Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish.

Amyloodinium ocellatum is among the most devastating protozoan parasites, causing huge economic losses in the mariculture industry. However, the pathogenesis of amyloodiniosis remains unknown, hindering the development of targeted anti-parasitic drugs. The A. ocellatum in vitro model is an indispensable tool for investigating the pathogenic mechanism of amyloodiniosis at the cellular and molecular levels. The present work developed a new cell line, ALG, from the gill of yellowfin seabream (Acanthopagrus latus). The cell line was routinely cultured at 28°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS). ALG cells were adherent and exhibited an epithelioid morphology; the cells were stably passed over 30 generations and successfully cryopreserved. The cell line derived from A. latus was identified based on partial sequence amplification and sequencing of cytochrome B (Cyt b). The ALG was seeded onto transwell inserts and found to be a platform for in vitro infection of A. ocellatum, with a 37.23 ± 5.75% infection rate. Furthermore, scanning electron microscopy (SEM) revealed that A. ocellatum parasitizes cell monolayers via rhizoids. A. ocellatum infection increased the expression of apoptosis and inflammation-related genes, including caspase 3 (Casp 3), interleukin 1 (IL-1), interleukin 10 (IL-10), tumour necrosis factor-alpha (TNF-α), in vivo or in vitro. These results demonstrated that the in vitro gill cell monolayer successfully recapitulated in vivo A. latus host responses to A. ocellatum infection. The ALG cell line holds great promise as a valuable tool for investigating parasite-host interactions in vitro.

Inhibiting thioredoxin glutathione reductase is a promising approach to controlling Cryptocaryon irritans infection in fish.

Marine cultured fish often suffer from Cryptocaryon irritans infection, which causes enormous mortality. C. irritans is resistant to oxidative damage induced by zinc. To develop an effective drug to control the parasite, a putative thioredoxin glutathione reductase (CiTGR) from C. irritans was cloned and characterized. CiTGR was designed as a target to screen for inhibitors by molecular docking. The selected inhibitors were tested both in vitro and in vivo. The results showed that CiTGR is located in the nucleus of the parasite, possesses a common pyridine-oxidoreductases redox active center, and lacks a glutaredoxin active site. Recombinant CiTGR exhibited high TrxR activity but low glutathione reductase activity. Shogaol was found to significantly suppress TrxR activity and enhance toxicity of zinc on C. irritans (P < 0.05). The abundance of C. irritans on the fish body decreased significantly after oral administration of shogaol (P < 0.05). These results implied that CiTGR could be used to screen for drugs that weaken the resistance of C. irritans to oxidative stress, which is critical for controlling the parasite in fish. This paper deepens the understanding of the interaction between ciliated parasites and oxidative stress.

Characterization and virulence of Streptococcus agalactiae deficient in SaeRS of the two-component system.

There are a variety of regulatory systems in bacteria, among which the two-component system (TCS) can sense external environmental changes and make a series of physiological and biochemical reactions, which is crucial for the life activities of bacteria. As a member of TCS, SaeRS is considered to be an important virulence factor in Staphylococcus aureus, but its function in tilapia (Oreochromis niloticus)-derived Streptococcus agalactiae remains unknown. To explore the role of SaeRS in regulating virulence in the two-component system (TCS) of S. agalactiae from tilapia, ΔSaeRS mutant strain and CΔSaeRS complementary strain were constructed by homologous recombination. The results showed that the abilities of growth and biofilm formation of ΔSaeRS strain were significantly decreased when cultured in a brain heart infusion (BHI) medium (P < 0.01). Also, the survival rate of the ΔSaeRS strain in blood was decreased when compared with the wild strain S. agalactiae THN0901. Under the higher infection dose, the accumulative mortality of tilapia caused by the ΔSaeRS strain was significantly decreased (23.3%), of which THN0901 and CΔSaeRS strains were 73.3%. The results of competition experiments in tilapia showed that the invasion and colonization abilities of the ΔSaeRS strain were also dramatically lower than those of the wild strain (P < 0.01). Compared with the THN0901, the mRNA expression levels of virulence factors (fbsB, sip, cylE, bca, etc.) in the ΔSaeRS strain were significantly down-regulated (P < 0.01). SaeRS is one of the virulence factors of S. agalactiae. It plays a role in promoting host colonization and achieving immune evasion during the infection of tilapia, which provides a basis for exploring the pathogenic mechanism of S. agalactiae infected with tilapia.

Glutathione metabolism in Cryptocaryon irritans involved in defense against oxidative stress induced by zinc ions.

BACKGROUND: Cryptocaryon irritans is a fatal parasite for marine teleosts and causes severe economic loss for aquaculture. Galvanized materials have shown efficacy in controlling this parasite infestation through the release of zinc ions to induce oxidative stress. METHODS: In this study, the resistance mechanism in C. irritans against oxidative stress induced by zinc ions was investigated. Untargeted metabolomics analysis was used to determine metabolic regulation in C. irritans in response to zinc ion treatment by the immersion of protomonts in ZnSO4 solution at a sublethal dose (20 µmol). Eight differential metabolites were selected to assess the efficacy of defense against zinc ion stimulation in protomonts of C. irritans. Furthermore, the mRNA relative levels of glutathione metabolism-associated enzymes were measured in protomonts following treatment with ZnSO4 solution at sublethal dose. RESULTS: The results showed that zinc ion exposure disrupted amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in C. irritans. Four antioxidants, namely ascorbate, S-hexyl-glutathione, syringic acid, and ubiquinone-1, were significantly increased in the Zn group (P < 0.01), while the glutathione metabolism pathway was enhanced. The encystment rate of C. irritans was significantly higher in the ascorbate and methionine treatment (P < 0.05) groups. Additionally, at 24 h post-zinc ion exposure, the relative mRNA level of glutathione reductase (GR) was increased significantly (P < 0.01). On the contrary, the relative mRNA levels of glutathione S-transferase (GT) and phospholipid-hydroperoxide glutathione peroxidase (GPx) were significantly decreased (P < 0.05), thus indicating that the generation of reduced glutathione was enhanced. CONCLUSIONS: These results revealed that glutathione metabolism in C. irritans contributes to oxidative stress resistance from zinc ions, and could be a potential drug target for controlling C. irritans infection.

Skin transcriptomic analysis and immune-related gene expression of golden pompano (Trachinotus ovatus) after Amyloodinium ocellatum infection.

Amyloodiniosis is a severe disease of marine and brackish water fish caused by Amyloodinium ocellatum. Golden pompano (Trachinotus ovatus) is often repeatedly infected by A. ocellatum, leading to extensive mortality. However, little is known about the immune response mechanisms of the T. ovatus following reinfection with A. ocellatum. In this study, an extensive analysis at the transcriptome level of T. ovatus skin was carried out at 24 h post-infection by A. ocellatum. During the transcriptomic analysis, 1367 differentially expressed genes (DEGs) in the skin of T. ovatus under A. ocellatum infection and control conditions were obtained. In Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotated analyses, the DEGs were significantly enriched in the immune-related pathways. To better understand the immune-related gene expression dynamics, a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the primary and secondary infection groups of T. ovatus at different stages (3 h, 12 h, 24 h, 48 h and, 72 h post-infection) of infection with A.ocellatum. The results showed that innate immunity-related genes [interleukin (IL-8), chemokine ligand 3 (CCL3), toll-like receptor 7 (TLR7), and G-type lysosome (LZM g)] and adaptive immunity-related gene [major histocompatibility complex (MHC) alpha antigen I and MHC alpha antigen II] expression levels in the primary and secondary infection groups were significantly increased compared to the control group. The expression of MHC I and MHC II was more rapidly upregulated in the secondary infection group compared with the primary infection group after A.ocellatum infection. However, no significant differences of A.ocellatum load were observed in primary and secondary infection groups. In addition, the serum of the primary infection group had significantly higher concentrations of triglyceride (TG), higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities than the control group. This study contributes to understanding the defense mechanisms in fish skin against ectoparasite infection.

Interferon-Induced Protein 6-16 (IFI6-16) from Litopenaeus vannamei Regulate Antiviral Immunity via Apoptosis-Related Genes.

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)-Vago-JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF-IFN-JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT-IFI6-16-caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.

Production of monoclonal antibody against grouper (Epinephelus coioides) CD4-1 and the distribution of CD4-1+ cells.

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1+ cells in the various tissues from grouper. Tissue distribution analyses revealed that the grouper CD4-1+ cells were expressed in all tissues tested in the healthy grouper, with greater localization in the thymus, head kidney, and spleen tissues. In addition, we tested the changes in the proportion of CD4-1+ cells in the thymus, head kidney, and the gills of grouper post the infection by C. irritans. Our data suggest that the CD4-1 mAb produced against grouper in the current study can be used as a tool to characterize CD4-1+ cells and to investigate the functions of the grouper CD4-1+ cells in the host response against pathogens infection.

Effects of dietary black soldier fly (Hermetia illucens Linnaeus) on the disease resistance of juvenile grouper (Epinephelus coioides).

An experiment was performed to study the effects of dietary levels of black soldier fly larva meal (BSFLM) on the growth performance, immunity and disease resistance of juvenile grouper (Epinephelus coioides). Four isoproteic and isoenergetic diets were formulated with dietary BSFLM levels of 0 g/kg (T0), 25 g/kg (T2.5), 50 g/kg (T5) and 100 g/kg (T10). Each diet was randomly fed to triplicate groups, each containing 40 fish. The results of the 30-day study indicated that fish growth performance was not affected in the T2.5 and T5 groups compared with the T0 group. In the group with a dietary BSFLM level of 100 g/kg, the feed coefficient was significantly higher than that in the other three groups. The superoxide dismutase, catalase, glutathione peroxidase, lysozyme activity, and malondialdehyde content in the liver, and the interleukin-1 beta (IL-1ß), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α) and heat shock protein 70 (HSP70) expression in the gills, head kidney, liver and spleen remained consistent in all groups. In addition, no significant differences in the cumulative mortality or parasite abundance in groupers after Vibrio harveyi and Cryptocaryon irritans infection were observed. These results suggested that BSFLM supplemented diets did not inhibit disease resistance in groupers.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Study on the characterization of grouper (Epinephelus coioides) immunoglobulin T and its positive cells.

Immunoglobulins (Igs) play a vital role in the adaptive immunity of gnathostomes. IgT, a particular Ig class in teleost fishes, receives much attention concerning the mucosal immunity. While, the characteristic and function of Epinephelus coioides IgT is still unknown. In our study, a polyclonal antibody was first prepared with grouper IgT heavy chain recombinant protein. IgT was revealed to be polymeric in serum and mucus. In normal groupers, IgT had high expression level in head kidney and spleen, while little amount in gills, thymus, gut and liver. The number of IgT-positive cells in different tissues was in line with their IgT expression. Furthermore, IgT could coat fractional bacteria in the mucus. In conclusion, this research revealed the protein characteristic, basal expression and bacterial coverage of grouper IgT. This is the first study to identify the characteristic of grouper IgT and demonstrate the capacity of coating microbes.

Expression profile of the fish immune enzyme l-amino acid oxidase (LAAO) after Streptococcus agalactiae infection in zebrafish (Danio rerio).

l-amino acid oxidase (LAAO) is a recently discovered novel fish immune enzyme. To explore the role of LAAO in the immune system of bony fishes, we cloned the full-length coding sequence (CDS) of LAAO of the zebrafish Danio rerio (ZF-LAAO), conducted bioinformatics analysis of ZF-LAAO, and analyzed its expression profile in zebrafish infected with the pathogen Streptococcus agalactiae. The CDS of ZF-LAAO was 1,515 base pairs long, and the encoded protein of ZF-LAAO contained an 18 amino acid signal peptide. ZF-LAAO contained the conserved domains of the LAAO family (dinucleotide binding motif and GG-motif), 2 N-glycosylation sites, and 2 O-glycosylation sites, and it was a stable hydrophilic exocrine protein. Similarity of the amino acid sequence of ZF-LAAO with LAAOs of 14 other bony fish species was >50% in all cases. The greatest similarity (79.45%) was with the LAAO of Anabarilius grahami, and these two LAAOs were grouped together in the phylogenetic tree. In wild-type zebrafish infected with S. agalactiae, changes in ZF-LAAO gene (zflaao) expression occurred mainly in the early stage of infection, and the changes in zflaao expression were more pronounced than those of the immune enzyme lysozyme (LYZ). The expression levels of both LYZ gene of zebrafish (zflyz) and zflaao were significantly elevated at 6 h after infection (p < 0.001), but zflyz expression in the spleen decreased at 12 h whereas zflaao expression in the liver and spleen peaked at 12 h. These results provided a reference for functional studies of the novel immune enzyme LAAO in bony fish.

Grouper (Epinephelus coioides) IRAK-4 regulates activation of NF-κB and expression of inflammatory cytokines in grouper spleen cells.

IRAK-4 is a serine/threonine kinase that can bind to interleukin-1 receptor induced by interleukin-1. It plays a key role in the Toll-like receptor signaling pathway and is involved in innate and adaptive immune responses. In this study, piscine IRAK-4 significantly activated nuclear factor (NF)-κB signaling in grouper spleen cells. Grouper (Epinephelus coioides) IRAK-4 (EcIRAK-4) co-localized with EcMyD88 and did not impair EcMyD88-dependent NF-κB activation. Different doses of EcIRAK-4 caused different degrees of nuclear translocation of the transcription factor NF-κB p65 subunit, and it induced transcription of multiple pro-inflammatory cytokines. Using expression vectors of deletion domains or mutations at important sites of EcIRAK-4, we found that the EcIRAK-4 kinase domain is necessary for its signal transduction function. The conserved amino acid sites performed functions similar to those in mammals, and grouper-specific amino acids such as E339 also played important roles. These findings provide information about the functional characteristics of IRAK-4 in lower vertebrates.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

IκB kinase α-1 and -2 regulate cytokine expression in the orange-spotted grouper (Epinephelus coioides).

IκB kinase (IKK) is the core regulator of the nuclear factor-κB (NF-κB) pathway, which is involved in cellular development and proliferation, as well as the inflammatory response. IKKα is an important subunit of the IKK complex. In this study, two IKKαs (EcIKKα-1 and -2) were characterized in E. coioides. Similar to IKKα of other species, EcIKKα-1 and -2 contained a kinase domain, a leucine zipper, a helix-loop-helix domain and a beta NF-κB essential modulator-binding domain. Sequence alignment indicated that EcIKKα-1 and -2 shared high degrees of sequence identity with IKKs from other species (about 63%-96%). EcIKKα-1 and -2 are widely expressed in all tissues, but have different expression profiles in normal groupers. Additionally, EcIKKα-1 and -2 responded rapidly to Cryptocaryon irritans infection at the local infection site (i.e., gill tissue), but there was no significant change in EcIKKα-2 expression. In GS cells, EcIKKα-1 was uniformly distributed in the cytoplasm, while EcIKKα-2 was observed uniformly both in the cytoplasm and nucleus. Both EcIKKα-1 and -2 were found to activate NF-κB, but the luciferase activity of EcIKKα-2 was twice that of EcIKKα-1. In addition, EcIKKα-1 and -2 can regulate the expression of immune-related cytokines (IL-1ß, IL-6, IL-8, IL-12 [p35 subunit], and TNF-α). These findings should prove helpful to further elucidate the innate immunity function of IKKα in fish.

Orange-spotted grouper (Epinephelus coioides) NADPH oxidase: Cloning and expression analysis after Cryptocaryon irritans infection.

Phagocytic cells are activated to produce a large amount of reactive oxygen species (ROS) that kill pathogens quickly and efficiently through oxidation. NADPH oxidase is the main source of intracellular ROS. In the present study, five subunits of the phagocytic NADPH oxidase complex were identified in orange-spotted grouper (Epinephelus coioides). The open reading frame of grouper gp91phox, p22phox, p67phox, p47phox, and p40phox were 1,698 bp, 564 bp, 1,497 bp, 1,290 bp, and 1,050 bp, respectively, and encoded 565, 187, 498, 429, and 349 amino acids. Evolutionary analysis indicated that these proteins are evolutionarily homologous to the corresponding proteins of other fish and mammals, and contain conserved functional domains and sites that are important in mammals. In addition, real-time polymerase chain reaction analysis showed that the expression of these five genes was higher in immune-related tissues in normal grouper, and that these genes were up-regulated in gill and spleen after C. irritans infection, which suggests that these genes may be involved in the defense against C. irritans infection.

Effects of short-term fasting on the resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus agalactiae infection.

Short-term feed deprivation or fasting is commonly experienced by aquaculture fish species and may be caused by seasonal variations, production strategies, or diseases. To assess the effects of fasting on the resistance of Nile tilapia to Streptococcus agalactiae infection, vaccinated and unvaccinated fish were fasted for zero, one, three, and seven days prior to infection. The cortisol levels of both vaccinated and unvaccinated fish first decreased and then increased significantly as fasting time increased. Liver glycogen, triglycerides, and total cholesterol decreased significantly after seven days of fasting, but glucose content did not vary significantly between fish fasted for three and seven days. Hexokinase (HK) and pyruvate kinase (PK) activity levels were lowest after seven days of fasting, while phosphoenolpyruvate carboxykinase (PEPCK) activity levels varied in opposition to those of HK and PK. Serum superoxide dismutase (SOD) and catalase (CAT) activity levels first increased and then decreased as fasting time increased; SOD activity was highest after three days of fasting. Interleukin-1beta (IL-1ß) and IL-6 mRNA expression levels first increased and then decreased significantly, peaking after three days of fasting. However, suppressor of cytokine signaling-1 (SOCS-1) mRNA expression levels were in opposition to those of IL-1ß and IL-6. Specific antibody levels did not vary significantly among unvaccinated fish fasted for different periods. Although specific antibody level first increased and then decreased in the vaccinated fish as fasting duration increased, there were no significant differences in the survival rates of fasted vaccinated fish after challenge with S. agalactiae. The final survival rates of vaccinated fish fasted for zero, one, three, and seven days were 86.67 ±â€¯5.44%, 80.00 ±â€¯3.14%, 88.89 ±â€¯6.28%, and 84.44 ±â€¯8.32%, respectively. Among the unvaccinated fish, the survival rate was highest (35.56 ±â€¯3.14%) in the fish fasted for three days and lowest (6.67 ±â€¯3.14%) in the fish fasted for seven days. Therefore, our results indicated that short-term fasting (three days) prior to an infection might increase the resistance of unvaccinated Nile tilapia to S. agalactiae.

Grouper (Epinephelus coioides) Mpeg1s: Molecular identification, expression analysis, and antimicrobial activity.

Macrophage expressed gene 1 (Mpeg1) is a molecule that can form pores and destroy the cell membrane of invading pathogens. In this study, we identified two Mpeg1 isoforms from the orange-spotted grouper (Epinephelus coioides) and named them EcMpeg1a and EcMpeg1b. Predicted proteins of the two EcMpeg1s contained a signal peptide, a conserved membrane attack complex/perforin (MACPF) domain, a transmembrane segment, and an intracellular region. Sequence alignment demonstrated that two EcMpeg1 proteins share a high sequence identity with that of other teleosts. Tissue distribution analysis showed that EcMpeg1s were expressed in all tissues tested in healthy grouper, with the highest expression in the head kidney and spleen. After infection with the ciliate parasite Cryptocaryon irritans, expression of the two EcMpeg1s was significantly upregulated in the spleen and gills. Furthermore, the recombinant EcMpeg1a showed antiparasitic and antibacterial activity against Gram-negative and -positive bacteria, whereas EcMpeg1b had an inhibitory effect only against Gram-positive bacteria. These results indicated that EcMpeg1s play an important role in the host response against invading pathogens.

Grouper (Epinephelus coioides) MyD88 adaptor-like (Mal): Molecular cloning, expression, and functionality.

Initiation of the innate immune response requires recognition of pathogen-associated molecular patterns by pathogen recognition receptors such as Toll-like receptors (TLRs). MyD88 adaptor-like (Mal) is an adaptor that responds to TLR activation and acts as a bridging adaptor for MyD88. In the present study, the open reading frame of Mal was identified in orange-spotted grouper (Epinephelus coioides), and named EcMal. It contained 831 bp encoding 276 aa, and was encoded by a 1299 bp DNA sequence with three exons and two introns. EcMal and the Mal sequence of other species shared different degrees of sequence identity, and clustered into the same group. EcMal was distributed in all tissues tested in healthy grouper, with the highest expression level in the head kidney. After infection with Cryptocaryon irritans, the expression level of EcMal was up-regulated in the gill and spleen. In addition, EcMal exhibited global cytosolic and nucleus localization, and could significantly activate NF-κB activity in grouper spleen cells.

First report of trypanosomiasis in farmed largemouth bass (Micropterus salmoides) from China: pathological evaluation and taxonomic status.

The aim of this study was to evaluate the effect of trypanosomes on cultured largemouth bass (Micropterus salmoides) and describe the taxonomic identification of the parasite. The effects of the parasite on M. salmoides were examined based on clinical symptoms, hemograms, histopathology, and serum biochemistry. Diseased fish showed typical clinical symptoms of trypanosomiasis, which included lethargy, anorexia, and histopathological lesions in the liver, head kidney, and spleen. The serum of diseased fish had significantly lower concentrations of glucose, triglyceride, and low-density lipoprotein, and significantly higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities. The morphology of the trypanosomes was also analyzed using light microscopy, and their 18S rDNA sequence was analyzed to establish genetic relationships with other known strains. We found that the trypomastigote form of the trypanosomes from M. salmoides was similar to those isolated from Pelteobagrus fulvidraco. The trypanosomes had a slender and narrow body with a relatively long free flagellum, not well-developed undulating membrane, and an oval kinetoplast located near the subterminal posterior end of the body. The 18S rDNA sequences of the trypanosome from M. salmoides had the highest similarity (99.8%) with that of P. fulvidraco, suggesting they are identical species. Based on the differences in morphological characteristics and 18S rDNA sequence compared to trypanosomes isolated from other freshwater fish, it is considered as a new species and we propose the name Trypanosoma micropteri n. sp.