Ke-Jian Wang

Trace elements in two marine fish cultured in fish cages in Fujian province, China

Two cultured marine fish, the Japanese seabass (Lateolabrax japonicus) and red seabream (Pagrus major) were collected from eight fish cage sites along the coast of Fujian province in China. The concentrations of Ag, As, Cd, Co, Cu, Fe, Mn, Se, and Zn in their muscle, stomach and liver tissue were quantified. The risk of these trace elements to humans through fish consumption was then assessed. The highest concentrations of As, Cd, Se and Zn in fish feed from fish cages were found in Dongshan Station. Moreover, the As levels in the muscles of both species at all sites were generally higher than Chinas national standard (>1.0 microg/g). Trace element concentrations in two marine fish followed the order of livers > stomachs > muscles. Although the As levels in two marine caged fish exceeded the permissible standards, the estimated daily intake of As did not exceed the reference dose guideline established by US EPA. For other trace elements examined in this study, their concentrations did not exceed the permissible concentrations of the international standards.

Identification of the up-regulated expression genes in hemocytes of variously colored abalone (Haliotis diversicolor Reeve, 1846) challenged with bacteria.

Variously colored abalone (Haliotis diversicolor Reeve, 1846), which is an important commercial aquatic species and has been widely cultured, frequently suffers from bacterial infection. Knowledge of the defense mechanism in this animal is still lacking and, so far few genes related to immune responses in abalones have been reported. In order to isolate differentially expressed genes in H. diversicolor challenged with bacteria, a forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified. A total of 435 clones in the SSH library were sequenced and 111 genes were recognized based on BLAST searches in NCBI and were categorized in association with different biological processes using AmiGO against the Gene Ontology database. Of the 111 cDNAs, 86 genes were identified for the first time in H. diversicolor. The up-regulated cDNAs screened in the SSH library were validated using quantitative real-time PCR and 78 genes showed differential expression patterns. A total of 34 genes were confirmed to be distinctly up-regulated in abalones after bacterial challenge, encoding proteins involved in cellular metabolic processes; cellular component organization and biogenesis; signal transduction and biological regulation; immune defense and response to stimuli; other functions and unknown functions. This is the first report to unveil multiple up-regulated genes with differential expression patterns involving various cellular processes in bacterially challenged H. diversicolor. The data obtained from this study will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.

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.

Immunomodulation in the marine gastropod Haliotis diversicolor exposed to benzo(a)pyrene

It has been reported that environmental pollutants in the aquatic ecosystem could weaken immune competence of organisms. The purpose of the present study was to investigate the effects of benzo(a)pyrene [B(a)P] on immunomodulation in marine gastropods and to see if these effects are caused by or related to the generation of reactive oxygen species. In our present study, the marine gastropod Haliotis diversicolor was exposed to sublethal concentrations (0.01, 0.02, 0.04 and 0.08 mg L(-1)) of B(a)P for 7d under laboratory conditions and the alterations of hematological parameters like haemocyte count, haemocyte viability, protein content and immune components like phenoloxidase, phagocytosis and superoxide anion generation were measured. In addition, the changes in lysozyme activity, antibacterial activity due to the effect of B(a)P on abalone were analysed. B(a)P was found to decrease significantly the total number of circulating haemocytes. Intracellular superoxide anion generation and phenoloxidase significantly increased on exposure to B(a)P, whereas phagocytic activity was decreased significantly at higher concentration. Significant alterations were found in the uptake of neutral red and the observed alterations of hematological parameters and immune components tested indicated the generation of immunotoxicological effects on abalone due to B(a)P exposure. The results demonstrate a possible relationship between B(a)P and the immunological parameters of abalone studied.

The in vitro immune modulatory effect of bisphenol A on fish macrophages via estrogen receptor α and nuclear factor-κB signaling

Bisphenol A (BPA) is a well-known environmental endocrine-disrupting chemical. Employing primary macrophages from head kidney of red common carp (Cyprinus carpio), the present study aimed to evaluate the immune modulatory effect of BPA and to explore its potential action mechanism associated with estrogen receptor (ER) and nuclear factor-κB (NF-κB) pathways. A dynamic response process was observed in macrophages upon various concentrations of BPA exposure, which significantly enhanced the antibacterial activity of macrophages at 0.1, 1, or 10 μg/L, but instead induced the apoptosis at 100, 1000, and 10,000 μg/L. A potential pro-inflammatory effect of BPA exposure was suggested, judging from the increased production of nitrite oxide and reactive oxygen species (ROS), the induction of interleukin-1β mRNA and protein, as well as NF-κB and other NF-κB-associated immune gene expression. Following BPA coexposure with the ER or NF-κB antagonist, the induction of ROS, ERα, and NF-κB-associated immune gene expression was significantly inhibited, implying interaction between those two pathways. This study thus indicated that low doses of BPA exposure alone could significantly disturb the immune response of fish primary macrophages in vitro, and for the first time revealed the synergistic action of ERα and NF-κB transcription factors in the BPA effect.

Differential gene expression profile from haematopoietic tissue stem cells of red claw crayfish, Cherax quadricarinatus, in response to WSSV infection.

White spot syndrome virus (WSSV) is one of the most important viral pathogens in crustaceans. During WSSV infection, multiple cell signaling cascades are activated, leading to the generation of antiviral molecules and initiation of programmed cell death of the virus infected cells. To gain novel insight into cell signaling mechanisms employed in WSSV infection, we have used suppression subtractive hybridization (SSH) to elucidate the cellular response to WSSV challenge at the gene level in red claw crayfish haematopoietic tissue (Hpt) stem cell cultures. Red claw crayfish Hpt cells were infected with WSSV for 1h (L1 library) and 12h (L12 library), respectively, after which the cell RNA was prepared for SSH using uninfected cells as drivers. By screening the L1 and L12 forward libraries, we have isolated the differentially expressed genes of crayfish Hpt cells upon WSSV infection. Among these genes, the level of many key molecules showed clearly up-regulated expression, including the genes involved in immune responses, cytoskeletal system, signal transduction molecules, stress, metabolism and homestasis related genes, and unknown genes in both L1 and L12 libraries. Importantly, of the 2123 clones screened, 176 novel genes were found the first time to be up-regulated in WSSV infection in crustaceans. To further confirm the up-regulation of differentially expressed genes, the semi-quantitative RT-PCR were performed to test twenty randomly selected genes, in which eight of the selected genes exhibited clear up-regulation upon WSSV infection in red claw crayfish Hpt cells, including DNA helicase B-like, multiprotein bridging factor 1, apoptosis-linked gene 2 and an unknown gene-L1635 from L1 library; coatomer gamma subunit, gabarap protein gene, tripartite motif-containing 32 and an unknown gene-L12-254 from L2 library, respectively. Taken together, as well as in immune and stress responses are regulated during WSSV infection of crayfish Hpt cells, our results also light the significance of cytoskeletal system, signal transduction and other unknown genes in the regulation of antiviral signals during WSSV infection.

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.

Gene cloning of a sigma class glutathione S-transferase from abalone (Haliotis diversicolor) and expression analysis upon bacterial challenge

Glutathione S-transferases (GSTs) are a multigene family of xenobiotic metabolizing phase II detoxification enzymes which take part in many pathological and physiological processes, and which can potentially be used as indicators and biomarkers for cancer diagnoses and organic or inorganic pollutant exposure. In this study, a full-length cDNA of a sigma class GST (abGSTsigma) (GenBank accession number EF546619) from variously colored abalone (Haliotis diversicolor) was identified. It was 1328bp containing an open reading frame of 624bp, encoding 208 amino acid residues with a predicted protein molecular weight of 23.67kDa and an estimated pI of 5.67. Sequence analysis showed that the predicted protein sequence of abGSTsigma cDNA contained the conserved domain of the GST_N_Sigma_like (PSSM: cd03039) and GST_C_Sigma_like (PSSM: cd03192). Alignment analysis demonstrated that the abGSTsigma of H. diversicolor was in a branch position with other known class sigma GSTs from different organisms. The abGSTsigma mRNA was distributed in multiple tissues tested and was highly demonstrated in the gill and mantle of normal abalones. In bacteria-challenged abalone, the abGSTsigma gene was significantly expressed in the hemocytes, gill, mantle and digestive gland and the total GSTs enzyme and SOD were also induced in the four tissues. The increased activities of SOD and GSTs can result in the elimination of reactive oxygen species (ROS) indicating antioxidant activities involved. The preliminary work revealed that the sigma class glutathione S-transferase gene abGSTsigma, a phase II detoxification enzyme, had a positive response to bacterial challenge, and that will lead to an insightful study on elucidating the interactions between immune responses and biotransformation exerted by abGSTsigma.