Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Development of matrix-specific standards for LA-ICP-MS zinc analysis in sand flathead (Platycephalus bassensis).

The use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to analyse soft tissues is limited because of the lack of suitable certified reference materials resulting in difficulties with calibration. In this study, several laboratory-prepared thin agarose sections were tested as matrix-specific standards. Our results showed 1 mm thin agarose sections were suitable as calibration standards for LA-ICP-MS Zn analyses in fish muscle, especially when the signal intensity of 66Zn was normalised to 13C. The thin agarose standard sections were used for LA-ICP-MS Zn analyses in the muscle of melanised and non-melanised sand flathead collected from a polluted estuary and assigned with different melanisation scores. Zn levels in melanised regions of fish muscle were determined to be significantly higher than non-melanised regions across all melanisation scores, though Zn levels in the melanised regions of muscle were not significantly different between sand flathead with different melanisation scores. In non-melanised regions of muscle from fish with melanisation and fish unaffected by melanisation, Zn levels were around baseline levels. Overall, this study has successfully developed matrix-specific standards for LA-ICP-MS analysis of soft tissues, thus allowing broader application of this analytical technique in future environmental pollution studies.

Dynamics of Cardicola spp. Infection in Ranched Southern Bluefin Tuna: First Observation of C. orientalis at Transfer.

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for the Southern Bluefin Tuna (Thunnus maccoyii, SBT) industry, where infections can lead to morbidity and mortality in ranched SBT populations. This study compared blood fluke infection in SBT from two companies during the 2021 ranching season. Both companies administered the same dosage of praziquantel approximately 5 weeks after transfer, feeding with frozen baitfish daily; the only difference in the company's practices was that the pontoons were located 2.5 km apart. Infection severity was measured as prevalence and intensity by quantifying adult C. forsteri in SBT heart and copy numbers of C. forsteri and C. orientalis ITS-2 DNA in SBT heart and gills. Data from the 2018 and 2019 harvests of SBT were used to make comparisons with 2021 harvest data. Cardicola orientalis was detected at transfer and no longer detected after treatment with praziquantel. Cardicola spp. were present in 83% of sampled SBT in 2021. Both companies demonstrated similar patterns of infection, and Company A had higher prevalence and intensity of Cardicola spp. infection. Based on C. forsteri ITS-2 DNA, infection intensity at harvest was significantly greater for both companies in 2021 when compared to 2018 and 2019. Continued monitoring of Cardicola spp. in SBT and improvements in diagnostics contribute to our understanding of Cardicola spp. epizootiology and the detection of changes in treatment efficacy.

Functional and molecular characterization of the Atlantic salmon gill epithelium cell line ASG-10; a tool for in vitro gill research.

Fish gills are not only the respiratory organ, but also essential for ion-regulation, acid-base control, detoxification, waste excretion and host defense. Multifactorial gill diseases are common in farmed Atlantic salmon, and still poorly understood. Understanding gill pathophysiology is of paramount importance, but the sacrifice of large numbers of experimental animals for this purpose should be avoided. Therefore, in vitro models, such as cell lines, are urgently required to replace fish trials. An Atlantic salmon gill epithelial cell line, ASG-10, was established at the Norwegian Veterinary institute in 2018. This cell line forms a monolayer expressing cytokeratin, e-cadherin and desmosomes, hallmarks of a functional epithelial barrier. To determine the value of ASG-10 for comparative studies of gill functions, the characterization of ASG-10 was taken one step further by performing functional assays and comparing the cell proteome and transcriptome with those of gills from juvenile freshwater Atlantic salmon. The ASG-10 cell line appear to be a homogenous cell line consisting of epithelial cells, which express tight junction proteins. We demonstrated that ASG-10 forms a barrier, both alone and in co-culture with the Atlantic salmon gill fibroblast cell line ASG-13. ASG-10 cells can phagocytose and express several ATP-binding cassette transport proteins. Additionally, ASG-10 expresses genes involved in biotransformation of xenobiotics and immune responses. Taken together, this study provides an overview of functions that can be studied using ASG-10, which will be an important contribution to in vitro gill epithelial research of Atlantic salmon.

Prediction of Feed Efficiency and Performance-Based Traits in Fish via Integration of Multiple Omics and Clinical Covariates.

Fish aquaculture is a rapidly expanding global industry, set to support growing demands for sources of marine protein. Enhancing feed efficiency (FE) in farmed fish is required to reduce production costs and improve sector sustainability. Recognising that organisms are complex systems whose emerging phenotypes are the product of multiple interacting molecular processes, systems-based approaches are expected to deliver new biological insights into FE and growth performance. Here, we establish 14 diverse layers of multi-omics and clinical covariates to assess their capacities to predict FE and associated performance traits in a fish model (Oncorhynchus tshawytscha) and uncover the influential variables. Inter-omic relatedness between the different layers revealed several significant concordances, particularly between datasets originating from similar material/tissue and between blood indicators and some of the proteomic (liver), metabolomic (liver), and microbiomic layers. Single- and multi-layer random forest (RF) regression models showed that integration of all data layers provide greater FE prediction power than any single-layer model alone. Although FE was among the most challenging of the traits we attempted to predict, the mean accuracy of 40 different FE models in terms of root-mean square errors normalized to percentage was 30.4%, supporting RF as a feature selection tool and approach for complex trait prediction. Major contributions to the integrated FE models were derived from layers of proteomic and metabolomic data, with substantial influence also provided by the lipid composition layer. A correlation matrix of the top 27 variables in the models highlighted FE trait-associations with faecal bacteria (Serratia spp.), palmitic and nervonic acid moieties in whole body lipids, levels of free glycerol in muscle, and N-acetylglutamic acid content in liver. In summary, we identified subsets of molecular characteristics for the assessment of commercially relevant performance-based metrics in farmed Chinook salmon.

Molecular evidence for stress, inflammation and structural changes in non-specific ulcers in skin of farmed Chinook salmon (Oncorhynchus tshawytscha).

Fish skin is critical to physical defence against pathogens and there is a need to understand the physiological processes impacting ulcers and their healing. Ulcers have been reported in farmed Chinook salmon in New Zealand. This study investigated stress, immune and structural gene expression in farmed Chinook salmon skin with and without ulcers from two sites in New Zealand sampled from February (higher temperature, late summer) to May (lower temperature, late autumn). Skin samples taken adjacent to non-specific ulcers in May and control fish in February demonstrated upregulation of heat shock protein 70 relative to control fish in May. Anterior gradient 2 expression was upregulated in fish with ulcers relative to control fish (both February and May), suggesting increased mucous cell activity. Based on the results of this study, fish with non-specific ulcers showed evidence of stress, inflammation, re-epithelisation, and delayed healing near the ulcer site, elucidating the importance of these processes in the pathogenesis of non-specific ulcers in farmed chinook salmon.

Relationship between gut microbiota and Chinook salmon (Oncorhynchus tshawytscha) health and growth performance in freshwater recirculating aquaculture systems.

Gut microbiota play important roles in fish health and growth performance and the microbiome in fish has been shown to be a biomarker for stress. In this study, we surveyed the change of Chinook salmon (Oncorhynchus tshawytscha) gut and water microbiota in freshwater recirculating aquaculture systems (RAS) for 7 months and evaluated how gut microbial communities were influenced by fish health and growth performance. The gut microbial diversity significantly increased in parallel with the growth of the fish. The dominant gut microbiota shifted from a predominance of Firmicutes to Proteobacteria, while Proteobacteria constantly dominated the water microbiota. Photobacterium sp. was persistently the major gut microbial community member during the whole experiment and was identified as the core gut microbiota for freshwater farmed Chinook salmon. No significant variation in gut microbial diversity and composition was observed among fish with different growth performance. At the end of the trial, 36 out of 78 fish had fluid in their swim bladders. These fish had gut microbiomes containing elevated proportions of Enterococcus, Stenotrophomonas, Aeromonas, and Raoultella. Our study supports the growing body of knowledge about the beneficial microbiota associated with modern salmon aquaculture systems and provides additional information on possible links between dysbiosis and gut microbiota for Chinook salmon.

Association between melanin deposits in gill tissue and microbiome across different hatchery reared Atlantic salmon.

AIMS: To investigate the relationship between microbial community profiles and gill pathology during a production cycle of Atlantic salmon in two commercial hatcheries. METHODS AND RESULTS: Relationships between gill histology, environmental conditions, and microbiome were determined using high-throughput data, including 16S rDNA amplicon sequencing data, histopathology data, and water quality parameters. Hatchery A used riverine water and operated a mixed system of recirculation aquaculture system (RAS) and flowthrough. Hatchery B was used bore water and operated a RAS. Melanin deposits, hyperplastic, and inflammatory lesions were observed histologically in the gills. A higher prevalence of melanin deposits was detected and correlated to a change in beta diversity of bacterial communities in early time points (fingerling and parr stages). High abundance of Sphaerotilus sp.,Pseudomonas sp.,Nitrospira sp.,Exiguobacterium sp.,Deinococcus sp.,and Comamonas sp. was correlated with a high prevalence of melanin in filaments. Bacterial diversity increased as the fish cohort transitioned from RAS to flowthrough in hatchery A. CONCLUSIONS: Under commercial conditions, the commensal community of gill bacteria was related to melanin prevalence.

Draft genome of the bluefin tuna blood fluke, Cardicola forsteri.

The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C. forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 > 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion (> 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover > 6%. Gene prediction identified 8,564 hypothetical polypeptides, > 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts.

Prevalence of epitheliocystis in freshwater Atlantic salmon reared in flow-through and recirculation aquaculture systems.

Epitheliocystis, an intracellular bacterial infection in the gills and skin epithelium, has been frequently reported in Atlantic salmon (Salmo salar) during freshwater production in a number of countries. This study describes the prevalence and intensity of a natural epitheliocystis infection present in the gills of two strains of Atlantic salmon reared in either a flow-through (FT) or a recirculation aquaculture system (RAS) in Ireland. Repeated sampling of gills prior to and throughout seawater transfer, histology and quantitative real-time PCR were used to determine infection prevalence and intensity. Despite no clinical gill disease, and minor histopathological changes, epitheliocystis lesions were identified in histology at all time points. Specific PCR confirmed the presence of Candidatus Clavichlamydia salmonicola in both strains and its number of copies was correlated with intensity of epitheliocystis lesions. A significant interaction between hatchery system and fish strain on the prevalence and intensity of gill epitheliocystis was found both using histological and molecular methods. Specifically, fish from FT had higher prevalence and intensity than RAS reared fish and within FT, the Irish cohort were more affected than Icelandic.

Urinary calculi in larvae of striped trumpeter, Latris lineata.

Urinary calculi are observed in some cultured marine fish larvae and may negatively impact larval health and survival. This study assessed urinary calculi in striped trumpeter Latris lineata larvae from hatching to 28 days post-hatching (dph). The prevalence of urinary calculi was variable over time and ranged from 15% to 50%, whereas the average size of calculi increased with larval age. Urinary calculi were semi-translucent, light cream to white colour with irregular morphology and a uniform internal structure. The calculi resulted in pressure atrophy causing a distended epithelium of the urinary bladder of fish with calculi. The calculi were predominantly formed of calcium hydrogen phosphate (CaHPO4 ). Routine assessment of the prevalence of urinary calculi in marine fish larvae is recommended, along with other parameters, to monitor larval quality and inform hatchery management actions.

Transcriptomic characterisation of a common skin lesion in farmed chinook salmon.

Little is known about host responses of farmed Chinook salmon with skin lesions, despite the lesions being associated with increased water temperatures and elevated mortality rates. To address this shortfall, a transcriptomic approach was used to characterise the molecular landscape of spot lesions, the most commonly reported lesion type in New Zealand Chinook salmon, versus healthy appearing skin in fish with and without spot lesions. Many biological (gene ontology) pathways were enriched in lesion adjacent tissue, relative to control skin tissue, including proteolysis, fin regeneration, calcium ion binding, mitochondrial transport, actin cytoskeleton organisation, epithelium development, and tissue development. In terms of specific transcripts of interest, pro-inflammatory cytokines (interleukin 1ß and tumour necrosis factor), annexin A1, mucin 2, and calreticulin were upregulated, while cathepsin H, mucin 5AC, and perforin 1 were downregulated in lesion tissue. In some instances, changes in gene expression were consistent between lesion and healthy appearing skin from the same fish relative to lesion free fish, suggesting that host responses weren't limited to the site of the lesion. Goblet cell density in skin histological sections was not different between skin sample types. Collectively, these results provide insights into the physiological changes associated with common spot lesions in farmed Chinook salmon.

Comparative transcriptome profiling of virulent and avirulent isolates of Neoparamoeba perurans.

Neoparamoeba perurans, the aetiological agent of amoebic gill disease, remains a persistent threat to Atlantic salmon mariculture operations worldwide. Innovation in methods of AGD control is required yet constrained by a limited understanding of the mechanisms of amoebic gill disease pathogenesis. In the current study, a comparative transcriptome analysis of two N. perurans isolates of contrasting virulence phenotypes is presented using gill-associated, virulent (wild type) isolates, and in vitro cultured, avirulent (clonal) isolates. Differential gene expression analysis identified a total of 21,198 differentially expressed genes between the wild type and clonal isolates, with 5674 of these genes upregulated in wild type N. perurans. Gene set enrichment analysis predicted gene sets enriched in the wild type isolates including, although not limited to, cortical actin cytoskeleton, pseudopodia, phagocytosis, macropinocytic cup, and fatty acid beta-oxidation. Combined, the results from these analyses suggest that upregulated gene expression associated with lipid metabolism, oxidative stress response, protease activity, and cytoskeleton reorganisation is linked to pathogenicity in wild type N. perurans. These findings provide a foundation for future AGD research and the development of novel therapeutic and prophylactic AGD control measures for commercial aquaculture.

Evaluation of Low Temperature and Salinity as a Treatment of Atlantic Salmon against Amoebic Gill Disease.

Amoebic gill disease (AGD) is a significant health issue for Atlantic salmon farmed in a marine environment. While the disease is currently managed using freshwater or hydrogen peroxide baths, there is a need to develop other treatments. The aims of this study were to examine the effect of salinity (0 ppt and 35 ppt) and temperature (3 °C and 15 °C) on attachment and survival of Neoparamoeba perurans in vitro over short exposure times (15 min and 2 h) and to assess the efficacy of reduced temperature (3 °C) as treatment for Atlantic salmon affected by AGD. In vitro freshwater 3 °C was at least as effective as freshwater 15 °C and the attachment was significantly lower after 2 h in freshwater 3 °C than freshwater 15 °C. In vivo there was no difference between the fish treated with freshwater 15 °C for 2 h or freshwater 3 °C. This study showed that despite exposure to low temperature reducing attachment of N. perurans to their substrate in vitro, 15 min cold-water bath treatment was not more effective at reducing AGD in Atlantic salmon than current commercial 2 h freshwater bath.

Size-dependent resistance to amoebic gill disease in naïve Atlantic salmon (Salmo salar).

Amoebic gill disease, caused by the protozoan ectoparasite Neoparamoeba perurans, remains a significant threat to commercial Atlantic salmon aquaculture operations worldwide, despite partial control afforded by selective breeding and therapeutic intervention. Anecdotal reports from commercial producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. Here, large (>350 g) and small (<200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L-1 without intervention. Progression and severity of AGD in challenged cohorts was evaluated through gill pathology, using gill score and histological examination, and quantification of gill-associated amoebae burden using qPCR. To determine the potential basis for differences in AGD susceptibility between cohorts, transcriptome analysis was conducted using RNA extracted from whole gill arches. Overall, the large Atlantic salmon cohort had significantly lower gill parasite burdens and reduced AGD-related gross pathology compared to the small cohort. Relative gill load of N. perurans appeared to be proportional to gill score in both size classes, with larger smolts typically observed to have comparatively reduced parasite burdens at a given gill score. Moreover, comparison between gene expression profiles of large and small smolts highlighted upregulation of genes consistent with elevated immune activity in large smolts. Combined, the results presented here provide strong evidence of size-dependent resistance to AGD in AGD-naïve Atlantic salmon.

Praziquantel use in aquaculture - Current status and emerging issues.

Parasitic diseases are major constraints in fish mariculture. The anthelmintic praziquantel (PZQ) can effectively treat a range of flatworm parasites in a variety of fish species and has potential for broader application than its current use in the global aquaculture industry. In this review we report on PZQ's current use in the aquaculture industry and discuss its efficacy against various flatworm parasites of fish. Routes of PZQ administration are evaluated, along with issues related to palatability, pharmacokinetics and toxicity in fish, while PZQ's effects on non-target species, environmental impacts, and the development of drug-resistance are discussed.

Cleaner seas: reducing marine pollution.

In the age of the Anthropocene, the ocean has typically been viewed as a sink for pollution. Pollution is varied, ranging from human-made plastics and pharmaceutical compounds, to human-altered abiotic factors, such as sediment and nutrient runoff. As global population, wealth and resource consumption continue to grow, so too does the amount of potential pollution produced. This presents us with a grand challenge which requires interdisciplinary knowledge to solve. There is sufficient data on the human health, social, economic, and environmental risks of marine pollution, resulting in increased awareness and motivation to address this global challenge, however a significant lag exists when implementing strategies to address this issue. This review draws upon the expertise of 17 experts from the fields of social sciences, marine science, visual arts, and Traditional and First Nations Knowledge Holders to present two futures; the Business-As-Usual, based on current trends and observations of growing marine pollution, and a More Sustainable Future, which imagines what our ocean could look like if we implemented current knowledge and technologies. We identify priority actions that governments, industry and consumers can implement at pollution sources, vectors and sinks, over the next decade to reduce marine pollution and steer us towards the More Sustainable Future. Supplementary Information: The online version contains supplementary material available at 10.1007/s11160-021-09674-8.

Profiling the glycome of Cardicola forsteri, a blood fluke parasitic to bluefin tuna.

Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host-parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.

Prevalence and Intensity of Cardicola spp. Infection in Ranched Southern Bluefin Tuna and a Comparison of Diagnostic Methods.

The parasitic blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna Thunnus maccoyii (SBT) ranched in Australia. In this study we compared the effect of treatment, company, and ranching year on blood fluke infections in ranched SBT. SBT were sampled during the 2018 and 2019 ranching seasons from praziquantel (PZQ) treated pontoons and untreated pontoons managed by two companies. Severity of infection was diagnosed by several criteria including adult fluke counts from hearts, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. PZQ treatment remains highly effective against C. forsteri infection. Prevalence and intensity of Cardicola spp. infection was lower in 2019 than 2018 for Company A in treated pontoons at week 12 and week 17 of ranching, and lower for Company A than Company B in untreated pontoons at month 5 of ranching. Results indicate re-infection may be less likely in the environment near Company A pontoons, and consistent years of treatment may have lowered the parasite load in the environment. qPCR demonstrated higher sensitivity when comparing diagnostic methods for C. forsteri in heart, and higher specificity when comparing diagnostic methods for Cardicola spp. in gills. Continuing to monitor blood fluke infections in ranched SBT can help to detect changes in drug efficacy over time and help industry to develop a best practice for treatment.