A multi-tissue de novo transcriptome assembly and relative gene expression of the vulnerable freshwater salmonid Thymallus ligericus.

Freshwater ecosystems are among the most endangered ecosystems worldwide. While numerous taxa are on the verge of extinction as a result of global changes and direct or indirect anthropogenic activity, genomic and transcriptomic resources represent a key tool for comprehending species' adaptability and serve as the foundation for conservation initiatives. The Loire grayling, Thymallus ligericus, is a freshwater European salmonid endemic to the upper Loire River basin. The species is comprised of fragmented populations that are dispersed over a small area and it has been identified as a vulnerable species. Here, we provide a multi-tissue de novo transcriptome assembly of T. ligericus. The completeness and integrity of the transcriptome were assessed before and after redundancy removal with lineage-specific libraries from Eukaryota, Metazoa, Vertebrata, and Actinopterygii. Relative gene expression was assessed for each of the analyzed tissues, using the de novo assembled transcriptome and a genome-based analysis using the available T. thymallus genome as a reference. The final assembly, with a contig N50 of 1221 and Benchmarking Universal Single-Copy Orthologs (BUSCO) scores above 94%, is made accessible along with structural and functional annotations and relative gene expression of the five tissues (NCBI SRA and FigShare databases). This is the first transcriptomic resource for this species, which provides a foundation for future research on this and other salmonid species that are increasingly exposed to environmental stressors.

Establishment of an in vitro model from the vulnerable fish species Coregonus maraena (maraena whitefish): Optimization of growth conditions and characterization of the cell line.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C. maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C. maranea.

The ecology and fishery of the vendace (Coregonus albula) in the Baltic Sea.

Brackish water ecosystems often have high primary production, intermediate salinities, and fluctuating physical conditions and therefore provide challenging environments for many of their inhabitants. This is especially true of the Baltic Sea, which is a large body of brackish water under strong anthropogenic influence. One freshwater species that is able to cope under these conditions in the northern Baltic Sea is the vendace (Coregonus albula), a small salmonid fish. Here, we review the current knowledge of its ecology and fishery in this brackish water environment. The literature shows that, by competing for resources with other planktivores and being an important prey for a range of larger species, C. albula plays a notable role in the northern Baltic Sea ecosystem. It also sustains significant fisheries in the coastal waters of Sweden and Finland. We identify the need to better understand these C. albula populations in terms of the predator-prey interactions, distributions of anadromous and sea spawning populations and other putative (eco)morphs, extent of gene exchange between the populations, and effects of climate change on their future. In this regard, we recommend strengthening C. albula-related research and management efforts by improved collaboration and coordination between research institutions, other governmental agencies, and fishers, as well as by harmonization of fishery policies across national borders.

Environment and genotype predict the genomic nature of domestication of salmonids as revealed by gene expression.

Billions of salmonids are produced annually by artificial reproduction for harvest and conservation. Morphologically, behaviourally and physiologically these fish differ from wild-born fish, including in ways consistent with domestication. Unlike most studied domesticates, which diverged from wild ancestors millennia ago, salmonids offer a tractable model for early-stage domestication. Here, we review a fundamental mechanism for domestication-driven differences in early-stage domestication, differentially expressed genes (DEGs), in salmonids. We found 34 publications examining DEGs under domestication driven by environment and genotype, covering six species, over a range of life-history stages and tissues. Three trends emerged. First, domesticated genotypes have increased expression of growth hormone and related metabolic genes, with differences magnified under artificial environments with increased food. Regulatory consequences of these DEGs potentially drive overall DEG patterns. Second, immune genes are often DEGs under domestication and not simply owing to release from growth-immune trade-offs under increased food. Third, domesticated genotypes exhibit reduced gene expression plasticity, with plasticity further reduced in low-complexity environments typical of production systems. Recommendations for experimental design improvements, coupled with tissue-specific expression and emerging analytical approaches for DEGs present tractable avenues to understand the evolution of domestication in salmonids and other species.

Diel migration pattern of pink salmon fry in small streams.

Downstream migration is a critical stage in the anadromous salmonid life cycle, but previous studies have shown different results between rivers or surveys for the diel downstream migration pattern of the fry of the pink salmon Oncorhynchus gorbuscha. We investigated the diel migration pattern of pink salmon fry in three small streams. Our results showed that pink salmon fry migrate mainly within a few hours after sunset; 89.9% of migration occurred between 18:00 and 23:00. Therefore, the results indicated that sunset time influences the diel migration pattern of pink salmon fry in small streams. This pattern could be a predator-avoidance behaviour.

Temperature, meal size and body size effects on the gastric evacuation of rainbow trout: modelling optimum and upper thermal limits.

This study examined the rate and course of gastric evacuation (GE) in rainbow trout Oncorhynchus mykiss in relation to meal size, body size and temperature. The GE experiments were performed on small (mean ± 95% C.I., 17.7 ± 0.5 cm total length), medium (22. 9 ± 0.2 cm) and large (28.3 ± 0.3 cm) rainbow trout fed meals of different sizes using commercial food pellets at water temperatures T ranging from 7.8°C to 19.2°C. Estimating the shape parameter of a general power function suggested that the square root function adequately described the GE in rainbow trout independently of meal size. The effects of total body length L and weight W on gastric evacuation rate (GER) were described by simple power functions. The square root function was further expanded by a temperature function with temperature optimum to describe the effect of temperature. The expanded square root function revealed a considerable effect of body size and temperature on the GER of rainbow trout. The GER increased exponentially with rising temperature, reached an optimum at c. 18.5°C and then declined abruptly to zero at c. 21°C. The GER of rainbow trout could thus be described by d S t d t = - 0.00152 L 0.75 e 0.08 T 1 - e 1.18 T - 20.9 S t (g h-1 ) or d S t d t = - 0.00440 W 0.26 e 0.08 T 1 - e 0.97 T - 21.1 S t (g h-1 ), where St is the current stomach content mass (g), and at post-prandial time t (h). These functions should prove useful to calculate total GE time as well as stomach fullness at different post-prandial times and therefore provide valuable information to develop optimal feeding strategies for farming of rainbow trout.

Social isolation affects intra-specific interaction behaviour and reduces the size of the cerebellar brain region in juvenile Atlantic salmon Salmo salar.

The social environment can affect the development of behavioural phenotypes in fish, and it is important to understand such effects when rearing fish in artificial environments. Here, the authors test the effects of spatial isolation on social interaction propensity and brain development in hatchery-reared Atlantic salmon Salmo salar L. Salmon reared in isolation generally stayed further away from a conspecific in a standardized intruder test than conspecifics reared together in groups. Isolated salmon also tended to be more active in an intruder test, albeit non-significantly so, but this pattern was not detected in open-field tests without an intruding conspecific. The cerebellar brain region was relatively smaller in isolated salmon, suggesting that the brain was developing differently in these fish. Therefore, some features of the behavioural and neural phenotype are affected by rearing in isolation. These effects should be considered when rearing salmon, particularly for experimental purposes as it may affect results of laboratory studies on behavioural expression and brain size.

Asymmetric competition over space use and territory between native brown trout (Salmo trutta) and invasive brook trout (Salvelinus fontinalis).

Interference competition over food and territory can shape population structure and habitat use within and between species. The introduction of invasive species often leads to novel competitive interactions over shared resources and invaders can eventually exclude the native species from preferred habitats. Invasive brook trout (Salvelinus fontinalis) introduced to northern Europe have excluded native brown trout (Salmo trutta) from numerous headwater streams. The fact that invasive brook trout can displace the more aggressive brown trout is puzzling. However, the earlier spawning and hatching of brook trout, compared to brown trout, may lead to unequal competition due to size advantage and prior resident status of brook trout at the fry stage. In this study, we examine the effect of competition between brown trout and brook trout using the natural size distribution of the two species. In two consecutive experiments, we first measured space use and feeding of a fry (age 0+) in the presence of a juvenile (age 1+). In experiment 2, we assessed territorial interactions between the species at the fry stage (age 0+) and if smaller brown trout could compensate the disadvantage by manipulating residence duration. Fry of brook trout feed sooner and spend more time close to the larger individual than brown trout fry. We also found that brook trout fry won most territorial contests against brown trout, and that increased residence duration led to longer and more aggressive interactions. The results suggest that smaller brown trout are displaced to suboptimal habitats in the presence of a larger brook trout. Therefore, the later emergence from gravel beds resulting in the naturally occurring size disadvantage of brown trout at the fry stage may lead to unequal territorial interactions that could explain why brown trout are displaced from preferred habitats in sympatry with brook trout.

Massive infection of Cystidicoloides ephemeridarum in brown trout Salmo trutta with skeletal deformities.

We investigated the cause of skeletal deformities found in brown trout from the Aspromonte mountain area in Reggio Calabria, Italy. Toxicological, histopathological and parasitological analyses were carried out on 14 fish with evident macro-morphological alterations from 2 different locations in the same river, and 4 control fish without morphological alterations from a different river (far from the first river but still within the area under study). Histopathological and radiological observations confirmed severe skeletal deformities in the specimens investigated. Parasitological examinations highlighted the presence of the nematode Cystidicoloides ephemeridarum, found only within the gastrointestinal tract of specimens showing deformities. Moreover, a direct correlation between parasite number and fish size was found. Given the low heavy metal levels and the presence of a massive parasitosis in teleosts showing deformities, we postulate a correlation between skeletal deformities and nematode infestation: the parasites caused a serious vitamin and mineral deficiency in the fish, which led to a dysplastic vertebral column. The low calcium levels found in malformed specimens compared with negative controls effectively confirm this hypothesis.

A comparison of avoidance to acoustic stimuli in fish with different auditory capabilities: juvenile chum salmon (Oncorhynchus keta) and common carp (Cyprinus carpio).

This study compared the repulsive effects of sound playbacks of intermittent 30, 150, 300, 600 and 900 Hz tones on two fish with different auditory capabilities: juvenile chum salmon (Oncorhynchus keta) and common carp (Cyprinus carpio). When 150 and 300 Hz tones were emitted from an underwater speaker, O. keta exhibited a moderate repulse reaction. Conversely, C. carpio exhibited a moderate repulse reaction to a tone with a frequency of 30 Hz, which indicates that a low-frequency component in complex broadband sound may be important for inducing a repulse reaction in cyprinids.

Response of European grayling, Thymallus thymallus, to multiple stressors in hydropeaking rivers.

Rivers of the large Alpine valleys constitute iconic ecosystems that are highly threatened by multiple anthropogenic stressors. This stressor mix, however, makes it difficult to develop and refine conservation and restoration strategies. It is, therefore, urgent to acquire more detailed knowledge on the consequences and interactions of prevalent stressors on fish populations, in particular, on indicator species such as the European grayling Thymallus thymallus. Here, we conducted a multi-river, multi-stressor investigation to analyze the population status of grayling. Using explorative decision-tree approaches, we disentangled the main and interaction effects of four prevalent stressor groups: flow modification (i.e., hydropeaking), channelization, fragmentation, and water quality alteration. Moreover, using a modified variant of the bootstrapping method, pooled bootstrapping, we determined the optimal number of characteristics that adequately describe fish population status. In our dataset, hydropeaking had the strongest single effect on grayling populations. Grayling biomass at hydrological control sites was around eight times higher than at sites affected by hydropeaking. The primary parameters for predicting population status were downramping rate and peak amplitude, with critical ranges of 0.2-0.4 cm min-1 and 10-25 cm. In hydropeaking rivers, river morphology and connectivity were the preceding subordinated parameters. Repeating the procedure with pooled bootstrapping datasets strengthened the hypothesis that the identified parameters are most relevant in predicting grayling population status. Hence, hydropeaking mitigation based on ecological thresholds is key to protect and restore already threatened grayling populations. In hydropeaking rivers, high river network connectivity and heterogenous habitat features can dampen the adverse effects of pulsed-flow releases by offering shelter and habitats for all life cycle stages of fish. The presented approach of explorative tree analysis followed by post-hoc tests of identified effects, as well as the pooled bootstrapping method, offers a simple framework for researchers and managers to analyze multi-factorial datasets and draw solid management conclusions.

Functional effect of vaterite - the presence of an alternative crystalline structure in otoliths alters escape kinematics of the brown trout.

The fast-start escape response is the main locomotor behaviour observed in fish to evade predatory attacks and thereby increase their probability of survival. Thus far, this high-speed sensory motor control has been extensively studied in relation to extrinsic factors. In contrast, there has been surprisingly little consideration of intrinsic individual factors that can mediate sensorial perception, such as inter-individual variability in mechanosensory systems. The inner ear of teleost fishes is composed of otoliths that play an important role in hearing and balance functions. While sagittal otoliths are normally composed of aragonite in many fish species, the inclusion of vaterite (an abnormal crystalline structure) has been reported in a number of individuals from different environments. There is currently strong theoretical and empirical evidence that vaterite deposition has a negative impact on auditory sensitivity in fishes. While the functional/behavioural implications of this defect on otolith-related hearing function has been hypothesised, it has remained largely untested experimentally. Here, using juvenile (0+ years) Salmo trutta originating from the wild in experimental conditions, we report for the first time that the deposition of calcium carbonate in its crystalline vateritic polymorph has significant pervasive effects on the escape kinematics of fish. The presence of an alternative crystalline structure in otoliths is likely to alter fish behaviour in ways that decrease survival. We also report that altered behaviour in individuals with vateritic otoliths is partially compensated for by the presence of a functional lateral line. Such functional compensation suggests more slight consequences, if any, in the wild.

The bite force-gape relationship as an avenue of biomechanical adaptation to trophic niche in two salmonid fishes.

All skeletal muscles produce their largest forces at a single optimal length, losing force when stretched or shortened. In vertebrate feeding systems, this fundamental force-length relationship translates to variation in bite force across gape, which affects the food types that can be eaten effectively. We measured the bite force-gape curves of two sympatric species: king salmon (Oncorhynchus tshawytscha) and pink salmon (Oncorhynchusgorbuscha). Cranial anatomical measurements were not significantly different between species; however, peak bite forces were produced at significantly different gapes. Maximum bite force was achieved at 67% of maximum gape for king salmon and 43% of maximum gape for pink salmon. This may allow king salmon to use greater force when eating large or elusive prey. In contrast, pink salmon do not require high forces at extreme gapes for filter feeding. Our results illustrate that the bite force-gape relationship is an important ecophysiological axis of variation.

Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia.

Sprmatogonial stem cells (SSCs) are valuable for preservation of endangered fish species, biological experimentation, as well as biotechnological applications. However, the rarity of SSCs in the testes has been a great obstacle in their application. Thus, establishment of an efficient in-vitro culture system to support continuous proliferation of SSCs is essential. The present study aimed to establish an efficient and simple method for in vitro culture of Caspian trout undifferentiated spermatogonial cells. Using a two-step enzymatic digestion, testicular cells were isolated from immature testes composed of mainly undifferentiated spermatogonial cells with gonadosomatic indices of <0.05%. The spermatogonial cells were purified by differential plating through serial passaging. The purified cells indicated high expression of type A spermatogonia-related genes (Ly75, Gfrα1, Nanos2, Plzf and Vasa). Proliferation of purified cells was confirmed by BrdU incorporation. Co-culture of purified cells with testicular somatic cells as a feeder layer, resulted in continuous proliferation of type A spermatogonia. The cultured cells continued to express type A spermatogonia-specific markers after one month culture. The cultured spermatogonia were successfully incorporated into the germline after being intraperitoneally transplanted into sterile triploid rainbow trout hatchlings. These results, for the first time, demonstrated that the somatic microenvironment of the rainbow trout gonad can support the colonization and survival of intraperitoneally transplanted cells derived from a fish species belonging to a different genus. Therefore, the combination of in vitro culture system and xenotransplantation can be considered as a promising strategy for conservation of Caspian trout genetic resources.

An updated geographic distribution of Myxobolus cerebralis (Hofer, 1903) (Bivalvulida: Myxobolidae) and the first diagnosed case of whirling disease in wild-caught trout in the south-eastern United States.

Myxobolus cerebralis (Bivalvulida: Myxobolidae), the aetiological agent of salmonid whirling disease, was detected in 2 river basins of North Carolina during 2015, which initiated the largest spatial-temporal monitoring project for the disease ever conducted within the south-eastern United States (focused mainly in eastern Tennessee and western North Carolina). A total of 2072 rainbow trout Oncorhynchus mykiss, 1,004 brown trout Salmo trutta and 468 brook trout Salvelinus fontinalis were screened from 113 localities within 7 river basins during June 2017 through October 2019. Infections were detected by pepsin-trypsin digest, microscopy and the species-specific nested polymerase chain reaction (PCR) in 19 localities across 6 river basins. Myxospore morphology was indistinguishable from the published literature. In 2019, five rainbow trout that symptomatic for whirling disease (sloping neurocranium and lordosis) were captured and processed for histopathology. Myxospores were detected in the calvarial cartilage of two deformed trout with associated erosion of the cartilage consistent with reported whirling disease lesions. This is the first report of M. cerebralis in Tennessee and the first histologically confirmed cases of whirling disease in southern Appalachian (south-eastern United States) rivers and streams and expands the distribution of M. cerebralis throughout western North Carolina and eastern Tennessee.

[Exon and Intron Variability in Salmonidae Growth Hormone Genes].

Gene duplication is one of the main mechanisms of formation of new genetic material in evolution. The occurrence of a gene duplication is believed to relax selection pressure on one of the copies. Consequently, this gene accumulates mutations at a higher rate, and over time it acquires a new function. As a result of several rounds of polyploidization, many genes in salmon are duplicated, including the growth hormone gene. The analysis of nucleotide diversity in the paralogous genes of growth hormone, gh1 and gh2, demonstrated that the level of variability in their introns was higher than in the exons. In addition, the variability of each exon weakly correlated with its length, and seems to be determined by the functional significance of the protein region encoded. The level of variability in the exons of the gh2 gene was higher than that in the gh1 one, which was probably due to the current process of gene subfunctionalization.

Sensitivity of Warm-Water Fishes and Rainbow Trout to Selected Contaminants.

Guidelines for developing water quality standards allow U.S. states to exclude toxicity data for the family Salmonidae (trout and salmon) when deriving guidelines for warm-water habitats. This practice reflects the belief that standards based on salmonid data may be overprotective of toxic effects on other fish taxa. In acute tests with six chemicals and eight fish species, the salmonid, Rainbow Trout (Oncorhynchus mykiss), was the most sensitive species tested with copper, zinc, and sulfate, but warm-water species were most sensitive to nickel, chloride, and ammonia. Overall, warm-water fishes, including sculpins (Cottidae) and sturgeons (Acipenseridae), were about as sensitive as salmonids in acute tests and in limited chronic testing with Lake Sturgeon (Acipenser fulvescens) and Mottled Sculpin (Cottus bairdi). In rankings of published acute values, invertebrate taxa were most sensitive for all six chemicals tested and there was no trend for greater sensitivity of salmonids compared to warm-water fish.

Polyunsaturated Fatty Acid Content in Muscle Tissue Is Associated with the Duration of Embryo Development in Salmonoid Fishes (Salmonoidei).

A hypothesis was advanced and grounded that the total content of eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids in fish muscle tissue is associated with the species-specific (taxon-specific) duration of embryo development. A meta-analysis of the original and published data was performed using fishes of the families Coregonidae and Salmonidae as an example. Fishes with longer embryo development times, which are observed at lower temperatures, were found to have significantly higher EPA + DHA contents in muscles as compared with the species that belong to the same families but have shorter embryo development times. This association was explained by the fact that an embryo forms more cells per unit tissue volume at lower temperatures, which requires a greater specific amount of cell membranes and, therefore, greater amounts of EPA and DHA to produce them.

Low adaptive potential for tolerance to ethynylestradiol, but also low toxicity, in a grayling population (Thymallus thymallus).

BACKGROUND: The presence of a novel pollutant can induce rapid evolution if there is additive genetic variance for the tolerance to the stressor. Continuous selection over some generations can then reduce the toxicity of the pollutant but also deplete the additive genetic variance for the tolerance and thereby slow down adaptation. One common pollutant that has been ecologically relevant for some time is 17alpha-ethynylestradiol (EE2), a synthetic compound of oral contraceptives since their market launch in the 1960s. EE2 is typically found in higher concentrations in rivers than in lakes. Recent experimental work revealed significant genetic variance for the tolerance to EE2 in two lake-spawning salmonid species but no such variance in river-spawning brown trout. We used another river-spawning salmonid, the European grayling Thymallus thymallus, to study the toxicity of an ecologically relevant concentration of EE2. We also used a full-factorial in vitro breeding design and singly rearing of 1555 embryos and larvae of 40 sib groups to test whether there is additive genetic variance for the tolerance to this pollutant. RESULTS: We found that exposure to EE2 reduced larval growth after hatching, but contrary to what has been found in the other salmonids, there were no significant effects of EE2 on embryo growth and survival. We found additive genetic variance for embryo viability, i.e. heritability for fitness. However, there was no significant additive variance for the tolerance to EE2. CONCLUSIONS: Our findings support the hypothesis that continuous selection has reduced the toxicity of EE2 and depleted genetic variance for tolerance to this synthetic stressor.

Vertebral fusions in farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand.

Vertebral fusions are an established economic concern in farmed Atlantic salmon, but have not been studied in detail in farmed Chinook salmon. Two radiographic studies of vertebral fusions were performed in farmed Chinook salmon. Sixteen of 1,301 (1.2%) smolt and 201 of 2,636 (7.6%) harvest fish had fusions. There were no significant differences in the number of fused vertebrae/fusion in smolt compared with harvest fish. Secondly, tagged fish were repeatedly radiographed to determine the progression of the fusions. Nineteen (4.4%), 23 (5.3%) and 39 (9.0%) fish had fusions as smolt, after 129 days in sea water, and at harvest, respectively. There were no significant differences in the average number of vertebra/fusion between the three time points. Of the fusions that were observed in smolt, additional vertebra did not become fused in 81% of the lesions. Within the rare fusions that did progress due to the involvement of adjacent vertebra, an average of 1.6 vertebrae were added per year. Fish with fusions were significantly lighter than non-affected fish at harvest. Fusions are common in farmed Chinook salmon; however, they are typically stable after development. As fish with fusions were lighter at harvest, reducing fusions may have an economic benefit.