What can we learn about fish neutrophil and macrophage response to immune challenge from studies in zebrafish.

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.

Acute immune responses in zebrafish and evasive behavior of a parasite - who is winning?

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation.

Validation of a QTL associated with resistance to Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Vibriosis is a bacterial disease in fish caused by the Gram negative bacterium Vibrio anguillarum with severe impact on rainbow trout (Oncorhynchus mykiss) farming. Sustainable control methods should be developed and we here show that marker assisted selective breeding of fish naturally resistant to the disease is feasible. We have validated the use of a single nucleotide polymorphism (SNP) marker SNP AX-89,945,921 (QTL on chromosome 21). The QTL was previously found associated with resistance to vibriosis and described following a genome wide association analysis (GWAS) of trout exposed to the bacterium. For this validation spawners were genotyped by use of the 57 K Axiom®Trout Microarray (Affymetrix) and homozygous male fish carrying the allele with the SNP AX-89,945,921 were then selected and used to fertilize eggs from outbred female trout resulting in fish all carrying the SNP (QTL-fish). Control fish (non-QTL fish) were produced by fertilizing the same batch of eggs by use of male parents negative for the SNP. The fish were exposed in freshwater to V. anguillarum (water bath infection) at 19 C°. A total of 900 fish were challenged in a common garden set-up in triplicate. A bacterial solution of V. anguillarum (serotype O1) was added to each of three freshwater fish tanks, each with 150 QTL and 150 non-QTL fish. Fish were tagged by tail fin cut (upper/lower) to discern the two groups, whereafter fish were monitored around the clock to detect disease signs and remove moribund fish. Clinical vibriosis developed within two days in non-QTL-fish (overall morbidity of 70%). QTL fish developed clinical signs later and the morbidity was significantly lower and did not reach 50%. Rainbow trout farming may benefit from using the QTL associated with higher resistance towards vibriosis. The effect may be optimized in the future by use of both male and female parents homozygous for the marker allele.

Toxicity of the antiparasitic lipopeptide biosurfactant SPH6 to green algae, cyanobacteria, crustaceans and zebrafish.

A lipopeptide with biosurfactant properties produced by the bacterium Pseudomonas H6 (SPH6) has antiparasitic effects and may serve as an alternative to chemotherapeutants against aquatic pathogens in aquaculture. We have elucidated its ecotoxicological potential by short-term standardized tests, including a growth rate inhibition test with algae (Raphidocelis subcapitata), a lethality test on the cyanobacteria Phormidium autumnale, a lethality test using crustaceans (Daphnia magna), a fish embryo acute toxicity test and a fish acute toxicity test using zebrafish (Danio rerio). The decrease of the biosurfactant concentration in zebrafish test water during 24 h was measured. The toxicity for crustaceans was highest (LC50 = 20 mg/L), followed by the test with the zebrafish embryo (LC50 = 27 mg/L). The juvenile zebrafish fish (complete mortality occurred between 40 and 80 mg/L), the cyanobacteria (LC50 = 80 mg/L) and the green algae (EC50 = 170 mg/L) showed higher tolerance. The determination of SPH6 concentrations in fish tank (up to 50% elimination over 24 h) suggested that the compound may become adsorbed to tank walls, absorbed by fish or degraded. Further studies should determine its impact under different environmental settings (e.g. temperature) relevant for different branches of the aquaculture sector.

Plastic nanoparticles cause mild inflammation, disrupt metabolic pathways, change the gut microbiota and affect reproduction in zebrafish: A full generation multi-omics study.

Plastic pollution has become a major concern on a global scale. The plastic is broken down into minuscule particles, which have an impact on the biosystems, however long-term impacts through an entire generation is largely unknown. Here, we present the first whole generation study exposing fish to a 500 nm polystyrene plastic particle at environmentally relevant concentrations. Short- and long-term adverse effects were investigated in the zebrafish model organism using a holistic multi-omics approach. The particles accumulated in the yolk sac of young larvae and short-term biological impacts included immune-relevant gene regulation related to inflammation and tolerance as well as disruption of metabolic processes, such as the fatty acid and lipid pathways. The long-term effects comprised gene regulations pointing towards skin and/or gill inflammation, dysbiosis of the gut microbiota, a tendency towards decreased condition factor in adult males as well as a lowered reproductive capability. From this study, it can be concluded that exposures to plastic nanoparticles have an impact on population as well as ecosystem level in fish and likely also in other vertebrates.

Whole-genome association study searching for QTL for Aeromonas salmonicida resistance in rainbow trout.

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, has extensive negative effects on wild and farmed salmonids worldwide. Vaccination induces some protection under certain conditions but disease outbreaks occur even in vaccinated fish. Therefore, alternative disease control approaches are required to ensure the sustainable expansion of rainbow trout aquaculture. Selective breeding can be applied to enhance host resistance to pathogens. The present work used genome-wide association study (GWAS) to identify quantitative trait loci (QTL) associated with A. salmonicida resistance in rainbow trout. A total 798 rainbow trout exposed to A. salmonicida by bath challenge revealed 614 susceptible and 138 resistant fish. Genotyping was conducted using the 57 K single nucleotide polymorphism (SNP) array and the GWAS was performed for survival and time to death phenotypes. We identified a QTL on chromosome 16 and located positional candidate genes in the proximity of the most significant SNPs. In addition, samples from exposed fish were examined for expression of 24 immune-relevant genes indicating a systematic immune response to the infection. The present work demonstrated that resistance to A. salmonicida is moderately heritable with oligogenic architecture. These result will be useful for the future breeding programs for improving the natural resistance of rainbow trout against furunculosis.

Inflammatory reactions in rainbow trout fins and gills exposed to biocides.

Several biocides are widely used in rainbow trout aquaculture against various ectoparasites and ectobionts, but the inflammation induced in treated fish is less well described. Dose-response studies were conducted to elucidate the effects on rainbow trout (gills and fins) induced by a series of biocides including formalin, hydrogen peroxide (H2O2), peracetic acid (PAA) and the surfactant SPH6, which was isolated from the bacterium Pseudomonas H6. The compounds have documented antiparasitic effects, but the specific effects on fish needs further documentation. This study was performed over 24 h, and inflammatory reactions were evaluated in gills and fins. A dose-dependent effect was noted for expression of immune genes encoding for IL-1ß, TNFα, IFNγ, IL-10, IL-8, lysozyme, serum amyloid A (SAA), hepcidin, precerebellin and complement factor C3. PAA induced the strongest upregulation of cytokine and acute phase reactant genes followed by H2O2 and formalin. SPH6 showed a lower effect, and in several cases the compound induced downregulation of several genes. Gills showed a stronger response compared to fins. The mucous cell density in fins showed a range of changes which varied by compound. PAA, and to a lesser degree H2O2 and formalin, initially induced mucous cell hyperplasia, whereas SPH6 immediately decreased the number of cells containing mucus.

A non-lethal method for detection of Bonamia ostreae in flat oyster (Ostrea edulis) using environmental DNA.

Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.

Immune gene expression and genome-wide association analysis in rainbow trout with different resistance to Yersinia ruckeri infection.

Selective breeding programmes involving marker assisted selection of innately pathogen resistant strains of rainbow trout rely on reliable controlled infection studies, extensive DNA typing of individual fish and recording of expression of relevant genes. We exposed juvenile rainbow trout (6 h bath to 2.6 × 105 CFU mL-1) to the fish pathogen Yersinia ruckeri serotype O1, biotype 2, eliciting Enteric Red Mouth Disease ERM, and followed the disease progression over 21 days. Cumulative mortality reached 42% at 12 days post challenge (dpc) after which no disease signs were recorded. All fish were sampled for DNA-typing (50 k SNP chip, Affymetrix®) throughout the course of infection when they showed clinical signs of disease (susceptible fish) or at day 21 when fish showed no clinical signs of disease (survivors - resistant fish). Genome-wide association analyses of 1027 trout applying single nucleotide polymorphisms (SNPs) as markers revealed an association between traits (susceptible/resistant) and certain regions of the trout genome. It was indicated that multiple genes are involved in rainbow trout resistance towards ERM whereby it is considered a polygenic trait. A corresponding trout group was kept as non-exposed controls and a comparative expression analysis of central innate and adaptive immune genes in gills, spleen and liver was performed for three fish groups: 1) moribund trout exhibiting clinical signs 7 dpc (CS), 2) exposed fish without clinical signs at the same sampling point (NCS) and 3) surviving fish at 21 dpc (survivors). Immune genes encoding inflammatory cytokines (IL-1ß, IL-2A, IL-6A, IL-8, IL-10A, IL-12, IL-17A/F2A, IL-17C1, IL-17C2, IL-22, IFNγ, TNFα), acute phase reactants (SAA, C3, cathelicidins, lysozyme) were expressed differently in CS and NCS fish. Correlation (negative or positive) between expression of genes and bacterial load suggested involvement of immune genes in protection. Down-regulation of adaptive immune genes including IgDm, IgDs, IgT and TCR-ß was seen primarily in CS and NCS fish whereas survivors showed up-regulation of effector molecule genes such as cathelicidins, complement and lysozyme suggesting their role in clearing the infection. In conclusion, SNP analyses indicated that ERM resistance in rainbow trout is a multi-locus trait. The gene expression in surviving fish suggested that several immune genes are associated with the trait conferring resistance.

Effects of pH on free-living stages of a Nordic strain of the economically important freshwater fish parasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a unicellular freshwater fish parasite and the causative agent of the globally distributed white spot disease. The fitness of the parasite depends on available hosts and abiotic factors such as temperature, salinity and pH. With climatic change these abiotic factors may be altered, thereby influencing the health of the parasite. In this study, the tolerance towards different pH values (2-11) was investigated on a Nordic strain of the parasite by recording tomont survival, release of theronts, theront size and theront survival. Tomonts were able to survive and release theronts in pH 5-10, however the number of released theronts was significantly lower at high and low pH. Theronts produced at pH 8 and exposed to the different pH values survived at pH 4-10 for 1 h, which may be sufficient time for the parasite to locate and infect new hosts. The release of theronts was slower at pH 10, and the size of theronts developed at higher pH was significantly increased (up to 73.5 µm in length). In conclusion, our study showed that the free-living stages of I. multifiliis were capable of surviving at a pH from 5 to10, and that high pH had an effect on the morphology and release of the parasites.

Gill amoebae from freshwater rainbow trout (Oncorhynchus mykiss): In vitro evaluation of antiparasitic compounds against Vannella sp.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose-response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml).

A Major QTL for Resistance to Vibrio anguillarum in Rainbow Trout.

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish - including survivors - and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.