Comparative Transcriptome Analysis Reveals the Light Spectra Affect the Growth and Molting of Scylla paramamosain by Changing the Chitin Metabolism.

Light is an essential ecological factor that has been demonstrated to affect aquatic animals' behavior, growth performance, and energy metabolism. Our previous study found that the full-spectrum light and cyan light could promote growth performance and molting frequency of Scylla paramamosain while it was suppressed by violet light. Hence, the purpose of this study is to investigate the underlying molecular mechanism that influences light spectral composition on the growth performance and molting of S. paramamosain. RNA-seq analysis and qPCR were employed to assess the differentially expressed genes (DEGs) of eyestalks from S. paramamosain reared under full-spectrum light (FL), violet light (VL), and cyan light (CL) conditions after 8 weeks trial. The results showed that there are 5024 DEGs in FL vs. VL, 3398 DEGs in FL vs. CL, and 3559 DEGs in VL vs. CL observed. GO analysis showed that the DEGs enriched in the molecular function category involved in chitin binding, structural molecular activity, and structural constituent of cuticle. In addition, the DEGs in FL vs. VL were mainly enriched in the ribosome, amino sugar and nucleotide sugar metabolism, lysosome, apoptosis, and antigen processing and presentation pathways by KEGG pathway analysis. Similarly, ribosome, lysosome, and antigen processing and presentation pathways were major terms that enriched in FL vs. CL group. However, only the ribosome pathway was significantly enriched in up-regulated DEGs in VL vs. CL group. Furthermore, five genes were randomly selected from DEGs for qPCR analysis to validate the RNA-seq data, and the result showed that there was high consistency between the RNA-seq and qPCR. Taken together, violet light exposure may affect the growth performance of S. paramamosain by reducing the ability of immunity and protein biosynthesis, and chitin metabolism.

Recirculating aquaculture system as microbial community and water quality management strategy in the larviculture of Scylla paramamosain.

The structure and function of the water microbial community can change dramatically between different rearing modes. Yet investigations into the relationships between microbial community and water quality remain obscure. We provide the first evidence that rearing modes alter bacterial community and water quality in the rearing water of the mud crab (Scylla paramamosain) larvae. The juveniles in the recirculating aquaculture system (RAS) had a higher viability than those in the water exchange system (WES). RAS had the significantly lower levels of total ammonia nitrogen (TAN), NH3, NO2--N, total nitrogen (TN), total dissolved solids (TDS), and chemical oxygen demand than those of WES. The number of significantly different amplicon sequence variants between rearing modes increased as the larvae developed. NH3, TAN, TDS, NO2--N, and TN were closely related to the late alterations in water bacterial community. Both the FAPROTAX tool and quantitative PCR analysis showed enhanced nitrogen cycling functional potential of water bacterial community of RAS. Random forest analysis identified the enriched water bacteria especially heterotrophic bacteria such as Phaeodactylibacter, Tenacibaculum, and Hydrogenophaga, which were vital in removing nitrogenous compounds via simultaneous nitrification and denitrification. Notably, RAS could save 18.5 m3 of seawater relative to WES in larviculture on the scale of 2.5 m3. Together, these data indicate that RAS could function as microbial community and water quality management strategy in the larviculture of crab.

Optimizing Anesthetic Practices for Mud Crab: A Comparative Study of Clove Oil, MS-222, Ethanol, and Magnesium Chloride.

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

Rise and metabolic roles of Vibrio during the fermentation of crab paste.

Microbial community may systematically promote the development of fermentation process of foods. Traditional fermentation is a spontaneous natural process that determines a unique nutritional characteristic of crab paste of Portunus trituberculatus, However, rare information is available regarding the development pattern and metabolic role of bacterial community during the fermentation of crab paste. Here, using a 16S rRNA gene amplicon sequencing technology, we investigated dynamics of bacterial community and its relationship with metabolites during the fermentation of crab paste. The results showed that bacterial community changed dynamically with the fermentation of crab paste which highlighted by consistently decreased α-diversity and overwhelming dominance of Vibrio at the later days of fermentation. Vibrio had a positive correlation with trimethylamine, hypoxanthine, formate, and alanine while a negative correlation with inosine and adenosine diphosphate. In contrast, most of other bacterial indicators had a reverse correlation with these metabolites. Moreover, Vibrio presented an improved function potential in the formation of the significantly increased metabolites. These findings demonstrate that the inexorable rise of Vibrio not only drives the indicator OTUs turnover in the bacterial community but also has incriminated the quality of crab paste from fresh to perished.

Adaptive changes of swimming crab (Portunus trituberculatus) associated bacteria helping host against dibutyl phthalate toxification.

The pollution of dibutyl phthalate (DBP) in aquatic environments is becoming an extensive environmental problem and detrimental to aquatic animals. Here, we quantified the response pattern of the bacterial community and metabolites of swimming crab (Portunus trituberculatus) juveniles exposed to 0.2, 2, and 10 mg/L DBP using 16 S rRNA gene amplicon sequencing coupled with metabolomic technique. The results showed that DBP changed the bacterial community compositions in a concentration-dependent pattern and decreased the Shannon index at the second developmental stage of the swimming crabs. The Rhodobacteraceae taxa were specifically enriched by crabs when challenged by 2 and 10 mg/L DBP, with an increased in Shannon index and enhanced drift in its assembly. Moreover, DBP changed the metabolic profiling of the swimming crab, highlighted by increased levels of lactate, valine, methionine, lysine, and phenylalanine in the 10 mg/L DBP-exposed crabs. Rhodobacteraceae presented the most considerable contribution to the metabolic potentials in phthalate and benzoate degradation, lactate production, and amino acid biosynthesis. Overall, our results indicated an adaptive change of crab-associated bacteria helped the host resist DBP stress. The findings extend our insights into the relationship between the microbiota and its host metabolism under DBP stress and reveal the potential microbiota modalities for DBP detoxification.

Time-Restricted Feeding Could Not Reduce Rainbow Trout Lipid Deposition Induced by Artificial Night Light.

Artificial night light (ALAN) could lead to circadian rhythm disorders and disrupt normal lipid metabolism, while time-restricted feeding (TRF) could maintain metabolic homeostasis. In mammals, TRF has been demonstrated to have extraordinary effects on the metabolic regulation caused by circadian rhythm disorders, but studies in lower vertebrates such as fish are still scarce. In this study, the impacts of ALAN on the body composition and lipid metabolism of juvenile rainbow trout were investigated by continuous light (LL) exposure as well as whether TRF could alleviate the negative effects of LL. The results showed that LL upregulated the expression of lipid synthesis (fas and srebp-1c) genes and suppressed the expression of lipid lipolysis (pparß, cpt-1a, and lpl) genes in the liver, finally promoting lipid accumulation in juvenile rainbow trout. However, LL downregulated the expression of genes (Δ6-fad, Δ9-fad, elovl2, and elovl5) related to long-chain polyunsaturated fatty acid (LC-PUFA) synthesis, resulting in a significant decrease in the proportion of LC-PUFA in the dorsal muscle. In serum, LL led to a decrease in glucose (Glu) levels and an increase in triglyceride (TG) and high-density lipoprotein cholesterol (H-DLC) levels. On the other hand, TRF (mid-dark stage feeding (D)) and mid-light stage feeding (L)) upregulated the expression of both the lipid synthesis (srebp-1c and pparγ), lipolysis (pparα, pparß, and cpt-1a), and lipid transport (cd36/fat and fatp-1) genes, finally increasing the whole-body lipid, liver protein, and lipid content. Meanwhile, TRF (D and L groups) increased the proportion of polyunsaturated fatty acid (PUFA) and LC-PUFA in serum. In contrast, random feeding (R group) increased the serum Glu levels and decreased TG, total cholesterol (T-CHO), and H-DLC levels, suggesting stress and poor nutritional status. In conclusion, ALAN led to lipid accumulation and a significant decrease in muscle LC-PUFA proportion, and TRF failed to rescue these negative effects.

ATP catabolism and bacterial succession in postmortem tissues of mud crab (Scylla paramamosain) and their roles in freshness.

The seafood microbiome is highly diverse and plays an essential role in the spoilage of seafood. Nevertheless, how such a diverse microbiome influences freshness of mud crab (Scylla paramamosain) remains unclear. Here we investigated the postmortem ATP catabolism and succession of the bacterial community in the hepatopancreas and muscle of S. paramamosain using a high-performance liquid chromatography method and 16S rRNA gene amplicon sequencing. Our results showed a tissue-dependent change in ATP catabolism determinized the differences in the changes of nucleotide freshness indices of hepatopancreas and muscle over postmortem time of mud crab. The muscle K value could be used as an optimal nucleotide freshness indicator for the freshness of mud crab, with a proposed threshold of 20%. From a microbiota perspective, a more significant bacterial community change was observed in the muscle than in the hepatopancreas. These changes could result in a close relationship between ATP and its catabolites and microbial taxa in the muscle. Moreover, Photobacterium, Peptostreptococcaceae, average path distance, OTU richness, and Shannon index of muscle bacterial community markedly contributed to K value. These findings suggest that the mud crab of 4 h postmortem at room temperature is still edible. Notably, the importance of microbial community composition and interaction for the spoilage of mud crab should be carefully considered.

Accumulation, detoxification, and toxicity of dibutyl phthalate in the swimming crab.

Dibutyl phthalate (DBP) is one of the most commonly used and toxic phthalate esters and has a variety of harmful effects on aquatic animals. However, there is still a lack of knowledge on the accumulation, detoxification, and toxicity of DBP in aquatic animals. In this study, we chose the swimming crab Portunus trituberculatus, an ecologically and economically important species, as the model and investigated the metabolism of DBP and its effects on the detoxification, antioxidation, survival and growth of the crab juveniles to better understand DBP-triggered molecular response over different time courses. As a result, DBP could be accumulated in the swimming crab in a concentration-dependent manner and metabolized to monobutyl phthalate (MBP) and phthalic acid (PA) through de-esterification. DBP exposure induced the different responses of three cytochrome P450 members and antioxidant enzyme genes, enhanced gene transcript and protein levels of glutathione-S-transferase and two heat stress proteins and malondialdehyde accumulation, decreased glutathione level, and inhibited antioxidant enzyme activities. Further, no significant effect of DBP was observed in crab survival, size, and weight but there was molting retardation. Therefore, DBP induced strong detoxification and antioxidative defense mechanisms to overcome detrimental effects of DBP on the swimming crab juveniles despite a molting retardation as a trade-off in fitness costs. The prevalent coexistence of DBP with MBP and PA during the whole exposure period is raising concerns on the combined action and ecological risk to aquatic animals.

Elevated pCO2 alters the interaction patterns and functional potentials of rearing seawater microbiota.

Mean oceanic CO2 values have already risen and are expected to rise further on a global scale. Elevated pCO2 (eCO2) changes the bacterial community in seawater. However, the ecological association of seawater microbiota and related geochemical functions are largely unknown. We provide the first evidence that eCO2 alters the interaction patterns and functional potentials of microbiota in rearing seawater of the swimming crab, Portunus trituberculatus. Network analysis showed that eCO2 induced a simpler and more modular bacterial network in rearing seawater, with increased negative associations and distinct keystone taxa. Using the quantitative microbial element cycling method, nitrogen (N) and phosphorus (P) cycling genes exhibited the highest increase after one week of eCO2 stress and were significantly associated with keystone taxa. However, the functional potential of seawater bacteria was decoupled from their taxonomic composition and strongly coupled with eCO2 levels. The changed functional potential of seawater bacteria contributed to seawater N and P chemistry, which was highlighted by markedly decreased NH3, NH4+-N, and PO43--P levels and increased NO2--N and NO3--N levels. This study suggests that eCO2 alters the interaction patterns and functional potentials of seawater microbiota, which lead to the changes of seawater chemical parameters. Our findings provide new insights into the mechanisms underlying the effects of eCO2 on marine animals from the microbial ecological perspective.

Effects of Elevated pCO2 on the Survival and Growth of Portunus trituberculatus.

Identifying the response of Portunus trituberculatus to ocean acidification (OA) is critical to understanding the future development of this commercially important Chinese crab species. Recent studies have reported negative effects of OA on crustaceans. Here, we subjected swimming crabs to projected oceanic CO2 levels (current: 380 µatm; 2100: 750 µatm; 2200: 1500 µatm) for 4 weeks and analyzed the effects on survival, growth, digestion, antioxidant capacity, immune function, tissue metabolites, and gut bacteria of the crabs and on seawater bacteria. We integrated these findings to construct a structural equation model to evaluate the contribution of these variables to the survival and growth of swimming crabs. Reduced crab growth shown under OA is significantly correlated with changes in gut, muscle, and hepatopancreas metabolites whereas enhanced crab survival is significantly associated with changes in the carbonate system, seawater and gut bacteria, and activities of antioxidative and digestive enzymes. In addition, seawater bacteria appear to play a central role in the digestion, stress response, immune response, and metabolism of swimming crabs and their gut bacteria. We predict that if anthropogenic CO2 emissions continue to rise, future OA could lead to severe alterations in antioxidative, immune, and metabolic functions and gut bacterial community composition in the swimming crabs through direct oxidative stress and/or indirect seawater bacterial roles. These effects appear to mediate improved survival, but at the cost of growth of the swimming crabs.

Effect of Rhizophora apiculata plantation for improving water quality, growth, and health of mud crab.

A deteriorated water quality is closely associated with disease outbreaks in aquaculture, where microorganisms play indispensable roles in improving water quality and aquatic animals' health. Mangrove is known to be a natural water quality filter and microbiological buffer of pathogen and prebiotics. However, it is unclear how and to what extent Rhizophora apiculata plantation is of benefits to the gut microbiota and growth over mud crab (Scylla paramamosain) aging. To address these concerns, we explored the bacterial communities in mud crab gut and rearing water at 45, 114, and 132 days after incubation, roughly corresponding to juvenile, pre-adult, and adult stages of mud crab. Results showed that 1-year R. apiculata plantation slightly increased the body weight of mud crab and improved water quality to a certain extent. Both bacterioplankton and gut bacterial communities were highly temporal dynamic, while the two communities were significantly distinct (ANOSIM r = 0.90, P = 0.0001). Relative abundances of dominant taxa in water and gut significantly varied between the plantation and the control conditions over mud crab aging. R. apiculata plantation promoted the stability of gut microbiota, as evidenced by more diverse core species. Furthermore, R. apiculata plantation led to the dominance of Verrucomicrobiae species in water and probiotic Bacteroidetes and Lactobacillales taxa in gut. A structural equation model revealed that water variables directly constrained gut microbiota, which in turn affected the body weight of mud crab (r = 0.52, P < 0.001). In addition, functional pathways facilitating immunity and lipid metabolism significantly increased in mud crab gut under the plantation, while those involved in infectious diseases exhibited the opposing trend. These findings greatly expand our understanding of the R. apiculata plantation effects on water quality, gut microbiota, and growth feature of mud crab. Overall, R. apiculata plantation is beneficial for mud crab growth and health. KEY POINTS: • A short-term R. apiculata plantation could potentially improve water quality. • Bacterioplankton is more sensitive than mud crab gut microbiota in response to R. apiculata plantation. • R. apiculata plantation enhances mud crab resistance against pathogen invasion. • R. apiculata plantation alters mud crab gut microbiota, which in turn promotes their body weight.

NMR-based metabolomics profiling of no-added-nitrite Chinese bacon (unsmoked) during processing.

Variations in the taste quality of no-added-nitrite Chinese bacon (unsmoked) during processing were investigated using 1 H-NMR and multivariate data analysis. The results showed that 21 metabolites were dominant during processing, which involved marinating, air-drying, fermentation, and baking, including amino acids, sugars, organic acids, nucleic acids and their derivatives, and alkaloids. The contents of isoleucine, leucine, valine, alanine, acetate, glutamate, succinate, glycine, sucrose, tyrosine, and phenylalanine increased continuously throughout the process. The lactate, creatine, carnosine, betaine, taurine, hypoxanthine, and AMP contents all significantly increased after baking; the inosine content significantly increased after fermentation and then decreased; the histamine content significantly increased after air-drying and then decreased; and the histidine content decreased. Each processing treatment promoted taste formation in no-added-nitrite Chinese bacon (unsmoked), especially baking. The baking point owned relatively higher levels of metabolites and sensory evaluation compared to other treatments. Sensory evaluation revealed that the ultimate taste of Chinese bacon (unsmoked) at the end of baking tended toward umami (glutamate), sweetness (AMP), and sourness (lactate). The first and second principal components explained 74.0% and 13.4% of the variables, respectively. These findings indicated the potential of NMR-based metabolomics for assessing the taste quality of no-added-nitrite Chinese bacon (unsmoked), which could contribute to a better understanding of taste compound changes in meat products. PRACTICAL APPLICATION: Nitrite is commonly used in Chinese bacon (unsmoked), but excessive intake is not good for human health. Nitrite has been replaced with nitrite substitutes to prepare no-added-nitrite Chinese bacon (unsmoked). The metabolites of no-added-nitrite Chinese bacon (unsmoked) were detected to determine the key treatment that contributes to the formation of taste during processing. This study determined the main taste components of no-added-nitrite Chinese bacon (unsmoked) and its formation process, which provides new insight into the production and characteristics of flavor in Chinese bacon (unsmoked).

Identification and characterization of a novel galectin from the mud crab Scylla paramamosain.

Galectins are a family of ß-galactoside-binding lectins that play key roles in the invertebrate innate immunity system, but no galectin genes have been identified in the mud crab (Scylla paramamosain) so far. The present study is the first to clone a galectin gene (SpGal) from S. paramamosain, by the rapid amplification of cDNA ends technique based on expressed sequence tags. The full-length cDNA of SpGal was 3142 bp. Its open reading frame encoded a polypeptide of 280 amino acids containing a GLECT/Gal-bind lectin domain and a potential N-glycosylation site. The deduced amino acid sequence and multi-domain organization of SpGal were highly similar to those of invertebrate galectins, and phylogenetic analysis showed that SpGal was closely related to galectin isolated from Portunus trituberculatus. The mRNA transcripts of SpGal were found to be constitutively expressed in a wide range of tissues, with its expression level being higher in the hepatopancreas, gill, and hemocytes. The mRNA expression level of SpGal increased rapidly after the crabs were stimulated by Vibrio alginolyticus, and the maximum expression appeared at 6 h after the challenge. The lipopolysaccharide-binding ability of SpGal was dependent on its concentration, and it also exhibited agglutination activity with three Gram-negative (Aeromonas hydrophila, Chryseobacterium indologenes and Vibrio alginolyticus) and three Gram-positive (Bacillus aquimaris, Staphylococcus aureus and Micrococcus lysodeik) bacterial strains. In addition, hemagglutination activity with rabbit erythrocytes was observed in the absence of d-galactose. These results indicate that SpGal in S. paramamosain acts as a pattern recognition receptor to recognize a broad spectrum of microbes. The findings together indicate that SpGal plays an important role in the innate immune mechanisms of S. paramamosain against pathogenic infection.

Characterization of PHB in the gonadal development of the swimming crab Portunus trituberculatus.

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities.

Molecular cloning, characterization, and expression of a C-type lectin from Scylla paramamosain, which might be involved in the innate immune response.

C-type lectins (CTLs) have characteristic carbohydrate recognition domains (CRDs) and play important roles in the immune system. In the present study, a new CTL, SpCTL5, was identified from the hepatopancreas of the mud crab Scylla paramamosain. The open reading frame of SpCTL5 comprised 762 bp, encoding a polypeptide of 253 amino acids with a putative signaling peptide of 20 amino acids. The predicted SpCTL5 protein contained a single CRD. SpCTL5 transcripts were distributed in all examined tissues, with the highest level being detected in the hepatopancreas. Upon challenging with Vibrio alginolyticus, the mRNA levels of SpCTL5 in the hepatopancreas were up-regulated. The recombinant protein of SpCTL5 could agglutinate three Gram-positive bacteria and three Gram-negative bacteria in the presence of Ca2+. Furthermore, hemagglutination analysis showed that the recombinant protein of SpCTL5 can agglutinate rabbit erythrocytes. This study indicated that SpCTL5 acts as a pattern recognition receptor for the innate immune response which protects S. paramamosain from bacterial infection. Moreover, these findings also provide information to further our understanding of the innate immunology of invertebrates.

An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge.

Anti-lipopolysaccharide factors are effective antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In the present study, a new sequence encoding for ALF (designated as PtALF8) was cloned by suppression subtractive hybridization method using ovary of swimming crab Portunus trituberculatus as material. The full-length cDNA of PtALF8 consisted of 531 bp with an ORF of 348 bp encoding a peptide of 115 amino acids containing a putative signal peptide of 19 amino acids. The mature PtALF8 had a predicted molecular weight (MW) of 11.28 kDa and theoretical isoelectricpoint (pI) of 5.11. The PtALF8 contains an MBT domain which was not found in the other 7 isoforms of ALF reported in P. trituberculatus. Unlike most ALFs expressed in hemocytes, PtALF8 transcript was predominantly detected in hepatopancreas. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF8 transcript in hemocytes reached the highest level at 3 h, then decreased to the lowest level at 24 h, and started to increase at 48 h. The recombinant protein showed antimicrobial and bactericidal activity against several bacteria, such as Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria, V. alginolyticus, indicated that the PtALF8 isoform might play protective function against invading bacteria in P. trituberculatus.

1H NMR and multivariate data analysis of the differences of metabolites in five types of dry-cured hams.

In order to distinguish the taste styles of dry-cured hams (Jinhua, Xuanwei, Country, Parma and Bama), we established a 1H nuclear magnetic resonance spectroscopy method to identify metabolites. Totally, 33 charged metabolites, including amino acids, organic acids, nucleic acids and their derivatives, sugars, alkaloids and others were identified. The abundant glutamate, lysine, alanine, leucine and lactate could be the major contributors of taste. Total variables were explained by PC1 (67.7%) and PC2 (16.0%) which showed that Parma and Xuanwei styles were close to each other (similar amino acids, peptide, organic acids and alkaloids contents). Bama style showed the highest PC1 and amino acids, organic acids and alkaloids contents. Country style was located on the left-most area of PC1 (the lowest amino acids, organic acids and peptide, but the highest sugars contents). Sensory evaluation revealed that Bama ham had the highest overall taste score, followed by Jinhua, Parma, Xuanwei and American Country ham. We concluded that the proportions and combinations of taste components explained the specific taste instead of any single component. These findings provided a better understanding of different metabolomics among hams.

Characterisation and functional analysis of an L-type lectin from the swimming crab Portunus trituberculatus.

L-type lectins are involved in glycoprotein secretion and are associated with immune responses. Herein, an L-type lectin was identified in swimming crab (Portunus trituberculatus). The 1347 bp PtLTL cDNA includes a 26 bp 5'-untranslated region (UTR), a 547 bp 3'-UTR with a poly(A) tail, and a 774 bp open reading frame encoding a 257 amino acid protein with a putative 21 residue signalling peptide. The protein includes an L-type lectin carbohydrate recognition domain containing four conserved cysteines. The 714 bp cDNA fragment encoding the mature peptide of PtLTL1 was recombined into pET-21a (+) with a C-terminally hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). Recombinant PtLTL1 caused agglutination of all three Gram-positive and Gram-negative bacterial strains tested. In addition, erythrocyte agglutination and LPS-binding activity were observed. PtLTL1 mRNA transcripts were most abundant in P. trituberculatus hepatopancreas and hemocytes, and expression was up-regulated in hemocytes challenged with Vibrio alginolyticus, suggesting PtLTL functions in the immune response against bacterial pathogens.

Transcriptomic analysis of Portunus trituberculatus reveals a critical role for WNT4 and WNT signalling in limb regeneration.

The swimming crab (Portunus trituberculatus) is among the most economically important seawater crustacean species in Asia. Despite its commercial importance and being well-studied status, genomic and transcriptomic data are scarce for this crab species. In the present study, limb bud tissue was collected at different developmental stages post amputation for transcriptomic analysis. Illumina RNA-sequencing was applied to characterise the limb regeneration transcriptome and identify the most characteristic genes. A total of 289,018 transcripts were obtained by clustering and assembly of clean reads, producing 150,869 unigenes with an average length of 956 bp. Subsequent analysis revealed WNT signalling as the key pathway involved in limb regeneration, with WNT4 a key mediator. Overall, limb regeneration appears to be regulated by multiple signalling pathways, with numerous cell differentiation, muscle growth, moult, metabolism, and immune-related genes upregulated, including WNT4, LAMA, FIP2, FSTL5, TNC, HUS1, SWI5, NCGL, SLC22, PLA2, Tdc2, SMOX, GDH, and SMPD4. This is the first experimental study done on regenerating claws of P. trituberculatus. These findings expand existing sequence resources for crab species, and will likely accelerate research into regeneration and development in crustaceans, particularly functional studies on genes involved in limb regeneration.

Molecular cloning of a C-type lectin from Portunus trituberculatus, which might be involved in the innate immune response.

C-type lectin plays an important role in the innate immune response of crustaceans including Portunus trituberculatus which is an important marine species. In the present study, we cloned the full length of a C-type lectin (designated as PtCTL4) from P. trituberculatus via 3'RACE. The full length of the nucleic acid sequence has a length of 654 bp including an open reading frame (ORF) of 480 bp. PtCTL4 possesses conserved CTL features, while containing a CRD domain with Ca2+ binding site 2 and six conserved cysteine residues. Quantitative RT-PCR analysis showed that PtCTL4 expression level was highest in the hepatopancreas, while it was relatively low in other tissues such as hemocytes, eyestalk, muscle, and gonad. The expression level of PtCTL4 reached a maximum at 3 h after challenge with Vibrio alginolyticus, then decreased to the lowest level at 12 h, and returned to normal level at 48 h. Hemagglutination analysis showed that the recombinant PtCTL4 (rPtCTL4) can agglutinate rabbit erythrocyte. The rPtCTL4 can agglutinate Gram-positive bacteria (Bacillus aquimaris, Micrococcus lysodeik, and Staphylococcus aureus) and Gram-negative bacteria (Aeromonas hydrophila, V. alginolyticus, and Chryseobacterium indologenes) in the presence of Ca2+. This study indicated that PtCTL4 acts as a pattern recognition receptor in the innate immune response of P. trituberculatus.