The function of the cytoplasmic dynein light chain PTKM23 in the transport of PTSMAD2 during spermatogenesis in Portunus trituberculatus.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFß signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-Tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-Tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis. This study provides a theoretical molecular biological basis for the breeding of P. trituberculatus.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus.

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.

The Functions of Pt-DIC and Pt-Lamin B in Spermatogenesis of Portunus trituberculatus.

Cytoplasmic Dynein is a multiple-subunit macromolecular motor protein involved in the transport process of cells. The Dynein intermediate chain (DIC) is one of the subunits of Dynein-1. In our previous studies, we showed that Pt-DIC may play an important role in the nuclear deformation of spermiogenesis in Portunus trituberculatus. Lamin B is essential for maintaining nuclear structure and functions. Surprisingly, Pt-Lamin B was expressed not only in the perinucleus but also in the pro-acrosome during spermiogenesis in P. trituberculatus. Studies have also shown that Dynein-1 can mediate the transport of Lamin B in mammals. Thus, to study the relationship of Pt-DIC and Pt-Lamin B in the spermatogenesis of P. trituberculatus, we knocked down the Pt-DIC gene in P. trituberculatus by RNAi. The results showed that the distribution of Pt-DIC and Pt-Lamin B in spermiogenesis was abnormal, and the colocalization was weakened. Moreover, we verified the interaction of Pt-DIC and Pt-Lamin B via coimmunoprecipitation. Therefore, our results suggested that both Pt-DIC and Pt-Lamin B were involved in the spermatogenesis of P. trituberculatus, and one of the functions of Dynein-1 is to mediate the transport of Lamin B in the spermiogenesis of P. trituberculatus.

The iTRAQ-based quantitative proteomics reveals metabolic changes in Scylla paramamosain under different light intensities during indoor overwintering.

Light intensity is one of the ecological factors that appreciably affects the metabolism of Scylla paramamosain during overwintering. This study adopted the isobaric tag for relative and absolute quantitation (iTRAQ) method to investigate metabolic changes of S. paramamosain under three illumination levels (0, 1.43 and 40.31 µmol m-2·s-1) for four months during indoor overwintering. The iTRAQ identified 3282 proteins, among which 267 exhibited significant differential expression (122 upregulated and 145 downregulated) in the low light group, and 299 with significant differential expression (252 upregulated and 47 downregulated) in the high light group. Analysis of these results showed that there were different metabolic regulatory patterns under different light intensities. Low light is more conducive to the survival of S. paramamosain, which needs to produce and consume relatively less energy to sustain physiological activities. Thus, the essential proteins associated with physiological activities were significantly upregulated, while those related to energy production were significantly downregulated. In contrast, high light exerts a certain stress on the survival of S. paramamosain and required more energy to cope with this stress, which forced a significant upregulation of proteins related to stress response and energy production. The findings of this study highlighted the metabolic regulatory mechanisms of S. paramamosain under different light intensities.

Transcriptome analysis of genes and pathways associated with metabolism in Scylla paramamosain under different light intensities during indoor overwintering.

BACKGROUND: Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla, which is commonly distributed along the coasts of China, Vietnam, and Japan. Genomic and transcriptomic data are scarce for the mud crab. Light intensity is one of the ecological factors that affect S. paramamosain during indoor overwintering. To understand the energy metabolism mechanism adapted to light intensity, we analyzed the transcriptome of S. paramamosain hepatopancreas in response to different light intensities (0, 1.43, 40.31 µmol·m- 2·s- 1). RESULTS: A total of 5052 differentially expressed genes were identified in low light group (LL group, 3104 genes were up-regulated and 1948 genes were down-regulated). A total of 7403 differentially expressed genes were identified in high light group (HL group, 5262 genes were up-regulated and 2141 genes were down-regulated). S. paramamosain adapts to different light intensity environments through the regulation of amino acids, fatty acids, carbon and energy metabolism. Different light intensities had a strong impact on the energy generation of S. paramamosain by influencing oxygen consumption rate, aerobic respiration, glycolysis/gluconeogenesis pathway, the citrate cycle (TCA cycle) and fatty acid degradation. CONCLUSION: Low light is more conducive to the survival of S. paramamosain, which needs to produce and consume relatively less energy to sustain physiological activities. In contrast, S. paramamosain produced more energy to adapt to the pressure of high light intensities. The findings of the study add to the knowledge of regulatory mechanisms related to S. paramamosain metabolism under different light intensities.

Antibacterial activities and mechanisms of action of a defensin from manila clam Ruditapes philippinarum.

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum.

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.

High-throughput sequencing reveals the core gut microbiota of the mud crab (Scylla paramamosain) in different coastal regions of southern China.

BACKGROUND: Scylla paramamosain is a commercially important mud crab. The microbiota is a community that inhabits the crab intestine, and is important for physiological functional and host health. RESULTS: Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria were the dominant phyla of the 36 representative phyla. Eleven genera of the 820 representative genera were considered as core gut microbiota and were distributed in the five dominant phyla. The core genus of the Proteobacteria included Arcobacter, Photobacterium, Vibrio, Shewanella and Desulfovibrio. The other four phyla contained one or two genera. Male and female crab samples had two different core genera, (male samples: Psychrilyobacter & Lactococcus; female samples: Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma). CONCLUSIONS: This is the first time core intestinal microbiota have been identified in crab from nine coastal regions of southern China. This study provides sequencing data related to the gut microbiota of S. paramamosain, and may contribute to probiotic development for S. paramamosain aquaculture industries.

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.

Transcriptomic Analysis of Marine Gastropod Hemifusus tuba Provides Novel Insights into Conotoxin Genes.

The marine gastropod Hemifusus tuba is served as a luxury food in Asian countries and used in traditional Chinese medicine to treat lumbago and deafness. The lack of genomic data on H. tuba is a barrier to aquaculture development and functional characteristics of potential bioactive molecules are poorly understood. In the present study, we used high-throughput sequencing technologies to generate the first transcriptomic database of H. tuba. A total of 41 unique conopeptides were retrieved from 44 unigenes, containing 6-cysteine frameworks belonging to four superfamilies. Duplication of mature regions and alternative splicing were also found in some of the conopeptides, and the de novo assembly identified a total of 76,306 transcripts with an average length of 824.6 nt, of which including 75,620 (99.1%) were annotated. In addition, simple sequence repeats (SSRs) detection identified 14,000 unigenes containing 20,735 SSRs, among which, 23 polymorphic SSRs were screened. Thirteen of these markers could be amplified in Hemifusus ternatanus and seven in Rapana venosa. This study provides reports of conopeptide genes in Buccinidae for the first time as well as genomic resources for further drug development, gene discovery and population resource studies of this species.

Identification and characterization of miRNAs in the gills of the mud crab (Scylla paramamosain) in response to a sudden drop in salinity.

BACKGROUND: The mud crab (Scylla paramamosain) is a euryhaline and commercially important species. MiRNAs participate in the regulation of many physiological activities. RESULTS: The miRNA transcriptome of the gills of S. paramamosain was used to investigate the expression profiles of miRNAs in response to a sudden drop in salinity. In total, seven known miRNAs and 43 novel miRNAs were identified, with 18 differentially expressed small RNAs. Fourteen thousand nine hundred fifty-one differentially expressed miRNAs target genes were screened by prediction. GO analysis of differentially expressed miRNAs target genes indicated that 578 genes associated with cellular processes, 523 associated with metabolic processes, and 422 associated with single-organism processes were the most strongly affected by a sudden drop in salinity from 23‰ to 3‰. KEGG pathway analysis showed 14 pathways were related to amino acid metabolism, which plays an important role in osmoregulation. Besides, several pathways were associated with starch and sucrose metabolism (ko00500), glycosaminoglycan degradation (ko00531), and galactose metabolism (ko00052). CONCLUSIONS: S. paramamosain regulated osmotic pressure and energy balance by regulating target genes to adapt to a sudden changes in salinity. These results provided a basis for further investigations of miRNA-modulating networks underlying the osmoregulation of S. paramamosain.

Transcriptomic analysis of adaptive mechanisms in response to sudden salinity drop in the mud crab, Scylla paramamosain.

BACKGROUND: Scylla paramamosain (Crustacea: Decapoda: Portunidae: Syclla De Hann) is a commercially important mud crab distributed along the coast of southern China and other Indo-Pacific countries (Lin Z, Hao M, Zhu D, et al, Comp Biochem Physiol B Biochem Mol Biol 208-209:29-37, 2017; Walton ME, Vay LL, Lebata JH, et al, Estuar Coast Shelf Sci 66(3-4):493-500, 2006; Wang Z, Sun B, Zhu F, Fish Shellfish Immunol 67:612-9, 2017). While S. paramamosain is a euryhaline species, a sudden drop in salinity induces a negative impact on growth, molting, and reproduction, and may even cause death. The mechanism of osmotic regulation of marine crustaceans has been recently under investigation. However, the mechanism of adapting to a sudden drop in salinity has not been reported. METHODS: In this study, transcriptomics analysis was conducted on the gills of S. paramamosain to test its adaptive capabilities over 120 h with a sudden drop in salinity from 23 ‰ to 3 ‰. RESULTS: At the level of transcription, 135 DEGs (108 up-regulated and 27 down-regulated) annotated by NCBI non-redundant (nr) protein database were screened. GO analysis showed that the catalytic activity category showed the most participating genes in the 24 s-tier GO terms, indicating that intracellular metabolic activities in S. paramamosain were enhanced. Of the 164 mapped KEGG pathways, seven of the top 20 pathways were closely related to regulation of the Na+ / K+ -ATPase. Seven additional amino acid metabolism-related pathways were also found, along with other important signaling pathways. CONCLUSION: Ion transport and amino acid metabolism were key factors in regulating the salinity adaptation of S. paramamosain in addition to several important signaling pathways.

Molecular characterization, expression, and antibacterial activity of a c-type lysozyme isolated from the manila clam, Ruditapes philippinarum.

Lysozyme is an antibacterial enzyme that is widely distributed in nature and plays an important role in the prevention of bacterial infections. In this study, a c-type lysozyme (designated as "RpCLYZ") was cloned and characterized from the manila clam, Ruditapes philippinarum. The full-length cDNA was 619 bp with an open reading frame (ORF) of 447 bp, and encoded a polypeptide of 148 amino acids with a calculated molecular mass of 17.0 kDa and an isoelectric point of 4.83. RpCLYZ was found to share high sequence similarity with c-type lysozymes from other invertebrates. The mRNA transcript of RpCLYZ was universally expressed in a wide range of tissues, especially in gills and mantle. Challenge with Vibrio anguillarum, significantly induced mRNA expression of RpCLYZ, which reached a maximum level 48 h after bacterial challenge. Recombinant RpCLYZ (rRpCLYZ) exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria. Additionally, the optimal pH and temperature for rRpCLYZ activity were determined to be 4.5 and 20 °C, respectively. These results suggest that RpCLYZ participates in innate immune responses against bacterial invasion.

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.

Effects of melanin from Sepiella Maindroni ink (MSMI) on the intestinal Microbiome of mice.

BACKGROUND: By the search for new natural compounds with beneficial health effects, cephalopod ink has been considered as an attempt to develop new drugs and functional foods, which is an especially active field in Asia, where cephalopods are a major fishery catch, for which ink sacs are a bi-product and where homeopathic medicine has deep roots. There is a demand to evaluate the safety and influence to the organism. The specific composition and relative abundance of the gut microbiota, which is potentially a major modulator of host metabolism, drives the interaction between functional foods and host health. We explore the effects of melanin from Sepiella Maindroni, most common cuttlefish in China, on the intestinal microbiome of mice. RESULTS: ICR mice were randomly divided four groups, which were normal group (S), low melanin dose group (D; 120 mg/kg), medium melanin dose group (Z; 240 mg/kg), and high melanin dose group (G; 480 mg/kg). Melanin was delivered for 28 consecutive days. Fecal samples were used to generate 7715 operational taxonomic units (OTUs) via high-throughput sequencing. There were significant shifts in relative abundance of the dominant taxa at the phylum, class, order, family, and genus levels following melanin treatment. CONCLUSIONS: MSMI had no significant effect on the structure of intestinal flora in mice. The main effect was in the proportion of dominant bacterial communities. The effect positively correlated with the dose. From a health point of view, the use of melanin does not cause intestinal flora disorder. Our results may have important implications for MSMI as functional food component and potential therapeutic for manipulating gut microbiota.

Molecular cloning and characterization of a hemocyanin from Sepiella maindroni.

Hemocyanins are respiratory proteins occurring freely dissolved in the hemolymph of many arthropods and molluscs. Hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity. In the present study, the full-length cDNA encoding hemocyanin in Sepiella maindroni (SmHc) was cloned and characterized. Bioinformatic analysis predicted that SmHc contains one open reading frame of 10,032 bp and encodes a polypeptide of 3343 amino acids. Sequence analysis showed that the predicted protein sequence of SmHc contained eight functional units (FUs). Phylogenic analysis revealed that SmHc clustered with the mollusc Hcs. Quantitative real-time PCR assay detected SmHc transcripts were in a wide range of tissues, but mainly distributed in gills. After hypoxia or bacterial challenge, the expression level of SmHc in the gills was significantly higher than that of the control group. These results suggested that SmHc might play important roles in oxygen transport and the modulation of immune response in S. maindroni.

Peroxiredoxin 1 from cuttlefish (Sepiella maindroni): Molecular characterization of development and its immune response against Vibrio alginolyticus.

The aim of this work was constructive to understand the function of peroxiredoxin (PRDX) family member Peroxiredoxin 1 in Sepiella maindroni (SmPrx1) through molecular mechanisms of reproduction, embryonic development and immune responses to Vibrio alginolyticus. The full-length cDNA of SmPrx1 was of 1062 bp, contains a 5' untranslated region (UTR) of 79bp, a 3' UTR of 359 bp, an open reading frame of 624 bp encoding 207 amino acids. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of SmPrx1; this indicated that it was a member of 2-Cys Prx. Quantitative real-time (qRT)-PCR assays revealed that SmPrx1 was ubiquitously expressed in all examined tissues, muscle, ink sac, liver, ovary, testis, intestine, gill and totally blood cells, and showed high levels in testis. SmPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in testis, hemocyte, liver and ovary. Moreover, the SmPrx1 gene transcript was detected at all five stages of embryonic development phases that were respectively the zygote stage, the pre-embryonic stage, the organogenesis stage, the morphological integrity stage, the pre-hatching stage. The general tendency of expression was gradually increased and rapidly decreased. High expressed in progenitive tissues and embryonic development exhibit the proliferation-associated protein characterization like in mammal. The expression levels of SmPrx1 in liver and hemocytes grew swiftly and quickly reached peak value after Vibrio alginolyticus challenge. As hours passed by, the expression level began to reduce and resumed to normal levels after 48 h. The antioxidant activity and peroxidase activity of SmPrx1 were 6.17 U/mg. The results showed that the recombined protein of SmPrx1 had antioxidant activity and was the importance part of the antioxidant system in Sepiella maindroni. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine cephalopod immunity.

Characterization and functional analysis of a novel C-type lectin from the swimming crab Portunus trituberculatus.

C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full-length cDNA of PtCTL1 was of 702 bp, containing a 5' untranslated region (UTR) of 91 bp, a 3' UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca2+-dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca2+-dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate.