Anatomy of teleost fish immune structures and organs.

The function of a tissue is determined by its construction and cellular composition. The action of different genes can thus only be understood properly when seen in the context of the environment in which they are expressed and function. We now experience a renaissance in morphological research in fish, not only because, surprisingly enough, large structures have remained un-described until recently, but also because improved methods for studying morphological characteristics in combination with expression analysis are at hand. In this review, we address anatomical features of teleost immune tissues. There are approximately 30,000 known teleost fish species and only a minor portion of them have been studied. We aim our review at the Atlantic salmon (Salmo salar) and other salmonids, but when applicable, we also present information from other species. Our focus is the anatomy of the kidney, thymus, spleen, the interbranchial lymphoid tissue (ILT), the newly discovered salmonid cloacal bursa and the naso-pharynx associated lymphoid tissue (NALT).

Effects of Size and Geographical Origin on Atlantic salmon, Salmo salar, Mucin O-Glycan Repertoire.

Diseases cause ethical concerns and economic losses in the Salmonid industry. The mucus layer comprised of highly O-glycosylated mucins is the first contact between pathogens and fish. Mucin glycans govern pathogen adhesion, growth and virulence. The Atlantic salmon O-glycome from a single location has been characterized and the interindividual variation was low. Because interindividual variation is considered a population-based defense, hindering the entire population from being wiped out by a single infection, low interindividual variation among Atlantic salmon may be a concern. Here, we analyzed the O-glycome of 25 Atlantic salmon from six cohorts grown under various conditions from Sweden, Norway and Australia (Tasmania) using mass spectrometry. This expanded the known Atlantic salmon O-glycome by 60% to 169 identified structures. The mucin O-glycosylation was relatively stable over time within a geographical region, but the size of the fish affected skin mucin glycosylation. The skin mucin glycan repertoires from Swedish and Norwegian Atlantic salmon populations were closely related compared with Tasmanian ones, regardless of size and salinity, with differences in glycan size and composition. The internal mucin glycan repertoire also clustered based on geographical origin and into pyloric cecal and distal intestinal groups, regardless of cohort and fish size. Fucosylated structures were more abundant in Tasmanian pyloric caeca and distal intestine mucins compared with Swedish ones. Overall, Tasmanian Atlantic salmon mucins have more O-glycan structures in skin but less in the gastrointestinal tract compared with Swedish fish. Low interindividual variation was confirmed within each cohort. The results can serve as a library for identifying structures of importance for host-pathogen interactions, understanding population differences of salmon mucin glycosylation in resistance to diseases and during breeding and selection of strains. The results could make it possible to predict potential vulnerabilities to diseases and suggest that inter-region breeding may increase the glycan diversity.

Frequency of vateritic otoliths and potential consequences for marine survival in hatchery-reared Atlantic salmon.

Otoliths are inner-ear structures of all teleost fish with functional importance for hearing and balance. The otoliths usually consist of aragonite, a polymorph of calcium carbonate, but may also take the form partly or entirely of vaterite, a different polymorph of calcium carbonate. Vateritic otoliths occur sporadically in wild fish, but with a higher frequency in hatchery-reared fish. Abnormal otoliths have direct consequences for the inner-ear functions of fish and may be a symptom of environmental stress. In this study, the authors assess the differences in the frequency of abnormal otoliths and degree of abnormality (% vaterite) for different groups of hatchery-reared Atlantic salmon (Salmo salar) smolt and adults. The groups differed in parental broodstock origin (number of generations in hatchery) and treatment temperature. Smolt from the same groups were also released to complete their ocean migration. The otoliths of the returning and recaptured adults were subsequently extracted to assess the difference in frequency and degree of abnormality between the adults and the smolt from corresponding groups. Return rate varied among groups (0.2%-2.6%). The frequency of vateritic otoliths was high (11.4%-64.4%) and differed among smolt groups. The lowest return rates corresponded with the highest frequency of abnormal otoliths for the groups, suggesting that abnormal otoliths may have negative consequences for marine survival. Furthermore, indications of an effect of fast growth on the formation of abnormal otoliths were found for only one of the experimental groups, and for none of the groups after correcting for Type 1 error. This contradicts previous reports, suggesting rapid growth as the main cause of abnormal otoliths. Adult return rates were generally low, but abnormal otoliths were common, with high coverage (% vaterite).

Genetic growth potential, rather than phenotypic size, predicts migration phenotype in Atlantic salmon.

Knowledge of the relative importance of genetic versus environmental determinants of major developmental transitions is pertinent to understanding phenotypic evolution. In salmonid fishes, a major developmental transition enables a risky seaward migration that provides access to feed resources. In Atlantic salmon, initiation of the migrant phenotype, and thus age of migrants, is presumably controlled via thresholds of a quantitative liability, approximated by body size expressed long before the migration. However, how well size approximates liability, both genetically and environmentally, remains uncertain. We studied 32 Atlantic salmon families in two temperatures and feeding regimes (fully fed, temporarily restricted) to completion of migration status at age 1 year. We detected a lower migrant probability in the cold (0.42) than the warm environment (0.76), but no effects of male maturation status or feed restriction. By contrast, body length in late summer predicted migrant probability and its control reduced migrant probability heritability by 50-70%. Furthermore, migrant probability and length showed high heritabilities and between-environment genetic correlations, and were phenotypically highly correlated with stronger genetic than environmental contributions. Altogether, quantitative estimates for the genetic and environmental effects predicting the migrant phenotype indicate, for a given temperature, a larger importance of genetic than environmental size effects.

A teleost structural analogue to the avian bursa of Fabricius.

The bursa of Fabricius is a primary and secondary lymphoid organ considered exclusively present in birds, and studies of this structure have been vital to our current understanding of the adaptive immune system of vertebrates. In this study, we reveal substantial lymphoepithelial tissue in a previously undescribed bursa in Atlantic salmon (Salmo salar), situated caudal to the urogenital papilla of the cloaca and thus analogous to the anatomical placement of the bursa of Fabricius. We investigated three groups of Atlantic salmon at different maturational stages and characterized the structure by applying dissection, radiology, scanning electron microscopy and histological techniques, including immunohistochemistry and in situ hybridization. We found that the epithelial anlage of the salmon cloacal bursa developed into substantial lymphoepithelial tissue and subsequently regressed following sexual maturation. Such a dynamic development is also a key characteristic of the avian bursa. The presence of intraepithelial lymphocytes was concomitant with expression of the leukocyte-attracting chemokine CCL19, indicative of lymphoid organ functions. We did not observe recombination or gene conversion in salmon bursal lymphocytes at any developmental stage, indicating the absence of primary lymphoid organ functions in contrast to the bursa of Fabricius. However, the possibility of the bursa to trap both enteric and environmental antigens, combined with the presence of several antigen-presenting cells residing within the lymphoepithelium, suggest the structure has secondary lymphoid organ functions. We present the discovery of a lymphoid organ in Atlantic salmon with striking topographical similarities to that of the bursa of Fabricius in birds. In addition, the age-dependent dynamics of its lymphoepithelium suggest functions related to the maturation processes of lymphocytes.

Length measurement accuracy of adaptive resolution imaging sonar and a predictive model to assess adult Atlantic salmon (Salmo salar) into two size categories with long-range data in a river.

Imaging sonars are used around the world for fish population monitoring. The accuracy of the length measurements has been reported in multiple studies for relatively short (<15 m) ranges and high image resolution. However, imaging sonars are often used at longer ranges (i.e., >15 m) where the images produced from sonar returns become less detailed. The accuracy of the length measurements from the Adaptive Resolution Imaging Sonar (ARIS) was tested by releasing n = 69 known-sized adult Atlantic salmon (Salmo salar) directly into the sonar field at ranges between 15 and 29 m, and measuring their echoes manually by four users and semi-automatically using a computer workflow in Echoview software. Overall, the length measurements were very variable: compared to true (fork) lengths, the mean of differences varied between -9.9 cm and 7.8 cm in the human-generated datasets, and between -42.8 cm and -20 cm in the computer-generated dataset. In addition, the length measurements in different datasets were only in poor or moderate agreement with each other (intraclass correlation <0.61). Contrary to our expectations, the distance from the transducer or the subjectively assessed echo quality did not have an effect on the measurement accuracy in most of the datasets and when it did, the effect was not systematic between the datasets. Therefore, a size class and length prediction model was implemented in a Bayesian framework to group salmon into two size categories: One-Sea-Winter (<63 cm) and Multi-Sea-Winter (≥63 cm) groups. The model correctly predicted the size category in 83% of the fish in the computer-generated dataset and ranged from 68% to 74% in the human-generated datasets. We conclude that fish length measurements derived from long-range imaging sonar data should be used with caution, but post-processing can improve the usefulness of the data for specific purposes, such as adult Atlantic salmon population monitoring.

Hierarchical analysis of wild Atlantic salmon (Salmo salar) fecundity in relation to body size and developmental traits.

Using data from wild Atlantic salmon Salmo salar returning to spawn in seven Scottish rivers, we developed a model of fecundity based on individual body size and key developmental traits. We used a novel approach to model selection which maximises predictive accuracy for application to target river stocks to select the best from a suite of Bayesian hierarchical models. This approach aims to ensure the optimal model within the candidate set includes covariates that best predict out-of-sample data to estimate fecundity in areas where no direct observations are available. In addition to body size, the final model included the developmental characteristics of age at smolting and years spent at sea. Using two independent long-term monitoring datasets, the consequences of ignoring these characteristics was revealed by comparing predictions from the best model with models that omitted them.

Population and size-specific distribution of Atlantic salmon Salmo salar in the Baltic Sea over five decades.

Population-specific assessment and management of anadromous fish at sea requires detailed information about the distribution at sea over ontogeny for each population. However, despite a long history of mixed-stock sea fisheries on Atlantic salmon, Salmo salar, migration studies showing that some salmon populations feed in different regions of the Baltic Sea and variation in dynamics occurs among populations feeding in the Baltic Sea, such information is often lacking. Also, current assessment of Baltic salmon assumes equal distribution at sea and therefore equal responses to changes in off-shore sea fisheries. Here, we test for differences in distribution at sea among and within ten Atlantic salmon Salmo salar populations originating from ten river-specific hatcheries along the Swedish Baltic Sea coast, using individual data from >125,000 tagged salmon, recaptured over five decades. We show strong population and size-specific differences in distribution at sea, varying between year classes and between individuals within year classes. This suggests that Atlantic salmon in the Baltic Sea experience great variation in environmental conditions and exploitation rates over ontogeny depending on origin and that current assessment assumptions about equal exploitation rates in the offshore fisheries and a shared environment at sea are not valid. Thus, our results provide additional arguments and necessary information for implementing population-specific management of salmon, also when targeting life stages at sea.

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.).

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non-aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non-aqueous-based fixatives; qualitative and semi-quantitative analysis revealed a greater preservation of the gill mucus using the non-aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid-Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn-fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus-embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.

Precision and accuracy of Dahl-Lea back-calculated smolt lengths from adult scales of Atlantic salmon Salmo salar.

Using tagged and recaptured Atlantic salmon Salmo salar (n = 106) the present analysis shows that the most commonly applied linear back-calculation method for estimating past length, the Dahl-Lea method, resulted in overestimation of the length of large smolts and underestimation of small smolts. A correction equation (y = 0.53x + 6.23) for estimating true smolt length (y) from lengths back-calculated from adult scale measures (x) to account for these systematic discrepancies is proposed.

Experimental investigation of the effects of temperature and feeding regime on scale growth in Atlantic salmon Salmo salar post-smolts.

Salmo salar post-smolts were reared in seawater under controlled laboratory conditions for 12 weeks. The fish were exposed to three constant temperature treatments (15, 10.5 and 6°C) and four feeding treatments (constant feeding, food withheld for 7 days, food withheld for 14 days and food withheld intermittently for four periods of 7 days). Scale growth was proportional to fish growth across all treatments, justifying the use of scale measurements as a proxy for growth during the early marine phase. The rate of circuli deposition was dependant on temperature and feeding regime and was generally proportional to fish growth but with some decoupling of the relationship at 15°C. Deposition rates varied from 4.8 days per circulus at 15°C (constant feeding) to 15.1 days per circulus at 6°C (interrupted feeding). Cumulative degree day (° D) was a better predictor of circuli number than age, although the rate of circuli deposition ° D-1 was significantly lower at 6°C compared with 15 and 10.5°C. Inter-circuli distances were highly variable and did not reflect growth rate; tightly packed circuli occurred during periods without food when growth was depressed, but also during periods of rapid growth at 15°C. The results further current understanding of scale growth properties and can inform investigations of declining marine growth in S. salar based on interpretations of scale growth patterns.

Implications for introgression: has selection for fast growth altered the size threshold for precocious male maturation in domesticated Atlantic salmon?

BACKGROUND: Mature male parr (MMP) represent an important alternative life-history strategy in Atlantic salmon populations. Previous studies indicate that the maturation size threshold for male parr varies among wild populations and is influenced by individual growth, environmental conditions, and genetics. More than ten generations of breeding have resulted in domesticated salmon displaying many genetic differences to wild salmon, including greatly increased growth rates. This may have resulted in domesticated fish with the potential to outgrow the size threshold for early maturation, or evolution of the size threshold of the trait itself. To investigate this, we performed a common-garden experiment under farming conditions using 4680 salmon from 39 families representing four wild, two wild-domesticated hybrid, and two domesticated strains. RESULTS: Domesticated salmon outgrew wild salmon 2-5-fold, and hybrids displayed intermediate growth. Overall, the numbers of MMP varied greatly among families and strains: averaging 4-12% in domesticated, 18-25% in hybrid, and 43-74% in the wild populations. However, when the influence of growth was accounted for, by dividing fish into lower and upper size modes, no difference in the incidence of MMP was detected among domesticated and wild strains in either size mode. In the lower size mode, hybrids displayed significantly lower incidences of mature males than their wild parental strains. No consistent differences in the body size of MMP, connected to domestication, was detected. CONCLUSIONS: Our data demonstrate: 1- no evidence for the evolution of the size threshold for MMP in domesticated salmon, 2- the vastly lower incidence of MMP in domesticated strains under aquaculture conditions is primarily due to their genetically increased growth rate causing them to outgrow the size threshold for early maturation, 3- the incidence of MMP is likely to overlap among domesticated and wild salmon in the natural habitat where they typically display overlapping growth, although hybrid offspring may display lower incidences of mature male parr. These results have implications for wild salmon populations that are exposed to introgression from domesticated escapees.

Stable Core Gut Microbiota across the Freshwater-to-Saltwater Transition for Farmed Atlantic Salmon.

Gut microbiota associations through habitat transitions are fundamentally important yet poorly understood. One such habitat transition is the migration from freshwater to saltwater for anadromous fish, such as salmon. The aim of the current work was therefore to determine the freshwater-to-saltwater transition impact on the gut microbiota in farmed Atlantic salmon, with dietary interventions resembling freshwater and saltwater diets with respect to fatty acid composition. Using deep 16S rRNA gene sequencing and quantitative PCR, we found that the freshwater-to-saltwater transition had a major association with the microbiota composition and quantity, while diet did not show significant associations with the microbiota. In saltwater there was a 100-fold increase in bacterial quantity, with a relative increase of Firmicutes and a relative decrease of both Actinobacteria and Proteobacteria Irrespective of an overall shift in microbiota composition from freshwater to saltwater, we identified three core clostridia and one Lactobacillus-affiliated phylotype with wide geographic distribution that were highly prevalent and co-occurring. Taken together, our results support the importance of the dominating bacteria in the salmon gut, with the freshwater microbiota being immature. Due to the low number of potentially host-associated bacterial species in the salmon gut, we believe that farmed salmon can represent an important model for future understanding of host-bacterium interactions in aquatic environments.IMPORTANCE Little is known about factors affecting the interindividual distribution of gut bacteria in aquatic environments. We have shown that there is a core of four highly prevalent and co-occurring bacteria irrespective of feed and freshwater-to-saltwater transition. The potential host interactions of the core bacteria, however, need to be elucidated further.

Mapping of quantitative trait loci associated with size, shape, and parr mark traits using first- and second-generation backcrosses between European and North American Atlantic salmon (Salmo salar).

Little is known about the genetic architecture of traits important for salmonid restoration ecology. We mapped quantitative trait loci (QTL) using single nucleotide polymorphisms (SNPs) for juvenile body length, weight, shape, and vertical skin pigmentation patterns (parr marks) within three hybrid backcross families between European and North American subspecies of Atlantic salmon. Amounts of variation in skin colour and pattern quantified in the two second-generation transAtlantic families exceeded the ranges seen in purebred populations. GridQTL analyses using low-density female-specific linkage maps detected QTL showing experiment-wide significance on Ssa02, Ssa03, Ssa09, Ssa11, Ssa19, and Ssa26/28 for both length and weight; on Ssa04 and Ssa23 for parr mark number; on Ssa09 and Ssa13 for parr mark contrast; and on Ssa05, Ssa07, Ssa10, Ssa11, Ssa18, Ssa23, and Ssa26/28 for geometric morphometric shape coordinates. Pleiotrophic QTL on Ssa11 affected length, weight, and shape. No QTL was found that explained more than 10% of the phenotypic variance in pigmentation or shape traits. Each QTL was approximately positioned on the physical map of the Atlantic salmon genome. Some QTL locations confirmed previous studies but many were new. Studies like ours may increase the success of salmon restoration projects by enabling better phenotypic and genetic matching between introduced and extirpated strains.

Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon.

Heart- and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large-scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV in vivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6 weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.

Effects of individual variation in length, condition and run-time on return rates of wild-reared Atlantic salmon Salmo salar smolts.

Groups of wild-reared Atlantic salmon Salmo salar smolts were captured during their seaward migration on a tributary of the River Conon, Scotland, U.K., from 1999 to 2014 and tagged with passive integrated transponders (PIT). Fish that subsequently returned to the river after growing at sea were recorded automatically by a PIT-detector in a fish pass. Return rate was related directly to length and condition and inversely to day of the year that the smolt was tagged. Over years, as the study progressed, there was a significant increase in the proportion of smolts returning after two or more years at sea and no trend in returns of salmon having spent one winter at sea. There was no trend in the date of return of salmon across the study period. Fish that had spent more winters at sea returned earlier in the year.

Is bigger really better? Towards improved models for testing how Atlantic salmon Salmo salar smolt size affects marine survival.

A general framework is presented that should enhance our understanding of how intrinsic factors, such as body size, and extrinsic factors, such as climate, affect the dynamics and demographics of fish populations. Effects of intrinsic factors, notably studies relating juvenile Atlantic salmon Salmo salar body size to their probability to return as an adult, are often context-dependent and anecdotal, due to data constraints. By merit of its flexible specification, this framework should admit datasets with a range of situation-specific nuances, collected using different approaches, and thereby deliver more general and robust findings for more effective population management.

Comparison between Atlantic salmon Salmo salar post-smolts reared in open sea cages and in the Preline raceway semi-closed containment aquaculture system.

The use of closed containment (CCS) or semi-closed containment systems (S-CCS) for Atlantic salmon Salmo salar aquaculture is under evaluation in Norway. One such system is the Preline S-CCS, a floating raceway system that pumps water from 35 m depth creating a constant current through the system. Exposing fish to moderate water currents is considered aerobic exercise and it is often perceived as positive for fish welfare, growth, food utilization, muscle development and cardiac health. The present study compared fish reared in the Preline S-CCS and in a reference open pen. Samples were taken in fresh water before being transferred to the seawater systems and after 1, 2 and 4 months in seawater and analysed for growth, mortality, muscle development and plasma insulin-like growth factor I (IGF-I) levels. Moreover, gene transcription were determined in the skeletal muscle [igf-I, insulin-like growth factor 1 receptor a (igf1ra) and insulin-like growth factor 1 binding protein 1a (igf1bp1a)] and cardiac transcription factors [myocyte-specific enhancer factor 2C (mef2c), gata4 and vascular endothelial growth factor (vegf)]. While the results suggest that post-smolts in Preline S-CCS were smaller than reference fish, fish from Preline S-CCS have less accumulated mortality at the end of the experiment and showed 2.44 times more small muscle fibres than the reference group fish after 4 months in seawater. These results confirmed what was previously observed in the second generation of Preline. Similar levels of big muscle fibres between Preline S-CCS and reference suggest a similar hypertrophy of muscle fibres even with lower IGF-I expression in the Preline S-CCS. Cardiac gene transcription suggests cardiac hypertrophy was observed after 4 months in seawater in the Preline S-CCS group. Altogether, Preline S-CCS is a promising technology able to produce more robust S. salar with a faster growth and lower mortality in the subsequent standard open cage system growth period.

Consistent melanophore spot patterns allow long-term individual recognition of Atlantic salmon Salmo salar.

The present study shows that permanent melanophore spot patterns in Atlantic salmon Salmo salar make it possible to use images of the operculum to keep track of individual fish over extended periods of their life history. Post-smolt S. salar (n = 246) were initially photographed at an average mass of 98 g and again 10 months later after rearing in a sea cage, at an average mass of 3088 g. Spots that were present initially remained and were the most overt (largest) 10 months later, while new and less overt spots had developed. Visual recognition of spot size and position showed that fish with at least four initial spots were relatively easy to identify, while identifying fish with less than four spots could be challenging. An automatic image analysis method was developed and shows potential for fast match processing of large numbers of fish. The current findings promote visual recognition of opercular spots as a welfare-friendly alternative to tagging in experiments involving salmonid fishes.

Evidence for long-term change in length, mass and migration phenology of anadromous spawners in French Atlantic salmon Salmo salar.

This study provides new data on Atlantic salmon Salmo salar life-history traits across France. Using a long-term recreational angling database (1987-2013) covering 34 rivers in three regions (genetic units), a decline in individual length, mass and a delayed adult return to French rivers was reported. Temporal similarities in trait variations between regions may be attributed to common change in environmental conditions at sea. The relative rate of change in phenotypic traits was more pronounced in early maturing fish [1 sea-winter (1SW) fish] than in late maturing fish (2SW fish). Such contrasted response within populations highlights the need to account for the diversity in life histories when exploring mechanisms of phenotypic change in S. salar. Such detailed life-history data on returning S. salar have not previously been reported from France. This study on French populations also contributes to reducing the gap in knowledge by providing further empirical evidence of a global pattern in S. salar across its distribution range. Results are consistent with the hypothesis that the observed changes in life-history traits are primarily associated with environmental changes in the North Atlantic Ocean. They also emphasize the presence of less important, but still significant contrasts between region and life history.