Xue-Ming Dan

Immune responses and immune-related gene expression profile in orange-spotted grouper after immunization with Cryptocaryon irritans vaccine

In order to elucidate the immune-protective mechanisms of inactivated Cryptocaryon irritans vaccine, different doses of C. irritans theronts were used to immunize orange-spotted grouper (Epinephelus coioides). We measured serum immobilization titer, blood leukocyte respiratory burst activity, serum alternative complement activity, and serum lysozyme activity weekly. In addition, the expression levels of immune-related genes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), major histocompatibility complexes I and II (MHC I and II), and transforming growth factor-β1 (TGF-β1) were determined in spleen and gills. The results showed that the immobilization titer, respiratory burst activity, and alternative complement activity of immunized fish were significantly increased, and the levels of the last two immune parameters in the high-dose vaccine group were significantly higher than in the low-dose vaccine group. Serum lysozyme activity in the high-dose vaccine group was significantly higher than in the PBS control group. Vaccination also regulated host immune-related gene expression. For example, at 2- and 3- weeks post immunization, IL-1β expression in the high-dose vaccine group spleen was significantly increased. At 4-weeks post immunization, the fish were challenged with a lethal dose of parasite, and the survival rates of high-dose vaccine group, low-dose vaccine group, PBS control group, and adjuvant control group were 80%, 40%, 0%, and 10% respectively. These results demonstrate that inactivated C. irritans vaccination improves specific and nonspecific immune responses in fish, enhancing their anti-parasite ability. These effects are vaccine antigen dose-dependent.

Immune-related genes expression profile in orange-spotted grouper during exposure to Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune‐related genes in antiparasitic immune responses in fish, we monitored the expression change of IL‐8, COX‐2, C‐type lectin and transferrin in local and systemic immune organs of orange‐spotted grouper post‐C. irritans infection. IL‐8 expression was up‐regulated during the course of infection in the skin, while COX‐2 and transferrin expression was up‐regulated in the gill. COX‐2 expression was significantly down‐regulated in the spleen (0·7–5% of its control) and head kidney (0·5–4% of its control) post‐primary infection. Transferrin expression was also down‐regulated in the spleen and head kidney from 6 h to 5 days post‐primary infection. However, C‐type lectin expression was up‐regulated in all tested organs post‐infection, with the exception of day 7 in the spleen post‐primary infection where the expression level was slightly down‐regulated (44% of its control). These results suggest that these four immune‐related genes play an important role in grouper anti‐C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides)

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.

Identification of IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent NF-κB activation

Interleukin 1 (IL-1) receptor-associated kinase (IRAK) family members are crucial signal transducer in the Toll-like receptor/IL-1R signal pathway, which mediates downstream signal cascades involved in the innate and adaptive immune responses. In this study, we identified an IRAK-4 protein (EcIRAK-4) in the orange-spotted grouper (Epinephelus coioides), with an N-terminal death domain, a proST domain, and a central kinase domain, similar to that of other fishes and mammals. A sequence alignment and phylogenic analysis demonstrated that full-length EcIRAK-4 shares a high degree of sequence identity with those of other fishes, especially the roughskin sculpin, and their death domains and kinase domains share greater identity than their proST domains. A conservation analysis indicated that most of the functional sites in mammalian IRAK-4 are conserved in IRAK-4 of the grouper and other fishes, with the exception of the sites of interaction with IRAK-2 and one autophosphorylation site within the activation loop. EcIRAK-4 is broadly expressed in all the tissues examined, with highest expression in the head kidney and liver. After infection with Cryptocaryon irritans, EcIRAK-4 expression was significantly upregulated, especially in the skin, which suggests that this molecule is involved in the hosts defense against parasitic infection. Surprisingly, after cotransfection with grouper MyD88, EcIRAK-4 significantly impaired the NF-κB activity induced by MyD88. EcIRAK-4 was uniformly distributed throughout the cytoplasm in HeLa cells. These findings suggest that although IRAK-4 is evolutionarily conserved between fish and mammals, its signal transduction function is markedly different.

Grouper (Epinephelus coioides) IL-34/MCSF2 and MCSFR1/MCSFR2 were involved in mononuclear phagocytes activation against Cryptocaryon irritans infection

MCSF and its well-known receptor MCSFR had been well studied in humans, regulating the differentiation, proliferation, and survival of the mononuclear phagocyte system. IL-34, which is an alternative ligand of MCSF receptor, was recently identified as a novel cytokine and functionally overlaps with MCSF. However, the functional study of these receptors and their ligands in fish are largely unknown. In the present study, the cDNA of two potential grouper MCSFR ligands have been cloned, EcIL-34 (657 bp) and EcMCSF2 (804 bp), as well as an additional copy of grouper MCSFR, EcMCSFR2 (3141 bp). Sequence analysis showed that these three molecules had higher identities with other fish counterparts compared to mammals and their conserved structures and important functional residues were also analyzed. Tissue distribution analysis showed that EcIL-34 is dominant in brain, gill and spleen compared to EcMCSF2, which is dominant in head kidney, trunk kidney, skin, heart and muscle. EcMCSFR1 was dominant in the most tissues except head kidney and liver compared to EcMCSFR2. The different tissue distribution patterns of these two grouper MCSF receptors and their two ligands indicate the different mononuclear phagocyte differentiation and activation modes in different tissues. In Cryptocaryon irritans infected grouper, EcIL-34 and EcMCSFR2 were the most strongly up-regulated ligand and receptor in the infected sites, gill and skin. Their up-regulation confirmed the proliferation and activation of phagocytes in C. irritans infected sites, which would improve the antigen presentation and elicit the host local specific immune response. In C. irritans infected grouper head kidney, both ligands EcIL-34 and EcMCSF2 (especially EcMCSF2) were up-regulated, but both receptors EcMCSFR1 and EcMCSFR2 were down-regulated, which indicated that the phagocytes differentiation and proliferation may have occurred in this hemopoietic organ, and after that they migrated to the infected cites. The down-regulation of EcIL-34 and EcMCSF2 and no significant change of EcMCSFR1 and EcMCSFR2 in most time point of grouper spleen showed it was less involved in phagocytes response to C. irritans infection.

Effects of Cryptocaryon irritans infection on the survival, feeding, respiratory rate and ionic regulation of the marbled rockfish Sebastiscus marmoratus.

To clarify the effects of a Cryptocaryon irritans infection on the physiological functions of the marbled rockfish Sebastiscus marmoratus, this study utilized C. irritans at concentrations of 2500; 5000; 7500; 10,000; 20,000; and 30,000 theronts/fish to infect marbled rockfish weighing 45 ± 3 g. The survival rate, food intake, respiratory rate, serum ion concentrations and gill Na+/K+-ATPase activity were determined. With the increase of the infection concentration and the passage of time, the survival rate of the rockfish gradually decreased. The groups infected with more than 5000 theronts/fish had stopped feeding within 4 days. The respiratory rates of the fish in the groups infected with 2500 and 5000 theronts/fish initially increased and then decreased. In contrast, the respiratory rate of the fish in the groups infected with more than 7500 theronts/fish was elevated to levels significantly higher than the control group after 12 h. The Na+/K+-ATPase activity and serum Na+ and Cl- concentrations increased with increasing infection concentration. In conclusion, the physiological functions of the fish infected with low concentrations of C. irritans can be effectively restored, whereas a high concentration infection induced severe stress. The declined food intake and accelerated respiratory rate could be useful for an early warning system as important indicators.

Immunological, ionic and biochemical responses in blood serum of the marine fish Trachinotus ovatus to poly-infection by Cryptocaryon irritans

To investigate the response of pompano fish (Trachinotus ovatus) to white spot disease, we used the protozoan Cryptocaryon irritans to infect live 450-g specimens at concentrations of 40,000 theronts/fish. We assessed the relative infection intensity (RII), serum immobilizing titer, and immunity-related enzyme activities (ACP, AKP, LZM), and assessed feeding, serum ion concentrations (Na(+), Cl(-), Ca(2+) and K(+)) and blood biochemistry (ALT, AST, LDH) of pompano. The fish were then treated with a lethal dose of C. irritans (70,000 theronts/fish) and the number of deaths was recorded. We found that the relative infection intensities of the control group, group I, and group II were 0, 0.630 ± 0.179, and 0.014 ± 0.006. Poly-infection induced a significant increase in the serum immobilizing titer (853.33 ± 295.60) of group II. In terms of the biochemical assessment, group II had significantly higher alkaline phosphatase and acid phosphatase activities than the other groups, and the lowest lysozyme activity (P < 0.05), compared to higher activity in the control group and the highest level in group I. Only the fishes of group I had stopped feeding after treatment. The concentrations of Na(+), Cl(-), and Ca(2+) in blood serum did not differ significantly among the three groups, but K(+) concentration increased with the increasing infection frequency. Alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities in fish of group II were significantly higher than those of the other groups. Survival of the fish subjected to the lethal dose of C. irritans was 0, 0, and 100 in groups control, I, and II, respectively. In conclusions, based on the food intake of group II, along with the results of relative infection intensity, serum immobilizing titer, and survival, we speculate that the fish in that group acquired high protective immunity following poly-infection by C. irritans, experiencing limited harm for pompano.

Growth, feed intake and immune responses of orange-spotted grouper (Epinephelus coioides) exposed to low infectious doses of ectoparasite (Cryptocaryon irritans)

To explore the effect of low-dose Cryptocaryon irritans infection on growth, feeding and antiparasitic immunity of orange-spotted grouper (Epinephelus coioides), this study utilized C. irritans at concentrations of 5500 theronts/fish (Group I, 1/10 of 96 h LC50) or 11,000 theronts/fish (Group II) to infect E. coioides weighing 38 g on average at week 0, 2 and 4, respectively. Food consumption was recorded daily; the fish were weighed weekly; serum immobilizing titer (SIT), and acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM) activity were recorded every 2 weeks; the fish were treated with lethal dose (70,000 theronts/fish) of C. irritans in the 8th week and death number were recorded. The result shows that in the 1st week after the first infection, the fishs weight gain (WG), length gain (LG), and specific growth rate (SGR) dropped as parasite dose increased, and WG, SGR values were negative; while, after the 2nd and the 3rd infection, no significant differences were detected among the three groups. These results indicated that the 1st infection affected the fish most, while the following infections were protected by some immunity. In the 3rd, 7th, and 8th week, condition factor (CF) increased with the increased infectious dose, indicating that the parasite affected body length more than body weight. As the experiment went on, accumulated food consumption (AFC) of all three groups steadily grew (control > Group I > Group II). But on the 2nd day after the first infection, daily food consumption (DFC) of Group I and II significantly dropped, the decline of Group II was greater than that of Group I, DFC recovered in the following week, with Group I earlier than Group II. After the 2nd infection, DFC of Group I and II dropped again, Group II still dropped more than Group I, and both groups recovered on the 3rd day after infection. The 3rd infection caused no significant difference in week food consumption (WFC). These results indicated that a higher dose of infection causes a greater drop in FC and a slower recovery. Weekly feed conversion ratio (WFCR) values of Group I and II in the 1st week was negative; in the 2nd week, WFCR was lower in the group infected by a higher dose of parasite; while in the 3rd and following weeks, no significant pattern was observed. Accumulate feed conversion ratio (AFCR) dropped as the infectious dose increased (control > Group I > Group II), AFCR of Group I and II reached above 0 in the 2nd and 4th week, respectively. From the 4th week on, the inter-group AFCR of the 3 groups still took on a declining trend with the increased infectious dose but the gap became smaller. One week after the first infection, SIT of Group I and Group II were 0; one week after the 2nd infection, SIT reached up to 8 (Group I) and 16 (Group II) respectively; and after the 3rd infection, SIT further increased and peaked in the 7th week. When challenged by lethal dose of C. irritans, fish of all 3 groups began to die since the 3rd day after infection, and the final deaths were 14, 12 and 8 for the control group, Group I and Group II, respectively. ACP activity in the 1st, 5th, 7th but the 3rd week was higher in the experiment group than that in the control group, but no significant difference was detected between Group I and II throughout the experiment. AKP activity increased as the infectious dose increased, but the difference among the three groups gradually became less obvious in latter infections, and no significant difference can be detected in the end. SOD activity increased with infection dose at each time point, while both group I and group II had their SOD activities first increased and then decreased as times of infection increased. The LZM activity of the two infection groups increased as the infectious times increased. Combining the results on growth and feeding, we speculated that the fishs physiological condition stabilized after 3 rounds of infection. To sum up, low-dose infection by C. irritans can induce the fishs immunity, but at the cost of decreasing food intake, decreased food conversion, and lagged growth.

Siganus oramin recombinant L-amino acid oxidase is lethal to Cryptocaryon irritans

A novel antimicrobial and antiparasitic protein (APP/SR-LAAO) isolated from serum of the rabbitfish (Siganus oramin) was confirmed to be lethal to Cryptocaryon irritans, an important marine parasitic ciliate that causes marine white spot disease in a variety of wild and cultured fish. In this study, a recombinant SR-LAAO (rSR-LAAO) was expressed on a large scale in Escherichia coli Rosetta-gami™(DE3)pLysS cells. rSR-LAAO was expressed as an inactive form in the inclusion bodies. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that after purification, refolding and ultrafiltration, rSR-LAAO had a significantly cytotoxic effect on C. irritans theronts. Using light microscopy, scanning electron microscopy and fluorescence microscopy, we found that theronts rapidly became weakly motile, cilia became detached, cells became rounded, membranes eventually lysed in different cell positions and cytoplasmic contents leaked out of the cell. These results suggested the recombinant SR-LAAO was significantly lethal to C. irritans and the death process of the parasite incubated with rSR-LAAO was remarkably similar compared to the SR-LAAO group as reported earlier.

Molecular identification and expression analysis of TLR5M and TLR5S from orange-spotted grouper (Epinepheluscoioides)

Abstract Toll‐like receptor 5 (TLR5) is an important receptor that interacts with bacterial flagellin and regulates host immune response in mammal. Recent studies demonstrate that piscine contains two types of TLR5, namely membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), and both of which perform crucial role in flagellin response. In the present study, a TLR5M and a TLR5S sequence was cloned from orange‐spotted grouper (Epinepheluscoioides), and their ORFs are respectively 2466 bp (821 aas) and 1935 bp (644 aas). EcTLR5M has the typical TLR structure of a LRR domain, a transmembrane region and a TIR domain, while EcTLR5S only contains a LRR domain like other species’ TLR5S. Both molecules have 23 LRR motifs, a LRR‐NT and a LRR‐CT in the LRR domain, similar to those of other species. Phylogenetic and sequence alignment indicated that both EcTLR5s respectively displayed closer relationship and higher sequence identity with those in other fish species. In healthy grouper, EcTLR5M was highly expressed in the skin, head kidney and spleen, while EcTLR5S was mainly detected in the liver. Ciliate Cryptocaryon irritans infection could significantly up‐regulate the expression level of EcTLR5s in the gill and spleen from day 1 to day 3, and higher expression fold change was observed in the spleen. Taken together, the present studies contributed to understanding the function of piscine TLR5M/S and clarify their possible role in fish immune response against ciliate infection. HighlightsBoth TLR5M and TLR5S were identified from the orange‐spotted grouper.EcTLR5M and EcTLR5S expression pattern was different in healthy tissues.Ciliate infection dramatically increased the expression of both EcTLR5s.