Comparative transcriptomic analysis provides insights into immune responses of lamprey larvae under three pathogens infections.

Based on the large number of deaths of lamprey larvae infected by pathogens in the culture process, the infection response of lamprey larvae to different pathogens was studied. Thus, high-throughput sequencing is firstly used to analyze the lamprey larvae infected by Aeromonas hydrophila, Vibrosplendidus, and Saprolegnia parasitica. A high quality of 338,628,426 Reads were obtained and 203,829 transcripts were assembled. A total of 158,001 Unigenes were annotated by Blast comparison, and a relatively comprehensive transcriptome database of lamprey larvae was established. Based on the transcriptome of lamprey larvae infected by three aquatic pathogens, classification results of COG and GO show that the genes classified into signal transduction mechanism accounted for the largest proportion, and 2316 differentially expressed genes were identified and screened out. After infection by the three pathogens, 16 genes and 19 genes expression were up-regulated and down-regulated, respectively. KEGG pathway analysis of differentially expressed genes showed that lamprey larvae were mainly affected their metabolism and oxidative phosphorylation pathways after infection by two gram-negative bacteria, Vibrosplendidus and Aeromonas hydrophila. As a fungus, Saprolegnia parasitica mainly affected ribosome synthesis in lamprey larvae. The results were validated by detecting the expression levels of 13 DEGs at 24 h after pathogens treatment using Q-PCR. The results provide valuable information for further research into the development of immunity and the innate immune response against pathogen invasion during the artificial propagation of lamprey larvae.

Molecular characterization and expression analysis of T cell receptor (TCR) γ and δ genes in dojo loach (Misgurnus anguillicaudatus) in response to bacterial, parasitic and fungal challenge.

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.

Molecular characterization and expression analysis of interleukin 15 (IL15) and interleukin-15 receptor subunit alpha (IL15Rα) in dojo loach (Misgurnus anguillicaudatus): Their salient roles during bacterial, parasitic and fungal infection.

Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma-IL15 and Ma-IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma-IL15, while a highly conserved sushi domain existed in Ma-IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma-IL15 and Ma- IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G4, I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma-IL15 and Ma-IL15Rα showed different expression patterns in different tissues during three infection models. Ma-IL15Rα mRNA was found to be more significantly elevated than Ma-IL15 after infection with F. columnare G4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma-IL15 in mucosal tissues including skin and gill, while it mainly increased Ma-IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma-IL15 and Ma-IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma-IL15 and Ma-IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection.

Role of pathogen-derived cell wall carbohydrates and prostaglandin E2 in immune response and suppression of fish immunity by the oomycete Saprolegnia parasitica.

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1ß1 [IL-1ß1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin [corrected] E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.

Do fathead minnows, Pimephales promelas Rafinesque, alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long-held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin-penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L(-1)). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.

Prevalence of serum antibodies against Saprolegnia parasitica in wild and farmed brown trout Salmo trutta.

The prevalence of serum antibodies against Saprolegnia parasitica in wild and farmed brown trout Salmo trutta from the province of Le6n (NW Spain) was studied by enzyme-linked immunosorbent assay (ELISA). Blood samples from healthy and Saprolegnia-infected brown trout were collected over 2 yr with a seasonal periodicity (January, April, July and October) from a hatchery and river with frequent presence of saprolegniosis (River Porma) and from a river in which the disease was rarely observed (River Omaña). The individual prevalence was 30.1%, but statistically significant differences were observed between the prevalence in trout from the hatchery (43.0%), from River Porma (31.8%) and from River Omaña (6.4%) and also between the prevalence observed in October (42.9%) and the values obtained in January (24.8%), April (22.7%) and July (27.5%). There was no difference between the seroprevalence in females (34.8%) and males (38.2%), but a positive correlation between raised serum antibody levels and larger (older) fish was found. The low prevalence of antibodies observed in Saprolegnia-infected trout (18.0%) suggests possible immune suppression and the lack of an effective specific immune response in fish with saprolegniosis.

A genetic component of resistance to fungal infection in frog embryos.

The embryo has traditionally been considered to completely rely upon parental strategies to prevent threats to survival posed by predators and pathogens, such as fungi. However, recent evidence suggests that embryos may have hitherto neglected abilities to counter pathogens. Using artificial fertilization, we show that among-family variation in the number of Saprolegnia-infected eggs and embryos in the moor frog, Rana arvalis, cannot be explained by maternal effects. However, analysed as a within-females effect, sire identity had an effect on the degree of infection. Furthermore, relatively more eggs and embryos were infected when eggs were fertilized by sperm from the same, compared with a different, population. These effects were independent of variation in fertilization success. Thus, there is likely to be a significant genetic component in embryonic resistance to fungal infection in frog embryos. Early developmental stages may show more diverse defences against pathogens than has previously been acknowledged.

Detection of 'long-haired' Saprolegnia (S. parasitica) isolates using monoclonal antibodies.

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts.

Brown trout Salmo trutta injected with antigenic extracts from a pathogenic isolate of Saprolegnia parasitica developed specific antibodies that were detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and Western blotting (WB), but not by immunodiffusion (ID). Three groups of five 2 yr old brown trout were injected intraperitoneally with 3 different antigenic extracts: small hyphal fragments (HF) and soluble extracts from sonicated mycelia grown in medium with or without beta-sytosterol (SEB and SE, respectively). In the 2 groups injected with SE and SEB, antibodies were found in 66.7 % of the serum samples by ELISA, 54.5% by IF and 48.5% by WB. In the group injected with HF, only 1 trout survived the experiment, and in this fish only 1 sample was positive by ELISA. The results obtained by ELISA and IF were similar and show that there is cross-reaction between the antigens used. By WB, the proteins most frequently recognised were 2 proteins of 25 and 29 kDa. No significant differences were found in the groups injected with SE or SEB.

Genome-wide survey of the gene expression response to saprolegniasis in Atlantic salmon.

Pathogenic saprolegniaceae species are among the major disease-causing agents in farmed salmonids and in freshwater fish in general. Recent studies have used high-throughput cDNA-based methods to identify new potential actors of fish defence systems against various bacteria and viruses. However, the response of fish to fungal or fungus-like pathogens is still poorly documented. Here, we used a 16,006-gene salmonid cDNA microarray to identify genes which transcription levels are modified in juvenile Atlantic salmon (Salmo salar) affected with saprolegniasis compared to healthy fish from the same families. Our results confirmed the importance of non-specific immunity in the response of fish to saprolegniaceae infections and identified both similarities and differences in their genome-wide transcriptional response to oomycetes compared with their responses to bacterial or viral infections. Moreover, several clones with no known homologues were shown to be over-transcribed in infected fish. These may represent as yet unidentified immune-relevant genes in fish.