Ar Bridle

Increased Elongase and Desaturase Gene Expression with Stearidonic Acid Enriched Diet Does Not Enhance Long-Chain (n-3) Content of Seawater Atlantic Salmon (Salmo salar L.)

Atlantic salmon (Salmo salar L.) can produce (n-3) long-chain (LC)-PUFA when fed biosynthetic precursors. This has potential for developing sustainable aquafeeds. Echium oil (EO) is rich in stearidonic acid [SDA; 18:4(n-3)] and bypasses the initial Delta6 desaturase (FAD6) step in the (n-3) LC-PUFA biosynthetic pathway. EO was fed to seawater Atlantic salmon for 12 wk and compared with fish fed a diet containing canola oil (CO), a source of alpha-linolenic acid [ALA; 18:3(n-3)] or fish oil (FO) that provides (n-3) LC-PUFA. Fatty acid (FA) composition of liver, white muscle, and whole fish was measured to show whether dietary precursors were endogenously biosynthesized to LC-PUFA. Gene expression of liver FA elongase and FAD5 was upregulated in EO fish compared with FO fish. Furthermore, dietary precursors affected the FA concentrations of direct biosynthetic products in all tissues. The increased gene expression in the EO fish was reflected by an increased FA concentration of eicosapentaenoic acid [20:5(n-3)] in the liver compared with the CO fish. However, the high concentrations of (n-3) LC-PUFA found in seawater Atlantic salmon fed diets rich in FO were not attained via biosynthesis from precursors (ALA or SDA) in diets.

Evidence of an Antimicrobial-Immunomodulatory Role of Atlantic Salmon Cathelicidins during Infection with Yersinia ruckeri

Cathelicidins are a family of antimicrobial peptides that act as effector molecules of the innate immune system with broad-spectrum antimicrobial properties. These evolutionary conserved cationic host-defence peptides are integral components of the immune response of fish, which are generally believed to rely heavily on innate immune defences to invading pathogens. In this study we showed that Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated peripheral blood leukocytes increasing the transcription of the chemokine interleukin-8. Further, functional differences were identified between the two cathelicidins. In the presence of serum, asCATH1 displayed greatly diminished host haemolytic activity, while the constitutively expressed asCATH2 had no haemolytic activity with or without serum. These findings support our hypothesis that fish cathelicidins exert their primary antimicrobial action at the site of pathogen invasion such as epithelial surfaces. Further, we hypothesise that like their mammalian counterparts in the presence of serum they act as mediators of the innate and adaptive immune response via the release of cytokines thus indirectly protecting against a variety of pathogens. We highlight the importance of this immunomodulatory role from the involvement of asCATHs during an infection with the fish pathogen Yersinia ruckeri. While we were able to demonstrate in vitro that asCATH1 and 2, possessed direct microbicidal activity against the fish pathogen, Vibrio anguillarum, and a common gram negative bacterium, Escherichia coli, little or no bactericidal activity was found against Y. ruckeri. The contribution of either asCATH in the immune response or as a potential virulence factor during yersiniosis is highlighted from the increased expression of asCATH1 and 2 mRNA during an in vivo challenge with Y. ruckeri . We propose that Atlantic salmon cathelicidins participate in the interplay between the innate and adaptive immune systems via the release of cytokines enabling a more effective response to invading pathogens.

In vitro cultured Neoparamoeba perurans causes amoebic gill disease in Atlantic salmon and fulfils Koch’s postulates

Amoebic gill disease (AGD) in marine farmed Atlantic salmon is of growing concern worldwide and remains a significant health issue for salmon growers in Australia. Until now the aetiological agent, Neoparamoeba perurans, has not been amenable to in vitro culture and therefore Kochs postulates could not be fulfilled. The inability to culture the amoeba has been a limiting factor in the progression of research into AGD and required the maintenance of an on-going laboratory-based infection to supply infective material. Culture methods using malt yeast agar with sea water overlaid and subculturing every 3-4 days have resulted in the establishment of a clonal culture of N. perurans, designated clone 4. Identity of the amoeba was confirmed by PCR. After 70 days in culture clone 4 infected Atlantic salmon, causing AGD, and was re-isolated from the infected fish. Diagnosis was confirmed by histology and the infectious agent identified by PCR and in situ hybridisation using oligonucleotide primers and probes previously developed and specific to N. perurans. This study has fulfilled Kochs postulates for N. perurans as a causative agent of AGD and illustrates its free-living and parasitic nature.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C(20)) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer , a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.

Identification of Surrogates of Protection against Yersiniosis in Immersion Vaccinated Atlantic Salmon

Simple cost-effective bacterins are the earliest and most successfully used commercial vaccines in fish. In particular, those prepared from Yersinia ruckeri have proven effective at controlling Enteric Red Mouth Disease (ERM) and yersiniosis in rainbow trout and Atlantic salmon, respectively. However, the emergence of outbreaks of ERM caused by atypical biotypes of Y. ruckeri and reports of vaccine failure resulting in mass mortality of hatchery Atlantic salmon has reinvigorated interest in vaccines against fish bacterial diseases. Therefore the objective of this study was to identify surrogates of protection against yersiniosis using cDNA microarray to characterise the response of host genes in the gills of unvaccinated and vaccinated Atlantic salmon challenged with Y. ruckeri. Differentially expressed genes were identified using two-way ANOVA and restricted to those with >2.5-fold change at P<0.05. Using cDNA microarray we identified the expression of 6 genes in response to infection and 4 genes associated with the protective host response to yersiniosis. Analysis by real-time PCR confirmed that three immunologically relevant genes, namely a cathelicidin (47-fold) and a C-type lectin (19-fold) increased in response to yersiniosis. Including collagenase (17-fold increase), an important tissue remodelling and repair enzyme, these genes represent 3 of 6 non-protective and/or pathological responses to yersiniosis. Genes associated with the protective host response included an immunoglobulin gene and a selenoprotein that showed significant fold changes (15-fold increases each), highlighting the importance of antibody-mediated protection against yersiniosis. These findings provide much needed knowledge of the host-pathogen interaction in response to bacterial infection and immunisation in fish. Significantly, we identified a transcriptional biosignature consisting of predominantly immune-relevant genes (14 up and 3 down-regulated) in the gills of Atlantic salmon after immersion vaccination and before bacterial challenge. This biosignature may be used as a surrogate of protection and therefore as a predictor of vaccine success against yersiniosis.

Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease

The external surfaces of fish, such as gill and skin, are covered by mucus, which forms a thin interface between the organism and water. Amoebic gill disease (AGD) is a parasitic condition caused by Neoparamoeba perurans that affects salmonids worldwide. This disease induces excessive mucus production in the gills. The host immune response to AGD is not fully understood, and research tools such as genomics and proteomics could be useful in providing further insight. Gill and skin mucus samples were obtained from Atlantic salmon (Salmo salar) which were infected with N. perurans on four successive occasions. NanoLC tandem mass spectrometry (MS/MS) was used to identify proteins in gill and skin mucus of Atlantic salmon affected by AGD. A total of 186 and 322 non-redundant proteins were identified in gill and skin mucus respectively, based on stringent filtration criteria, and statistics demonstrated that 52 gill and 42 skin mucus proteins were differentially expressed in mucus samples from AGD-affected fish. By generating protein-protein interaction networks, some of these proteins formed part of cell to cell signalling and inflammation pathways, such as C-reactive protein, apolipoprotein 1, granulin, cathepsin, angiogenin-1. In addition to proteins that were entirely novel in the context in the host response to N. perurans, our results have confirmed the presence of protein markers in mucus that have been previously predicted on the basis of modified mRNA expression, such as anterior gradient-2 protein, annexin A-1 and complement C3 factor. This first proteomic analysis of AGD-affected salmon provides new information on the effect of AGD on protein composition of gill and skin mucus. Future research should focus on better understanding of the role these components play in the response against infection with N. perurans.

SYBR, TaqMan, or both: Highly sensitive, non-invasive detection of Cardicola blood fluke species in Southern Bluefin Tuna (Thunnus maccoyii)

Three species of blood fluke from the genus Cardicola are known to parasitize and cause disease in Bluefin Tunas--C. forsteri, C. orientalis, and C. opisthorchis. Although initially believed to be separated by geography and host specificity, recent identification of at least two Cardicola spp. concurrently present within all three Bluefin species has raised questions concerning pathogenicity, relative abundance, and distribution of these parasites within Bluefin populations. Here, we present sensitive and differential real-time qPCR nucleic acid detection of these Cardicola spp. by targeting the ITS2 region of the parasite rDNA for PCR amplification. A limit of sensitivity of 1-5 genome copy equivelents was achieved for each of the three Cardicola species tested without cross-species or host genomic amplification. Similar sensitivity was further achieved in the presence of up to 20 ng/μL non-target host gDNA using SYBR Green chemistry alone, or in the presence of up to 160 ng/μL host gDNA through the utilization of a TaqMan probe common-reporter detection system. These methods were subsequently used to positively identify both C. forsteri and C. orientalis DNA in preserved samples of serum, gill, and heart from ranched Southern Bluefin Tuna Thunnus maccoyii. Both methods were more sensitive for positively and differentially identifying the presence of Cardicola spp. than either histological or heart-flush microscopy techniques previously employed, and also possess the ability to be applied in non-lethal blood sampling of these highly valued fish. This is the first report for rapid and differential molecular quantitative detection of Cardicola, and opens the potential for effective monitoring of infection in cultured bluefin populations. Further, it is anticipated that the use of SYBR Green for melt-curve analyses in conjunction with a common-reporter TaqMan assay will present a flexible, accurate, and cost-effective approach for differential detection of a variety of other pathogens in future.

Sesamin modulation of lipid class and fatty acid profile in early juvenile teleost, Lates calcarifer, fed different dietary oils

Sesamin, a major sesame seed lignan, has diverse biological functions including the modulation of molecular actions in lipid metabolic pathways and reducing cholesterol levels. Vertebrates have different capacities to biosynthesize long-chain PUFA from dietary precursors and sesamin can enhance the biosynthesis of ALA to EPA and DHA in marine teleost. Early juvenile barramundi, Lates calcarifer, were fed for two weeks on diets rich in ALA or SDA derived from linseed or Echium plantagineum, respectively. Both diets contained phytosterols and less cholesterol compared with a standard fish oil-based diet. The growth rates were reduced in the animals receiving sesamin regardless of the dietary oil. However, the relative levels of n-3 LC-PUFA in total lipid, but not the phospholipid, increased in the whole body by up to 25% in animals fed on sesamin with ALA or SDA. Sesamin reduced the relative levels of triacylglycerols and increased polar lipid, and did not affect the relative composition of phospholipid subclasses or sterols. Sesamin is a potent modulator for LC-PUFA biosynthesis in animals, but probably will have more effective impact at advanced ages. By modulating certain lipid metabolic pathways, sesamin has probably disrupted the body growth and development of organs and tissues in early juvenile barramundi.

Immunity to Amoeba

Amoebic infections in fish are most likely underestimated and sometimes overlooked due to the challenges associated with their diagnosis. Amoebic diseases reported in fish affect either gills or internal organs or may be systemic. Host response ranges from hyperplastic response in gill infections to inflammation (including granuloma formation) in internal organs. This review focuses on the immune response of Atlantic salmon to Neoparamoeba perurans, the causative agent of Amoebic Gill Disease (AGD).

Coping with sub-optimal water temperature: Modifications in fatty acid profile of barramundi as influenced by dietary lipid

Metabolic responses to sub-optimal temperature deplete lipid depots, remodel membrane lipid and alter the fatty acid profile in the whole body and tissues of ectothermic vertebrates including fish. The magnitude of these changes may depend on dietary history including oil sources with different fatty acid compositions. Barramundi, Lates calcarifer (Perciformes, Latidae), a tropical ectothermic fish, was fed on diets either rich in dietary long-chain (≥C(20)) polyunsaturated fatty acids (LC-PUFA) from fish oil, rich in stearidonic and γ-linolenic acid (SDA and GLA, respectively) from Echium plantagineum, or rapeseed oil deficient in LC-PUFA. Following 5 weeks at the optimum temperature of 30 °C when growth rates were comparable amongst dietary treatments, water temperature was dropped to 20 °C for 1 week for half of the animals and maintained at 30 °C for the other half. Decreased temperature increased the liver and skeletal muscle content of LC-PUFA in fish fed on echium oil compared with rapeseed oil, while dietary LC-PUFA depots in fish oil fed-fish depleted rapidly in the week of sub-optimal temperature. The lipid unsaturation index of cellular membrane in the liver and muscle increased under low temperature at the same rate regardless of dietary oil. Therefore, rapid exposure of an ectothermic vertebrate to a lower and sub-optimal temperature caused significant modulation in fatty acid composition. We propose that the tolerance of barramundi, a representative of tropical farmed fish, to sub-optimal temperature will be enhanced when fatty acid substrates closer to the LC-PUFA are available in their diet.