Expression In Gill Research Articles

Identification and expression analysis of Dicer2 in black tiger shrimp (Penaeus monodon) responses to immune challenges

Dicer is a key initiative protein of the RNA interference (RNAi) pathway that produces small interfering RNAs (siRNAs) or micro RNAs (miRNA), which then leads to RNA-directed gene regulation or viral immunity. In the present study, we identified and characterized a Dicer2 cDNA from black tiger shrimp Penaeus monodon (designated as PmDcr2). The full length cDNA of PmDcr2 contains a 5′ untranslated region (UTR) of 109 bp, an open reading frame (ORF) of 4509 bp and a 3′ UTR of 842 bp. The molecular weight (MW) of predicted PmDcr2 protein is 171.7 KDa with the theoretical isoelectric point of 6.23. PmDcr2 amino acid shared the highest similarity of 91.8% and 90.7% with Dicer2 of Litopenaeus vannamei and Marsupenaeus japonicas, respectively. Phylogenic analysis showed PmDcr2 was clustering with shrimp Dicer2, and closed to the insect group including Tribolium castaneum Dicer2. Real-time quantitative PCR showed that PmDcr2 was widely expressed in almost all examined tissues except muscle, with high expression in gill, hemocyte and lymph. The expression of PmDcr2 in hepatopancreas was up-regulated by Vibrio vulnificus and White Spot Syndrome Virus (WSSV), but not by Staphylococcus aureus. Furthermore, the viral nucleotide homologue dsRNA poly (I:C) and ssRNA R484 also remarkably induced PmDcr2 mRNA expression more efficient and stronger. These data reflect that PmDcr2 is not only response to the gram negative bacteria infection, but also specially to the viral infection in black tiger shrimp.

Two molluscan BCL-2 family members from Manila clam, Ruditapes philippinarum: Molecular characterization and immune responses

Apoptosis based immune responses are important component of host defense in mollusks. In this study, we have identified two novel molluscan BCL-2 cDNAs from Manila clam, Ruditapes philippinarum and named as RpBCL-2A and RpBCL-2B. There were four and three highly conserved BCL-2 homology (BH) regions in RpBCL-2A and RpBCL-2B, respectively suggesting these two genes could be different isoforms of anti-apoptotic BCL-2 family. Phylogenetic results revealed that Manila clam BCL-2 genes were clustered closely with invertebrate BCL-2 members. It gives evidence of their common origin and conserved features of invertebrate BCL-2 family. RpBCL-2A and 2B were expressed in tissue-specific manner showing the highest and lowest level of expression in gills and hemocytes, respectively. However there was no clear expression profile difference between two genes. After Vibrio tapetis challenge, transcriptional responses of RpBCL-2A and RpBCL-2B were induced in gills and hemocytes with high variation that could be due to effects of immune reactions of other host defense molecules.

Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia

We investigated relationships among branchial unidirectional Na(+) fluxes, ammonia excretion, urea excretion, plasma ammonia, plasma cortisol, and gill transporter expression and function in three freshwater fish differing in their sensitivity to high environmental ammonia (HEA). The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia-sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed chronically (12-168 h) to 1 mmol l(-1) ammonia (as NH4HCO3; pH 7.9). During HEA exposure, carp and goldfish elevated ammonia excretion (JAmm) and Na(+) influx rates ( ) while trout experienced higher plasma ammonia (TAmm) and were only able to restore control rates of JAmm and . All three species exhibited increases in Na(+) efflux rate ( ). At the molecular level, there was evidence for activation of a 'Na(+)/NH4(+) exchange metabolon' probably in response to elevated plasma cortisol and TAmm, though surprisingly, some compensatory responses preceded molecular responses in all three species. Expression of Rhbg, Rhcg (Rhcg-a and Rhcg-b), H(+)-ATPase (V-type, B-subunit) and Na(+)/K(+)-ATPase (NKA) mRNA was upregulated in goldfish, Rhcg-a and NKA in carp, and Rhcg2, NHE-2 (Na(+)/H(+) exchanger) and H(+)-ATPase in trout. Branchial H(+)-ATPase activity was elevated in goldfish and trout, and NKA activity in goldfish and carp, but NKA did not appear to function preferentially as a Na(+)/NH4(+)-ATPase in any species. Goldfish alone increased urea excretion rate during HEA, in concert with elevated urea transporter mRNA expression in gills. Overall, goldfish showed more effective compensatory responses towards HEA than carp, while trout were least effective.

Zebrafish ABCC5 gene expression in relation to metallic contamination and presence of Tubifex worms

The impact of contaminated sediment with copper and cadmium on the expression of ABCC5 gene expression in Zebrafish Danio rerio was studied after seven days of exposure. Also the effect of Tubifex worms as bioturbator organism on the gene expression was investigated. Expression of ABCC5 gene was estimated in Zebrafish brain, gills, muscles and digestive tract tissues in four different experimental conditions. The highest expression for ABCC5 gene was found in brain samples of all tested groups, while the gene expression in gills didn’t show any significant change in all groups. Presence of Cu and Cd had led to a significant increase in the expression of ABCC5 gene in brain and digestive tract samples. Surprisingly, in presence of Tubifex worms, expression of the same gene was higher in zebrafish tissues than expression in normal condition in absence of Tubifex worms. The only exception was in gills tissues, where a lower expression was detected.

Характеристика и анализ транскрипции нового CC-хемокина, связанного с врожденным иммунитетом кобии (Rachycentron canadum)

Chemokines are small, secreted cytokine peptides, known principally for their ability to induce migration and activation of leukocyte populations under both pathological and physiological conditions. On the basis of previously constructed express sequence tags (ESTs) of the head kidney and spleen cDNA library of the perciform marine fish Rachycentron canadum (common name cobia). We used bi-directional rapid amplification of cDNA ends (RACE) and obtained a full-length cDNA of a new CC chemokine gene (designated RcCC3). The RcCC3 putative peptide exhibits sequence similarity to the group of CCL19/21/25 CC chemokines. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used in transcript expression studies of RcCC3. We examined the constitutive expression of the transcripts in 12 tissues of non-stressed cobia; RcCC3 transcripts were detected in all tissues examined, with the highest expression in gill and liver, following by head kidney, kidney, spleen, skin, intestine, muscle, stomach, heart, blood and brain. Transcript expression of RcCC3 was examined in immune-related organs, including head kidney, spleen and liver, following intraperitoneal injection of phosphate-buffered saline control, polyriboinosinic polyribocytidylic acid (poly(I:C)) and formalin-killed Vibrio carchariae (bacterial vaccine). The transcripts in these tissues were quickly up-regulated by the injection of poly(I:C) and bacterial vaccine at early time points, although with different expression profiles. These results indicate RcCC3 represents an important component of innate immunity in cobia.

White shrimp Litopenaeus vannamei catalase: Gene structure, expression and activity under hypoxia and reoxygenation

Catalase (EC 1.11.1.6) is an antioxidant enzyme involved in redox equilibrium, regulating hydrogen peroxide (H(2)O(2)) concentration, a harmful reactive oxygen species (ROS) that is produced during hypoxia. Hypoxia occurs commonly in aquatic environments and in shrimp farms. We studied the catalase gene of the shrimp Litopenaeus vannamei and tested its expression and enzyme activity during hypoxia (1.5mg/L O(2); 6 and 24h) and reoxygenation (1h after hypoxia). The complete gene is 2974bp long and has four introns of 821, 223, 114 and 298bp, respectively. The first intron has tree microsatellites, with GT and (T)AT(GT) repeated sequences. L. vannamei catalase is part of an invertebrate clade including crustaceans and rotifers. Catalase expression and activity is different in gills and hepatopancreas. Expression in gills increased 3.2 and 3-fold in response to hypoxia and reoxygenation (6 and 24h hypoxia, followed by 1h reoxygenation) compared to normoxia, while no differences were detected in the expression and activity in hepatopancreas. Catalase activity in gills had a contrary response to expression in hypoxia and reoxygenation.

Immune response-related gene expression profile of a novel molluscan IκB protein member from Manila clam (Ruditapes philippinarum)

Mollusks lack an adaptive immune system and rely solely on the innate immune response. The nuclear factor-kappa B (NF-κB) signaling pathway is one of the most important components of the innate immune system and its activity is regulated by physical interaction with the inhibitor of NF-κB (IκB) protein. The manila clam, Ruditapes philippinarum (Rp), is a key species of the world's aquaculture industry, and recent pathogenic threats, such as the Gram-negative lipopolysaccharide (LPS)-expressing Vibrio tapetis bacteria, have produced severe adverse economic impacts. Here, we describe identification, characterization and immune responses of novel IκB (Rp-IκB) in the manila clam. The Rp-IκB cDNA is comprised of a 1,032bp open reading frame, which encodes 343 amino acid residues and has a predicted molecular mass of 38kDa. The Rp-IκB protein exhibits typical structural features of IκB family members, including the IκB degradation motif, PEST sequence, and six ankyrin repeats. Phylogenetic analysis showed that manila clam and other known molluscan IκB proteins grouped together in the invertebrate cluster. Analysis of the tissue expression distribution revealed that Rp-IκB was ubiquitously expressed. However, immune challenge with V. tapetis and purified LPS endotoxin induced significant up-regulation of Rp-IκB expression in gill and hemocytes. These results indicated that Rp-IκB may play an important role in manila clam defense against bacterial infection.

Identification and expression analysis of a CC chemokine from cobia (Rachycentron canadum)

Chemokines are small, secreted cytokine peptides known principally for their ability to induce migration and activation of leukocyte populations and regulate the immune response mechanisms. The cobia (Rachycentron canadum), a marine finfish species, has a great potential for net cage aquaculture in the South China Sea. We isolated and characterized a CC chemokine cDNA from cobia-designated RcCC2. Its cDNA is 783bp in length and encodes a putative protein of 110 amino acids. Homology and phylogenetic analysis revealed that the RcCC2 gene, which contains four conserved cysteine residues, shares a high degree of similarity with other known CC chemokine sequences and is closest to the CCL19/21 clade. The mRNA of RcCC2 is expressed constitutively in all tested tissues, including gill, liver, muscle, spleen, kidney, head kidney, skin, brain, stomach, intestine and heart, but not blood, with the highest level of expression in gill and liver. The reverse transcription quantitative polymerase chain reaction was used to examine the expression of the RcCC2 gene in immune-related tissues, including head kidney, spleen and liver, following intraperitoneal injection of the viral mimic polyriboinosinic polyribocytidylic acid, formalin-killed Vibrio carchariae (bacterial vaccine) and phosphate-buffered saline as a control. RcCC2 gene expression was up-regulated differentially in head kidney, spleen and liver during 12h after challenge. These results indicate that the RcCC2 gene is inducible and is involved in immune responses, suggesting RcCC2 has an important role in the early stage of viral and bacterial infections.

Cloning of the IL-1β3 gene and IL-1β4 pseudogene in salmonids uncovers a second type of IL-1β gene in teleost fish

To date two closely related interleukin-1β genes (IL-1β1 and IL-β2) have been found in salmonids. The cloning of trout and salmon IL-1β3, and a salmon IL-1β4 pseudogene reveals that two types of IL-1β genes exist in teleost species. Type I teleost IL-1β genes, including salmonid IL-1β3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1β genes, e.g. salmonid IL-1β1–2, lack one or two coding exons at their 5′-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1β genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1β3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1β3 also has a role in inflammation and host defence.

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 306bp 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 8.4Kb, 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 3.5μmol/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 6h 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.

Effect of zinc sacrificial anode degradation on the defence system of the Pacific oyster, Crassostrea gigas: Chronic and acute exposures

Two types of exposures were performed to assess the effects of zinc released from sacrificial anode degradation: a chronic exposure, in which oysters were exposed to 0.53±0.04mgZnL−1 for 10weeks, and an acute exposure, where oysters were exposed to 10.2±1.2mgZnL−1 for 1week. At the end of the acute exposure experiment, 81.8% mortality was recorded. In contrast, no mortality was detected after 10weeks exposure. Moreover, all of the immune system biomarkers studied, except the number of circulating haemocytes, were stimulated by a moderate level of zinc and inhibited by a high level. Our exposure conditions did not induce SOD or MXR mRNA expression in gills and digestive gland. However, an increase of MT mRNA is observed in these tissues. The results indicate that oysters are sensitive to acute zinc toxicity but are only moderately affected by a mild zinc concentration.

Atorvastatin up-regulate toxicologically relevant genes in rainbow trout gills

There are large and increasing discharges of statins into the aquatic environment. Statins are cholesterol-lowering pharmaceuticals, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an enzyme in the cholesterol synthesis pathway. Earlier studies have shown that statins will affect the expression of a range of genes in mammalian tissues and this group of pharmaceuticals has also been shown to affect membrane transporters. Changes in gene expression and ion transport in aquatic organisms may have dramatic consequences for the individual. The aim of the present study was to clarify whether waterborne exposure to a selected statin, atorvastatin, would affect gene expression in rainbow trout (Oncorhynchus mykiss) gill or liver or ion regulation in gills. Juvenile rainbow trout were exposed to two atorvastatin acid and atorvastatin lactone concentrations for 7 days (nominal concentrations 200 ng L(-1) and 10 μg L(-1)). The exposures caused up-regulated gene expression in gill, not liver, and only at the lowest concentration. Genes involved in membrane transport (pgp, mrp1), oxidative stress response (sod, mt), apoptosis (bax) and biotransformation (sult2b) were differentially expressed whereas the expression of genes involved in cholesterol biosynthesis (hmgr, fdps) or peroxisomal proliferation (ppar) were not affected. There were no significant changes in gill Na(+)/K(+) ATPase activity following exposure to atorvastatin. The pattern of differentially expressed genes in rainbow trout gills differ from responses previously observed in mammalian tissues following statin exposure.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone (Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

Cloning, mRNA expression and bioinformatic analysis of full length type collagen alpha 1 Ⅰ cDNA from grass carp (Ctenopharyngodon idellus)

TypeⅠcollagen, as a number of collagen family, is the most abundant collagen and major components of the extracellular matrices of all metazoan life, and plays crucial roles in differentiation, formation of collagen fibers and tissue remodeling after injury, etc. Type Ⅰ collagen alpha 1(COL1A1) cDNA of grass carp (Ctenopharyngodon idella) was isolated through the RT-PCR and RACE approaches. The cDNA was 5 772-bp in length, including a 4 347-bp CDS (coding sequence) and encoded a polypeptide of 1 449 aa. The homology of COL1A1 amino acid with relative species (zebrafish, goldfish, etc.) was as high as 93% with zebra fish and goldfish. The protein peptide molecular weight was 137.2 ku and theoretical pI was 5.44 using ProtParam software on line. The protein peptides of COL1A1 possessed 6 α-helixes, 12 β-sheets, others of ruleless coil regions, and 18 regions of triple helical repeats, 22 low complexity regions, 17 function domains. There were two calcium-binding sites and one zinc-binding site in the COL1A1 protein peptide. COL1A1 mRNA was determined in all the tested 8 tissues (muscle, intestine, hepatopancreas, gill, skin, fin, kidney and spleen) of grass carp by semi- quantitative RT-PCR, and the mRNAs expression in gill, kidney, skin and fin significantly higher than other tissues (P0.05). The structure and bioinformatics characteristics of the COL1A1 from grass carp may help to further understand the function of COL1A1 gene in the repair process of damaged tissue in the grass carp.

Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

BackgroundThe prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited.ResultsHere we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue.ConclusionSuppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.

Identification of a cobia ( Rachycentron canadum) CC chemokine gene and its involvement in the inflammatory response

The chemokines regulate immune cell migration under inflammatory and physiological conditions. We investigated a CC chemokine gene ( RcCC1) from cobia ( Rachycentron canadum). The full-length RcCC1 cDNA is comprised 673 nucleotides and encodes a four-cysteine arrangement 99-amino-acid protein typical of known CC chemokines. The genomic DNA of RcCC1 consists of three exons and two introns. Phylogenetic analysis showed that RcCC1 was closest to the MIP group of CC chemokines. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed RcCC1 was constitutively expressed in all tissues examined, with relative strong expression in gill, blood, kidney, spleen, and head kidney. The RcCC1 transcripts in the head kidney, spleen, and liver were quickly up-regulated after stimulation with formalin-inactivated Vibrio carchariae (bacterial vaccine) or polyriboinosinic polyribocytidylic acid (poly I:C). These results indicate RcCC1 not only plays a role in homeostasis, but also may be involved in inflammatory responses to bacterial and viral infection.

Two catalase homologs are involved in host protection against bacterial infection and oxidative stress in Crassostrea hongkongensis

Catalase is one of the key antioxidant enzymes and it appears to be involved in protection against immune infection and oxidative stress. Here, two catalase cDNAs ( ChCat-1 and ChCat-2) were isolated from hemocytes of Crassostrea hongkongensis using SSH and RACE. The full-length cDNAs of ChCat-1 and ChCat-2 are 1913 and 2466 bp in length, encoding proteins of 515 and 511 amino acids, respectively. Multiple alignments of amino acid sequences revealed that both ChCat-1 and ChCat-2 possess several characteristic features of the catalase family of enzymes, including one proximal active site signature, one heme-ligand signature, and three catalytic amino acid residues (His 72, Asn 145 and Tyr 355). Phylogenetic analysis indicates that these two catalases may share a common ancestral gene and result from a gene duplication event following the divergence of bivalves and gastropods. Constitutive expression of ChCat-1 and ChCat-2 was observed in all tissues studied, with highest levels of expression in gill and muscle, respectively. The expression of both genes was inducible by bacterial infection, and reached the maximum at 8 h (9.0-fold) and 12 h (2.3-fold) post-infection, respectively. Furthermore, both the purified ChCat-1 and ChCat-2 protein displayed a strong catalase activity, and S2 cells carrying ChCat-1 or ChCat-2 showed a higher degree of resistance to H 2O 2 than that of control cells. In a word, this is the first report of the presence of two catalase genes in a single marine bivalve, and our results highlight the involvement of both ChCat-1 and ChCat-2 in host protection against pathogen infection and oxidative stress in C. hongkongensis.

Identification of a novel P450 gene belonging to the CYP4 family in the clam Ruditapes philippinarum, and analysis of basal- and benzo(a)pyrene-induced mRNA expression levels in selected tissues

A novel full-length cDNA encoding a CYP4 protein was initially cloned from the clam, Ruditapes philippinarum. The nucleotide sequence contained an open reading frame coding for 442 amino acids and the deduced amino acid sequence showed 42.6–49.1% identity with other species CYP4s. The phylogenetic analysis demonstrated that the clam CYP4 was clustered within the CYP4s branch. The clam CYP4 mRNA expression was detected in gill, digestive gland, adductor muscle and mantle, and highest transcription level was observed in digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that there was no notable change in CYP4 mRNA expression in gill of R. philippinarum exposure to benzo(a)pyrene (BaP), while the mRNA expression was induced significantly in the digestive gland of the clam by 0.2 ppb (μg L −1) BaP ( p < 0.05). The results suggest that CYP4 of the clam may serve as a useful biomarker of marine environmental PAH pollution.

Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.

Molecular cloning and characterization of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-5 in the chondrostean paddlefish Polyodon spathula and their phylogenetic importance in the Osteichthyes

The interferon regulatory factor (IRF) with its 10 members is a very important gene family related to innate immunity. Currently, most fish IRFs reported are from bony fish (teleosts). Cloning and sequencing of IRFs from chondrosteans, the so-called “ancient fish” including sturgeon, paddlefish, bichir and gar, are absent from the literature. In this study, three IRF genes PsIRF-1, PsIRF-2 and PsIRF-5, were cloned and characterized from the paddlefish ( Polyodon spathula). PsIRF-1 includes an open reading frame (ORF) of 972 bp that encodes a putative protein of 324 amino acids; PsIRF-2 includes an ORF of 1023 bp encoding 341 amino acids and PsIRF-5 includes an ORF of 1491 bp that encodes 497 amino acids. The PsIRF-5 gene structure is similar to those in mammals but differs from those in teleosts in the first and second exons. Phylogenetic studies of the putative amino acid sequences of PsIRF-1, PsIRF-2 and PsIRF-5 based on the neighbor-joining and Bayesian inference method for Osteichthyes found widely accepted inter-relationships among actinopterygians and tetrapods. Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis of PsIRF-1, PsIRF-2 and PsIRF-5 in different paddlefish tissues shows higher levels of expression in gill, spleen and head kidney. Poly (I: C) (polyinosinic-polycytidylic acid) stimulation in vivo up-regulated PsIRF-1 and PsIRF-2 expression, while PsIRF-5 gene expression did not respond to the challenge of Poly (I: C).