Comparative toxicity of Corexit® 9500, oil, and a Corexit®/oil mixture on the eastern oyster, Crassostrea virginica (Gmelin).

Given their particle feeding behavior, sessile nature, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit® 9500, crude oil (high-energy water accommodated fraction; HEWAF), and a Corexit®/oil mixture (chemically-enhanced water accommodated fraction; CEWAF) to evaluate potential toxic effects on immunological (phagocytosis and respiratory burst), physiological (feeding rate), and histological endpoints. Phagocytosis was significantly increased following CEWAF exposure only. Respiratory burst was significantly decreased following Corexit® exposure, but significantly increased following exposure to the highest concentration of CEWAF. Oyster feeding rates were significantly decreased following exposure to Corexit®, HEWAF, and CEWAF, and were most sensitive to CEWAF exposure. These modulations of important immunological and physiological functions could result in serious health outcomes for oysters, such as increased parasitism and decreased growth. Our experiments showed that subtle, sub-lethal effects occurred following acute in vivo exposure to Corexit®, HEWAF, and CEWAF, though oysters were not equally sensitive to the three components. Data from this study can be used for more accurate risk assessment concerning the impact of oil and Corexit® on the health of oysters.

Identification and functional characterization of NEMO in Crassostrea gigas reveals its crucial role in the NF-κB activation.

NEMO (NF-κB essential modulator) is one of the important regulatory subunits of the IκB kinase (IκK) complex that controls the activation of the NF-κB signaling pathway. Here, we have identified the homolog of NEMO from the pacific oyster Crassostrea gigas. CgNEMO harbors the conserved the IκK binding region, NEMO ubiquitin binding domain and Zinc finger domain. In terms of tissue distribution, CgNEMO is expressed in various tissues with an observed highest expression in the hemocytes. Furthermore, infection by two related Vibrio strains significantly increased CgNEMO expression in the hemocytes. Cell culture based luciferase reporter assays showed that CgNEMO activates the NF-κB reporter in a dose-pendent manner. Moreover, CgNEMO was also found to counter the IkB-dependent inhibitory effect on NF-κB activation, providing a plausible mechanism of NF-κB activation by CgNEMO. Meanwhile, site-directed mutagenesis demonstrated that the putative ubiquitination site K535 is required for the activation of NF-κB, implying that ubiquitination of NEMO may be involved in regulating its activity. Finally, RNAi mediated knockdown of CgNEMO in vivo significantly compromised the bacterial induction of key cytokines TNF-α and IL-17, strongly suggesting a role for CgNEMO in acute immune defense in oyster. In conclusion, this study provides new insights into our understanding about the evolution of NEMO mediated NF-κB activation and the induction of cytokine. Our findings may provide valuable information about diseases control and management in oyster aquaculture.

CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca2+ increased significantly (p < 0.05). But, this increasing of Ca2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response.

The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas.

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

Allograft inflammatory factor-1 stimulates hemocyte immune activation by enhancing phagocytosis and expression of inflammatory cytokines in Crassostrea gigas.

Allograft inflammatory factor-1 (AIF-1) is a calcium-binding cytokine associated with immune cell activation and inflammatory response. Presently, we have identified and characterized an AIF-1 in a marine bivalve mollusk, Crassostrea gigas, and designated it as CgAIF-1. The full-length CgAIF-1 cDNA is 794 bp, encoding a protein of 149 amino acids with two conserved EF hand Ca(2+)-binding motifs. CgAIF-1 is constitutively expressed in various tissues with enriched expression in hemocytes. Moreover, CgAIF-1 transcription is induced by multiple Pathogen-Associated Molecular Patterns (PAMPs), including poly (I: C), LPS, PGN, HKLM and HKVA, but is limited by 1,3-ß-glucan. Furthermore, recombinant CgAIF-1 can specifically stimulate phagocytic ability of granulocytes, but not of intermediate cells and hyalinocytes. CgAIF-1 also enhances mRNA levels of MIF, TNF and IL-17. These results provide the first functional evidence that CgAIF-1 is involved in hemocyte activation in C. gigas, revealing conserved functions of AIF-1 in host defense from mollusks to mammals.

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(2)O(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.

Two superoxide dismutase (SOD) with different subcellular localizations involved in innate immunity in Crassostrea hongkongensis.

SODs are ubiquitous metalloenzymes that can scavenge superoxides in response to various stresses. In the present study, full-length cDNAs of two SOD genes were isolated from Crassostrea hongkongensis (designated ChMnSOD and ChCuZnSOD). The cDNAs are 997 and 918 bp in length with ORFs of 675 and 468 bp (encoding 225 and 156 amino acids), respectively. Sequence analysis revealed a conserved Sod_Fe domain in ChMnSOD, and a Sod_Cu_Zn domain in ChCuZnSOD. Subcellular localization of ChMnSOD is mitochondrial while intracellular expression of ChCuZnSOD is detected. Although their expression overlaps in a wide range of tissues, ChMnSOD mRNA expression is high in gonad while ChCuZnSOD's is strong in adductor muscle. After infection by Vibrio alginolyticus, ChMnSOD mRNA was up-regulated 5 fold (p < 0.05) at 4 h, but returned to normal level 6 h post-infection. The expression of ChCuZnSOD gene showed a slight delay to the infection challenge and was elevated roughly 4 fold after 8 h (p < 0.05), returning to normal at 12 h post-infection. The elevated transcript levels of the two SOD genes in response to V. alginolyticus infection highlights their important functions in eliminating toxic reactive oxygen species (ROS) and protecting organisms from bacterial invasion in C. hongkongensis.

Enhanced immunological and detoxification responses in Pacific oysters, Crassostrea gigas, exposed to chemically dispersed oil.

The aim of this study was to evaluate the effects of chemically dispersed oil on an economically and ecologically important species inhabiting coasts and estuaries, the Pacific oyster Crassostrea gigas. Studies were carried out with juveniles, known to generally be more sensitive to environmental stress than adults. A set of enzyme activities involved in immune defence mechanisms and detoxification processes, i.e. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), catecholase-type phenoloxidase (PO), laccase-type PO and lysozyme were analysed in different oyster tissues, i.e. the gills, digestive gland and mantle, and in the plasma and the haemoycte lysate supernatant (HLS) of the haemolymph. Results indicated that total PAH body burdens were 2.7 times higher in the presence than in the absence of the chemical dispersant. After 2 days of exposure to chemically dispersed oil, alkylated naphthalenes accounted for 55% of the total PAH body burden, whereas alkylated fluorenes and alkylated dibenzothiophenes accounted for 80% when the chemical dispersant was absent. Importantly, a higher number of enzyme activities were modified when oil was chemically dispersed, especially in the plasma and gills. Moreover, independently of the presence or absence of chemical dispersant, oil exposure generally inhibited enzyme activities in the gills and plasma, while they were generally activated in the mantle and haemocytes. These results suggest that the gills and plasma constitute sensitive compartments in C. gigas, and that the mantle and haemocytes may play an important role in protection against xenobiotics. Among the six enzyme activities that were analysed in these body compartments, five were modulated in the chemical dispersion (CD) treatment while only half of the enzyme activities were modulated in the mechanical dispersion treatment. Furthermore, CD treatment effects were often observed following exposure, but also during depuration periods. These results suggest that immune and/or detoxification responses are likely to be affected when dispersants are used to treat oil spills in shallow waters.

In vivo effects of LCO soluble fraction on immune-related functions and gene transcription in the Pacific oyster, Crassostrea gigas (Thunberg).

The effects of a soluble fraction of light cycle oil (LCO) on haemocyte parameters, phenoloxidase (PO) activity and mRNA expression of immune-related genes, in the Pacific oyster, Crassostrea gigas, were tested after seven days of exposure and two weeks of recovery period. Five polycyclic aromatic hydrocarbons (PAHs) out of ten detected in tank water had bioaccumulated at the end of the contamination period. The concentration of PAHs in oyster tissues decreased during the recovery period and 14 days after the exposure, 69% of bioaccumulated PAHs were detected in contaminated oysters. The exposure induced severe oyster mortality (21%), external and internal green colouration of the shell and a significant decrease of PO activity. The mRNA expression of several genes was altered. As a conclusion, a modulation of immune-related parameters was demonstrated using three different approaches, namely cellular (flow cytometry), biochemical (spectrophotometry) and genomics (gene transcription) in oysters after contact with soluble fraction of LCO.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.