Eastern king prawn Penaeus plebejus stock enhancement-Genetic evidence that hatchery bred prawns have survived in the wild after release.

Eastern king prawn (Penaeus plebejus) is endemic to eastern Australia and is of high commercial and recreational value. As part of a recreational fisheries enhancement initiative, hatchery reared juveniles from Queensland were released into two, more Southern New South Wales (NSW) estuaries between 2014 and 2015. Responsible stock enhancement programs rely on knowledge of the population structure of the released species. Previously, in consideration of fisheries data, it was assumed the king prawn populations in Australia are one single breeding stock. In the present study, our first aim was to test this posit of no genetic differentiation using mtDNA control region (mtCR) sequences from the wild samples collected from four estuaries ranging from Queensland/NSW border (source of the stocked animals) to Southern NSW. The second objective was to test for signals of hatchery-released animals in the two stocked estuaries. All four surveyed populations had an extremely high level of haplotype diversity (average h = 99.8%) and low level of haplotype sharing between populations. Estimates of PhiPT values were <0.01 or close to zero and AMOVA test did not indicate any significant differences among populations. Further, phylogenetic analysis and principal coordinate analysis did not support division of samples by population. Collectively these results suggest that eastern king prawn populations along the NSW coast can be considered as a single stock and stocking from the Queensland samples will not necessarily impact the genetic composition of the overall stock. After stocking of two estuaries, sharing of haplotypes was moderate to very high in the stocked sites (>80% in some collections) but negligible in the two unstocked estuaries (≤2%, which is assumed to be background coancestry unrelated to the hatchery). Moreover, some haplotypes present in the hatchery broodstock were detected in stocked sites, but not in unstocked sites. The highest stocking signal was detected in the estuary which becomes isolated from the sea by sand barrier suggesting such "lakes" maybe more favourable for stocking than estuaries directly open to the sea. Findings in the current study should assist in designing and implementation of future prawn stocking programs.

First High-Density Linkage Map and Single Nucleotide Polymorphisms Significantly Associated With Traits of Economic Importance in Yellowtail Kingfish Seriola lalandi.

The genetic resources available for the commercially important fish species Yellowtail kingfish (YTK) (Seriola lalandi) are relative sparse. To overcome this, we aimed (1) to develop a linkage map for this species, and (2) to identify markers/variants associated with economically important traits in kingfish (with an emphasis on body weight). Genetic and genomic analyses were conducted using 13,898 single nucleotide polymorphisms (SNPs) generated from a new high-throughput genotyping by sequencing platform, Diversity Arrays Technology (DArTseqTM) in a pedigreed population comprising 752 animals. The linkage analysis enabled to map about 4,000 markers to 24 linkage groups (LGs), with an average density of 3.4 SNPs per cM. The linkage map was integrated into a genome-wide association study (GWAS) and identified six variants/SNPs associated with body weight (P < 5e-8) when a multi-locus mixed model was used. Two out of the six significant markers were mapped to LGs 17 and 23, and collectively they explained 5.8% of the total genetic variance. It is concluded that the newly developed linkage map and the significantly associated markers with body weight provide fundamental information to characterize genetic architecture of growth-related traits in this population of YTK S. lalandi.

Genomic prediction using DArT-Seq technology for yellowtail kingfish Seriola lalandi.

BACKGROUND: Genomic prediction using Diversity Arrays Technology (DArT) genotype by sequencing platform has not been reported in yellowtail kingfish (Seriola lalandi). The principal aim of this study was to address this knowledge gap and to assess predictive ability of genomic Best Linear Unbiased Prediction (gBLUP) for traits of commercial importance in a yellowtail kingfish population comprising 752 individuals that had DNA sequence and phenotypic records for growth traits (body weight, fork length and condition index). The gBLUP method was used due to its computational efficiency and it showed similar predictive performance to other approaches, especially for traits whose variation is of polygenic nature, such as body traits analysed in this study. The accuracy or predictive ability of the gBLUP model was estimated for three growth traits: body weight, folk length and condition index. RESULTS: The prediction accuracy was moderate to high (0.44 to 0.69) for growth-related traits. The predictive ability for body weight increased by 17.0% (from 0.69 to 0.83) when missing genotype was imputed. Within population prediction using five-fold across validation approach showed that the gBLUP model performed well for growth traits (weight, length and condition factor), with the coefficient of determination (R2) from linear regression analysis ranging from 0.49 to 0.71. CONCLUSIONS: Collectively our results demonstrated, for the first time in yellowtail kingfish, the potential application of genomic selection for growth-related traits in the future breeding program for this species, S. lalandi.

Regional genetic diversity for NNV grouper viruses across the Indo-Asian region - implications for selecting virus resistance in farmed groupers.

Grouper aquaculture around Asia is impacted by the nervous necrosis virus (NNV) and, in response, host resistance to this infection is being considered as a trait for selection. However efficient selection may be confounded if there are different genetic strains of NNV within and between regions and over years. This study uses statistical approaches and assessment of "characteristic attributes" (i.e. nucleotide positions that discriminate among strains) to assess whether published and new NNV RNA2 cds sequences show genetic differentiation over geography, host species and years. Rather clear evidence was found for regional strains of NNV. Interestingly, most of the geographic defining "characteristic attributes" were in codon position three, and not translated into differences for the protein capsid (i.e. they were synonymous variations), suggesting that while NNV strains were geographically isolated and had diverged in different regions for RNA sequences, selection had largely conserved the protein sequences among regions. The apparent selection constraint on the capsid protein may mitigate the risk that despite geographic subdivision, NNV strain variability will confound genetic selection for host resistance. The existence of regional Asian NNV strains may suggest that hatcheries are at risk from NNV not only from imported material but also from endemic reservoirs.

Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence.

Captive breeding programs and aquaculture production have commenced worldwide for the globally distributed yellowtail kingfish (Seriola lalandi), and captive bred fingerlings are being shipped from the Southern Hemisphere to be farmed in the Northern Hemisphere. It was recently proposed that Pacific S. lalandi comprise at least three distinct species that diverged more than 2 million years ago. Here, we tested the hypothesis of different "species" in the Pacific using novel genomic data (namely single nucleotide polymorphisms and diversity array technology markers), as well as mtDNA and DNA microsatellite variation. These new data support the hypothesis of population subdivision between the Northeast Pacific, Northwest Pacific and South Pacific, and genetic divergence indicates restriction to the gene flow between hemispheres. However, our estimates of maximum mtDNA and nuclear DNA divergences of 2.43% and 0.67%, respectively, were within the ranges more commonly observed for populations within species than species within genera. Accordingly our data support the more traditional view that S. lalandi in the Pacific comprises three distinct populations rather than the subdivisions into several species.

Molecular cloning, expression and functional characterization of a teleostan cytokine-induced apoptosis inhibitor from rock bream (Oplegnathus fasciatus).

Apoptosis plays a key role in the physiology of multicellular organisms and is regulated by different promoting and inhibitory mechanisms. Cytokine-induced apoptotic inhibitor (CIAPI) was recently identified as a key factor involved in apoptosis inhibition in higher vertebrate lineages. However, most of the CIAPIs of lower vertebrate species are yet to be characterized. Herein, we molecularly characterized a teleostan counterpart of CIAPI from rock bream (Oplegnathus fasciatus), designating as RbCIAPI. The complete coding region of RbCIAPI was consisted of 942 nucleotides encoding a protein of 313 amino acids with a predicted molecular mass of ~33 kDa. RbCIAPI gene exhibited a multi-exonic architecture, consisting 9 exons interrupted by 8 introns. Protein sequence analysis revealed that RbCIAPI shares significant homology with known CIAPI counterparts, and phylogenetic reconstruction confirmed its closer evolutionary relationship with its fish counterparts. Ubiquitous spatial distribution of RbCIAPI was detected in our quantitative real time polymerase chain reaction (qPCR) analysis, where more prominent expression levels were observed in the blood and liver tissues. Moreover, the RbCIAPI basal transcription level was found to be modulated by different bacterial and viral stimuli, which could be plausibly supported by our previous observations on the transcriptional modulation of the caspase 3 counterpart of rock bream (Rbcasp3) in response to the same stimuli. In addition, our in vitro functional assay demonstrated that recombinant RbCIAPI could detectably inhibit the proteolysis activity of recombinant Rbcasp3. Collectively, our preliminary results suggest that RbCIAPI may play an anti-apoptotic role in rock bream physiology, likely by inhibiting the caspase-dependent apoptosis pathway. Therefore, RbCIAPI potentially plays an important role in host immunity by regulating the apoptosis process under pathogenic stress.

Oligo-microarray analysis and identification of stress-immune response genes from manila clam (Ruditapes philippinarum) exposure to heat and cold stresses.

Thermal stress regulates the complex system of gene expression and downstream biochemical and physiological responses in aquatic species. To identify genes involved in heat stress responses in manila clam (Ruditapes philippinarum), microarray analysis was conducted using clam transcriptome generated by pyrosequencing of cDNA library. Manila clams were exposed to heat (30 ± 1 °C) and cold (4 ± 1 °C) stresses and compared with control animals (18 ± 1 °C). Heat stressed animals have changed greater number of transcripts (8,306) than cold stress (7,573). Results of both heat and cold exposure has shown that over 2-fold up-regulated or down regulated (>2-or <2-fold) transcripts were higher at 24 h than at 6 h. It suggests that silent and constitutive express genes can activate at critical stage of thermal stress which could be between 6 and 24 h post stresses. We identified wide range of stress-immune response genes such as transcription factors, heat shock proteins, antioxidant and detoxification enzymes, inflammatory and apoptosis related genes, cell adhesion molecules, cytokines, and IFN regulatory proteins. Histological results revealed that non-specific cellular alterations such as lesions, hypertrophy, and necrosis in stressed gills could be due to decrease of gas exchange rate which may cause hypoxia.

Identification of a novel molluscan short-type peptidoglycan recognition protein in disk abalone (Haliotis discus discus) involved in host antibacterial defense.

Peptidoglycan recognition proteins (PGRPs) are a widely studied group of pattern recognition receptors found in invertebrate as well as vertebrate lineages, and are involved in bacterial pathogen sensing. However, in addition to this principal role, they can also function in multiple host defense processes, including cell phagocytosis and hydrolysis of peptidoglycans (PGNs). In this study, a novel invertebrate short-type PGRP was identified in disk abalone (Haliotis discus discus) designated as AbPGRP. The complete coding sequence of AbPGRP was 534 bp, encoding a 178-amino acid protein with a predicted molecular mass of 20 kDa. The AbPGRP gene had a bipartite arrangement consisting of two exons separated by a single intron. Homology analysis revealed that AbPGRP shares conserved features, including amino acid residues critical for substrate and ion binding as well as for its amidase activity, with homologs of other species. Phylogenetic analysis of AbPGRP revealed that it likely evolved from a common ancestor of invertebrates, having significant homology with other molluscan PGRPs. Recombinant AbPGRP exhibited detectable, dose-dependent PGN-hydrolyzing activity with the presence of Zn(2+), and strong antibacterial activity against Vibrio tapetis, consistent with the functional properties previously reported for PGRPs in other mollusks. Moreover, AbPGRP transcription was induced upon treatment of healthy abalones with bacterial peptidoglycan and lipopolysaccharide, although the expression profiles differed with treatment, suggesting a capacity for discriminating between bacterial pathogens through molecular pattern recognition. Collectively, the findings of this study indicate that AbPGRP is a true homolog of invertebrate PGRPs and likely plays an indispensable role in host immunity.

Genomic structure and immunological response of an STAT4 family member from rock bream (Oplegnathus fasciatus).

The Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a critical role in host defense against viral and bacterial infections. STAT proteins are a group of transcription factors that translocate into the nucleus and are critical for the induction of many genes crucial for the allergic cascade and immune defense. In the present study, a member of the STAT4 family was identified from rock bream (RbSTAT4) at the genomic level, and its transcriptional regulation in response to different pathological stimuli under in vivo conditions was investigated. The genomic sequence of RbSTAT4 is approximately 15.6 kb in length, including a putative core promoter region and 24 exons interrupted by 23 introns. Bioinformatics analysis of RbSTAT4 identified the presence of typical and conserved features of the STAT4 family, including the STAT_int domain, STAT alpha domain, STAT bind domain, linker domain, SH2 domain, and transcriptional activation domain. According to the phylogenetic analysis, RbSTAT4 exhibited the closest evolutionary proximity with the STAT4 member from mandarin fish (Siniperca chuatsi). The RbSTAT4 transcript in healthy rock breams was detected to have ubiquitous expression in 11 different tissues examined, where liver and spleen tissues showed moderate expressions compared with the highest expression level detected in gill tissue. The time-course in vivo immune stimulation of rock bream with lipopolysaccharide, poly I:C, live Edwardsiella tarda, and rock bream iridovirus caused significant transcriptional regulation of the RbSTAT4 expression in gill, head kidney, and spleen tissues, suggesting that RbSTAT4 is involved in immune regulation mechanisms and/or signaling cascades, orchestrating against both bacterial and viral pathogens.

A teleostean counterpart of ferritin M subunit from rock bream (Oplegnathus fasciatus): an active constituent in iron chelation and DNA protection against oxidative damage, with a modulated expression upon pathogen stress.

Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin "M" which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H2O2-mediated DNA damage.

Marine teleost ortholog of catalase from rock bream (Oplegnathus fasciatus): molecular perspectives from genomic organization to enzymatic behavior with respect to its potent antioxidant properties.

Catalases are well known antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3303 bp) of RbCat is comprised of a 1581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor-binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important amino acids present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity.

Structural characterization and expression analysis of a beta-thymosin homologue (Tß) in disk abalone, Haliotis discus discus.

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tß) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates.

Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress.

Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense.

Molluskan fasciclin-1 domain-containing protein: molecular characterizationand gene expression analysis of fasciclin 1-like protein from disk abalone (Haliotis discus discus).

Cell-to-cell contacts play a key role in multicellular systems and organisms. Fasciclin-1 (FAS-1) is a lipid-linked membrane associated glycoprotein that is a member of a newly recognized family of cell adhesion molecules sharing features with the immunoglobulins, cadherins, integrins, and selectins. Here, we report the identification and molecular characterization of a novel FAS-1 domain-containing cDNA from disk abalone (Haliotis discus discus), including its gene expression profile and immune response to bacterial stimuli and tissue injuries. Designated as Abfac1, the 909bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33kDa and isoelectric (pI) value of 4.9. The aa sequence contains two FAS-1 domains and three conserved regions, FRa motif, H-box, and FRb motif. Phylogenetic analysis showed the closest relation to Jellyfish cell adhesion protein. In healthy abalone, Abfac1 expression is highest in hepatopancreas followed by mantle and lowest in digestive gland. In immune-stimulated abalones, relative Abfac1 mRNA expression was increased in hemocytes by ~11-fold at 48h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6h after the Listeria monocytogenes infection and by ~9-fold at 6h after the LPS injection. Similarly, tissue injuries caused significant increase of relative mRNA expression by 3.5-fold in hemocytes and by ~10-fold in mantle at 12h post-injury. These results suggest that the novel member of the FAS-1 domain-containing protein family, Abfac1, may be involved in immune response and cell adhesion in disk abalone.

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties.

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 µmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein.

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) µg µL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.

Caspase 3 from rock bream (Oplegnathus fasciatus): genomic characterization and transcriptional profiling upon bacterial and viral inductions.

Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. In this study, caspase 3 was identified (Rbcasp3) and characterized from rock bream (Oplegnathus fasciatus). The full-length cDNA of Rbcasp3 is 2683 bp and contains an open reading frame of 849 bp, which encodes a 283 amino acid protein with a calculated molecular mass of 31.2 kDa and isoelectric point of 6.31. The amino acid sequence resembles the conventional caspase 3 domain architecture, including crucial amino acid residues in the catalytic site and binding pocket. The genomic length of Rbcasp3 is 7529 bp, and encompasses six exons interrupted by five introns. Phylogenetic analysis affirmed that Rbcasp3 represents a complex group in fish that has been shaped by gene duplication and diversification. Many putative transcription factor binding sites were identified in the predicted promoter region of Rbcasp3, including immune factor- and cancer signal-inducible sites. Rbcasp3, excluding the pro-domain, was expressed in Escherichia coli. The recombinant protein showed a detectable activity against the mammalian caspase 3/7-specific substrate DEVD-pNA, indicating a functional role in physiology. Quantitative real time PCR assay detected Rbcasp3 expression in all examined tissues, but with high abundance in blood, liver and brain. Transcriptional profiling of rock bream liver tissue revealed that challenge with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA whereas, Edwardsiella tarda (E. tarda) stimulated a late-phase significant transcriptional response. Rock bream iridovirus (RBIV) up-regulated Rbcasp3 transcription significantly at late-phase, however polyinosinic-polycytidylic acid (poly(I:C)) induced Rbcasp3 significantly at early-phase. Our findings suggest that Rbcasp3 functions as a cysteine-aspartate-specific protease and contributes to immune responses against bacterial and viral infections.