Jun Bo

The marine medaka Oryzias melastigma – A potential marine fish model for innate immune study

The objective of this study is to develop the marine medaka Oryzias melastigma as a potential marine fish model for innate immune and immunotoxicological studies. Hepcidin plays an important role in innate immune system. Two hepcidin genes (OM-hep1 and OM-hep2) were identified and characterized in the O. melastigma, which were highly conserved with other reported hepcidins. During embryogenesis, significant elevation of OM-hep1 and OM-hep2 transcripts were coincided with liver development in the embryos. In adult medaka, differential tissue expressions of both hepcidin transcripts were evident: high in liver, moderate in spleen and low in non-immune tissues. After bacterial challenge, the two hepcidin mRNAs were rapidly and remarkably induced in liver and spleen, suggesting the two OM-hepcidins in O. melastigma play a complementary role in innate defense. Gender difference in time of induction and extent of the two hepcidin mRNAs elevation in infected O. melastigma should be considered in immunotoxicological studies.

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.

Effect of 17β-estradiol on the immunocompetence of Japanese sea bass (Lateolabrax japonicus).

Environmental contaminants can interfere with hormonal regulation in both vertebrates and invertebrates, and these contaminants may disrupt the endocrine system of human and other organisms. Evidence is growing that contaminants may be partly responsible for the observed increase of disease in marine organisms by adversely affecting their immunity. Fish are commonly used as sentinel organisms in vertebrate immunotoxicology; however, to date, studies have been undertaken only on a single size group of fish (juvenile/adult) and for acute exposure. In the present study, Lateolabrax japonicus fingerlings and juveniles were exposed to two sublethal concentrations (200 and 2,000 ng/L) of 17beta-estradiol (E2) for 30 d under laboratory conditions, and alterations in immune parameters comprising differential leukocyte count, respiratory burst, myeloperoxidase, immunoglobulin levels, serum lysozyme, and bactericidal activity were investigated to establish whether estrogen produced immunomodulation and to understand the effects of long-term exposure on these immune parameters in fish fingerlings and juveniles. The results revealed a significant elevation of respiratory burst activity, myeloperoxidase, immunoglobulin levels, and differential leukocyte counts of the fish exposed to estrogen compared to the control. The remaining parameters were significantly reduced in the experimental groups when compared to the control. The results indicated that sublethal E(2) exposure induced immunomodulation in both fingerling and juvenile L. japonicus, and the changes caused by estrogen might affect the function of immune system in fish.

Antioxidant enzymes from the crab Scylla paramamosain: Gene cloning and gene/protein expression profiles against LPS challenge

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551bp with an open reading frame of 1551bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

Identification of differentially expressed genes and quantitative expression of complement genes in the liver of marine medaka Oryzias melastigma challenged with Vibrio parahaemolyticus

The innate immune system of fish is the primary defense against acute diseases. The marine medaka Oryzias melastigma has been shown to be a potential marine fish model for ecotoxicology, but little is known about the innate immune system of this small fish. In this study, suppression subtractive hybridization (SSH) was used to identify differentially expressed immune genes in the liver of O. melastigma infected with Vibrio parahaemolyticus. Among the 396 genes identified, based on NCBI BLAST search of the 1279 sequenced clones in the SSH libraries, 38 (9.6%) were involved in the immune process. Besides, genes involved in biological regulations (5.6%); cellular metabolism (24.7%); general response to stimuli (4.8%); cellular component organization (2.3%); signal transduction (2.5%) and transport process (2.8%) were also obtained. Ten complement component genes involved in four activation pathways were quantified (using q-PCR) and exhibited different patterns of transcription between the control and challenged individuals. The results reported upon here support the feasibility of developing O. melastigma as a marine model fish to understand the basic biological processes related to immune function and for immunotoxicological research. Findings of this study established a genetic platform for studying immune function using O. melastigma.

17β estradiol induced ROS generation, DNA damage and enzymatic responses in the hepatic tissue of Japanese sea bass.

The importance of endocrine disrupting chemicals and their effects on fish has been documented in recent years. However, little is known about whether the estrogenic compound 17β estradiol (E2) causes oxidative stress in the hepatic tissue of fish. Therefore, this work tested the hypothesis that E2 might cause oxidative stress in the Japanese sea bass Lateolabrax japonicus liver. To test this hypothesis, its effects on reactive oxygen species (ROS) production, DNA damage, antioxidants and biotransformation enzyme were investigated in two different size groups (fingerling and juvenile groups) following 30 days exposure. Results showed that there was a good relationship between the E2 exposure concentration, plasma E2 level and ROS generation. In addition ROS production correlated negatively with 7-ethoxyresorufin-O-deethylase activity and positively with DNA damage and lipid peroxidation (LPO). Antioxidant enzymes such as superoxide dismutase and catalase did not show any significant relation with ROS, LPO and DNA damage. In contrast, glutathione mediated enzymes showed a good relationship with the above parameters suggesting that the glutathione system in fish might be responsible for protection against the impact of E2 and also indicating a possible adaptive response during exposure periods. In addition, it was observed that fingerling was more susceptible to E2 exposure than juvenile fish. The present study provided strong evidence that the ROS level increased significantly in the liver of E2 exposed fish, and that ROS might serve as a biomarker to indicate estrogen contamination.

Quantitative gene expression and in situ localization of scygonadin potentially associated with reproductive immunity in tissues of male and female mud crabs, Scylla paramamosain

Scygonadin (Scy) is an important antimicrobial peptide which was first isolated from the seminal plasma of Scylla serrata (now renamed as Scylla paramamosain). Elucidation of the Scy expression pattern in tissues will help in understanding its potential function associated with the reproductive immunity. In our study, Scy mRNA transcripts and its protein were found widely distributed in mature male and female crabs. Scy mRNA transcripts were significantly demonstrated in the ejaculatory duct and hemocytes of males but were much less expressed in the other tissues tested. In addition, Scy mRNA transcripts were discerned in a number of cells in the glandular epithelium of the inner wall and in the secretion inside the ejaculatory duct using the in situ hybridization method. In females, Scy mRNA transcripts were obviously demonstrated in the hemocytes and gills but weakly detected in other tissues tested. The copy number of scygonadin mRNA transcripts in the ejaculatory duct of males was greatly higher than those in other tissues, in particular, was over 60,000 fold that in the hemocytes of females. Using immunohistochemistry, the Scy protein was found at higher levels in male tissues than in female ones, particularly in the reproductive duct of males. It was also interesting to note that Scy gene expression was not significantly induced with lipopolysaccharide challenge. However, it was highly expressed in the ejaculatory duct and the seminal vesicle of pre-copulatory males and in the spermathecae of post-copulatory females under mating conditions. The results suggested that Scy, as an important antimicrobial component, probably performed more functions in males, and was likely to be involved in a function associated with crab fertilization and reproduction in both males and females during mating.

Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP)

Hepcidin is known to be expressed in fish with bacterial challenge and iron overload. Here we first report the hepcidin expression induced in the developmental stages from embryo to fry of red sea bream (Pagarus major) and in juvenile black porgy (Acanthopagrus schlegelii B.) upon continuous waterborne exposure to BaP. The gene expression of CYP1A1 and IgL (immunoglobulin light chain) were both measured. Expression of the Pagarus major hepcidin gene (PM-hepc) was increased in post hatch fry at 24 h and 120 h exposure to BaP at concentrations of 0.1, 0.5 and 1.0 microg/l, respectively. The gene expression pattern was comparable to that of CYP1A1 but different from that of IgL. In addition, a high number of AS-hepc2 transcripts (Acanthopagrus schlegelii B. hepcidin gene) were detected in the liver upon exposure to 1.0 microg/l BaP. This study demonstrates that hepcidin gene expression is significantly induced in BaP-exposed red sea bream and black porgy.

Benzo[a]pyrene modulation of acute immunologic responses in red Sea bream pretreated with lipopolysaccharide

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM‐hepc) gene and the activities of some immune‐associated parameters in the lipopolysaccharide (LPS)‐challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 μg L−1) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM‐hepc mRNA expression at 3, 6, and 12‐h post‐LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM‐hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM‐hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS‐challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM‐hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune‐associated parameters.

Benzo[a]pyrene modulates the biotransformation, DNA damage and cortisol level of red sea bream challenged with lipopolysaccharide

In animals, biotransformation and the immune system interact with each other, however, knowledge of the toxic mechanism of benzo[a]pyrene (BaP) on these two systems is not well known. The present study investigated the toxic effects of BaP on the biotransformation system, cortisol level and DNA integrity of red sea bream (Pagrus major). The results showed that cortisol level was induced under the challenge of lipopolysaccharide (LPS). Short-term exposure (96 h) of BaP at environmental concentration significantly increased the cortisol level, hepatic EROD activity and CYP1A1 mRNA expression. When P. major was exposed to BaP for 14 d followed by LPS challenge this increased the cortisol level, EROD activity and hepatic DNA damage except CYP1A1 mRNA expression. Combined with our previous data, which showed that BaP exposure can modulate the immunologic response in P. major challenged with LPS, a hypothetical adverse outcome pathway of BaP on fish was suggested.