins. [ABSTRACT FROM AUTHOR]

Mud crabs ( Scylla spp.) are known to exist sympatrically in the wild. However, information on their population dynamics and the influence of climate parameters and lunar phase, especially along the equatorial region, are limited. Four sampling stations representing three seas (the Strait of Malacca, South China Sea and Sulu Sea) along the equator were selected. Mud crabs were collected using baited traps during spring tides from April 2012 to July 2013. All three Scylla species, S. olivacea, S. tranquebarica and S. paramamosain live in sympatry in the three seas. Scylla olivacea is the most prevalent species in the Strait of Malacca and South China Sea, whereas S. paramamosain dominates the Sulu Sea. The total crab abundance was not affected by rainfall or temperature. The abundance of S. tranquebarica in Strait of Malacca was negatively correlated with temperature and positively correlated with rainfall whereas the abundance of S. paramamosain positively correlated with temperature only at South China Sea. Scylla tranquebarica was the largest in terms of body size and it showed interchanging abundance trends with S. paramamosain . The average body size of S. paramamosain did not differ significantly with that of S. tranquebarica and S. olivacea . This decrease is most likely attributed to overfishing. Significant seasonal fluctuations in mean carapace width were detected in S. tranquebarica and S. paramamosain , but not in S. olivacea . The monthly sex ratio of all three species occasionally fluctuates above the equal sex ratio value. Lunar phase did not affect species abundance, but males and females were significantly heavier during full moon. These findings serve as a baseline of seasonal variation in crab population dynamics that are useful in mud crab fisheries and resource management. [ABSTRACT FROM AUTHOR]

Lysozymes are widely distributed immune effectors exerting muramidase activity against the peptidoglycan of the bacterial cell wall to trigger cell lysis. However, some invertebrate-type (i-type) lysozymes deficient of muramidase activity still exhibit antimicrobial activity. To date, the mechanism underlying the antimicrobial effect of muramidase-deficient i-type lysozymes remains unclear. Accordingly, this study characterized a novel i-type lysozyme, Sp lys-i, in the mud crab Scylla paramamosain . Sp lys-i shared the highest identity with the Litopenaeus vannamei i-type lysozyme ( Lv lys-i2, 54% identity) at the amino acid level. Alignment analysis and 3D structure comparison show that Sp lys-i may be a muramidase-deficient i-type lysozyme because it lacks the two conserved catalytic residues (Glu and Asp) that are necessary for muramidase activity. Sp lys - i is mainly distributed in the intestine, stomach, gills, hepatopancreas, and hemocytes, and it is upregulated by Vibrio harveyi or Staphylococcus aureus challenge. Recombinant Sp lys-i protein (r Sp lys-i) can inhibit the growth of Gram-negative bacteria ( V. harveyi , Vibrio alginolyticus , Vibrio parahemolyticus , and Escherichia coli ), Gram-positive bacteria ( S. aureus , Bacillus subtilis , and Bacillus megaterium ), and the fungus Candida albicans to varying degrees. In this study, two binding assays and a bacterial agglutination assay were conducted to elucidate the potential antimicrobial mechanisms of Sp lys-i. Results demonstrated that r Sp lys-i could bind to all nine aforementioned microorganisms. It also exhibited a strong binding activity to lipopolysaccharide from E. coli and lipoteichoic acid and peptidoglycan (PGN) from S. aureus but a weak binding activity to PGN from B. subtilis and β -glucan from fungi. Moreover, r Sp lys-i could agglutinate these nine types of microorganisms in the presence of Ca 2+ at different protein concentrations. These results suggest that the binding activity and its triggered agglutinating activity might be two major mechanisms of action to realize the muramidase-deficient antibacterial activity. In addition, r Sp lys-i can hydrolyze the peptidoglycan of some Gram-positive bacteria because it exhibits weak isopeptidase activities in salt and protein concentration-dependent manner. This result indicates that such an isopeptidase activity may contribute to the muramidase-deficient antimicrobial activity to a certain degree. In conclusion, Sp lys-i is upregulated by pathogenic bacteria, and it inhibits bacterial growth by binding and agglutination activities as well as isopeptidase activity, suggesting that Sp lys-i is involved in immune defense against bacteria through several different mechanisms of action. [ABSTRACT FROM AUTHOR]

In crustaceans, anti-lipopolysaccharide factors (ALFs) are important immune effectors that have sequence diversity and exhibit broad antimicrobial activities. In this study, we characterized a novel ALF homolog Sp ALF6 from mud crab Scylla paramamosain and its variant Sp ALF6-V, which was generated by mutations of two amino acids (H46 to R and A110 to P) due to the presence of two single nucleotide polymorphisms (SNPs). Sp ALF6 was an anionic peptide with isoelectric point (pI) 6.79, whereas Sp ALF6-V was a cationic protein with pI 7.98. These two proteins shared a common lipopolysaccharide (LPS)-binding domain (LBD) with pI 6.05. SpALF6 was expressed mainly in hemocytes and up-regulated by Vibrio parahaemolyticus or Staphylococcus aureus challenge, indicating that SpALF6 may participate in the antibacterial immune responses. To investigate the likely functional differences between Sp ALF6 and Sp ALF6-V and elucidate the underlying mechanisms, a single amino acid mutant Sp ALF6-M (from H46 to R, outside but very close to LBD), which had the same pI as Sp ALF6-V, was harvested by a fusion PCR. Then, both Sp ALF6 and Sp ALF6-M were overexpressed and purified to test antimicrobial activity and binding activity to microbial cells or polysaccharides. Sp ALF6-M exhibited more potent antimicrobial and cell-binding activity on Gram-positive bacteria and fungi than Sp ALF6. Furthermore, Sp ALF6-M possessed stronger lipoteichoic acid (LTA)-binding activity than Sp ALF6, demonstrating that this particular positively charged amino acid outside but close to LBD contributed to the increase in Sp ALF6-M antibacterial activity. In addition, Sp ALF6 LBD peptide and its biotin-labeled form were synthesized in this study. Results showed that this anionic LBD peptide itself did not exhibit any significant antimicrobial activity against 10 kinds of microorganisms but it possessed strong binding activity to LPS, LTA, and peptidoglycan. These findings suggested that this anionic LBD was still an important active center and required collaboration with some particular positively charged amino acids outside LBD to exhibit antibacterial activity. Thus, Sp ALF6-M antimicrobial activity was increased by the mutation of H46 to R instead of A110 to P, which did not change the protein charge, suggesting that Sp ALF6-V may have more potent antimicrobial activity than Sp ALF6 and play more important roles in antibacterial immunity. This study provided a new insight into the mechanisms of how ALF amino acid sequence diversity resulted in their functional divergence. [ABSTRACT FROM AUTHOR]