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.

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.

Histology and Ultrastructure of the Nephron and Kidney Interstitial Cells in the Atlantic Salmon (Salmo salar Linnaeus 1758) at Different Stages of Life Cycle.

This article presents data on the mesonephros histology and ultrastructure in the Atlantic salmon from the Baltic Sea and Barents Sea populations, with an emphasis on comparisons between the following ontogenetic stages: parr, smolting, adult life at sea, the adults' return to their natal river to spawn, and spawning. The ultrastructural changes in the renal corpuscle and cells of the proximal tubules of the nephron occurred as early as the smolting stage. Such changes reflect fundamental alterations during the pre-adaptation to life in saltwater. In the Barents Sea population, the adult salmon sampled in the sea had the smallest diameters of the renal corpuscle and proximal and distal tubules, the most narrow urinary space, and the thickest basement membrane. In the group of salmon that entered the mouth of the river and spent less than 24 h in freshwater, the structural rearrangements occurred only in the distal tubules. Better development of the smooth endoplasmic reticulum and a greater abundance of mitochondria in the tubule cells were observed in the adult salmon from the Barents Sea compared to those from the Baltic Sea. Cell-immunity activation was initiated during the parr-smolt transformation. Another pronounced innate-immunity response was registered in the adults returning to the river to spawn.

Polystyrene microparticles can affect the health status of freshwater fish - Threat of oral microplastics intake.

Plastic waste pollution is considered one of the biggest problems facing our planet. The production and use of these materials has led to huge amounts of plastic waste entering the aquatic environment and affecting aquatic life. In our experiment, the effect of polystyrene microparticles (PS-MPs; 52.5 ± 11.5 µm) on individual juvenile rainbow trout (Oncorhynchus mykiss) was tested at three different dietary concentrations of 0.5, 2 and 5 % for six weeks. At the end of the experiment, various health parameters of exposed organisms were compared with the control group. The haematological profile revealed an immune response by a decrease in lymphocyte count with a concurrent increase in the number of neutrophil segments at the highest concentration of PS-MPs (5 %). Biochemical analysis showed significant reductions in plasma ammonia in all tested groups, which may be related to liver and gill damage, as determined by histopathological examination and analysis of inflammatory cytokines expression. In addition, liver damage can also cause a significant decrease in the plasma protein ceruloplasmin, which is synthesized in the liver. PS-MPs disrupted the antioxidant balance in the caudal kidney, gill and liver, with significant changes observed only at the highest concentration. In summary, PS-MPs negatively affect the health status of freshwater fish and represent a huge burden on aquatic ecosystems.

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.

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.

Salmonidae Genome: Features, Evolutionary and Phylogenetic Characteristics.

The salmon family is one of the most iconic and economically important fish families, primarily possessing meat of excellent taste as well as irreplaceable nutritional and biological value. One of the most common and, therefore, highly significant members of this family, the Atlantic salmon (Salmo salar L.), was not without reason one of the first fish species for which a high-quality reference genome assembly was produced and published. Genomic advancements are becoming increasingly essential in both the genetic enhancement of farmed salmon and the conservation of wild salmon stocks. The salmon genome has also played a significant role in influencing our comprehension of the evolutionary and functional ramifications of the ancestral whole-genome duplication event shared by all Salmonidae species. Here we provide an overview of the current state of research on the genomics and phylogeny of the various most studied subfamilies, genera, and individual salmonid species, focusing on those studies that aim to advance our understanding of salmonid ecology, physiology, and evolution, particularly for the purpose of improving aquaculture production. This review should make potential researchers pay attention to the current state of research on the salmonid genome, which should potentially attract interest in this important problem, and hence the application of new technologies (such as genome editing) in uncovering the genetic and evolutionary features of salmoniforms that underlie functional variation in traits of commercial and scientific importance.

Salmincola markewitschi or S. carpionis (Copepoda: Lernaeopodidae)? A requirement for taxonomic revision due to their high morphological variations.

Salmincola markewitschi Shedko et Shedko, 2002 (Copepoda: Lernaeopodidae) is an ectoparasitic copepod mainly infecting the buccal cavities of white-spotted charr Salvelinus leucomaenis (Pallas) (Salmonidae). This species has only been recorded from Northeast Asia, where a morphologically similar congener Salmincola carpionis (Krøyer, 1837) is also distributed, using the same host species. These copepods are hard to distinguish from each other because of their similarities. We thus examined the newly collected specimens morphologically and genetically from five populations of white-spotted charr in Japan. Most of the specimens were morphologically consistent with S. markewitschi but showed great variations in the numbers of spines on the exopods of the antennae, shape of the maxilliped myxal palps, and the bulla diameter. Consequently, some specimens shared characteristics with S. carpionis. In addition to the mophological continuities, genetic analyses of 28S rDNA and COI mitochondrial DNA confirmed that all specimens belong to a single species. Further taxonomic revisions are required to draw conclusions of whether S. markewitschi is a valid species different from S. carpionis, by collecting samples from across their wide distributional ranges, such as Europe, North America, and Northeast Asia. A key to identification of species of Salmincola Wilson, 1915 occurring in Japan is also provided.

Interactions of semiconductor Cd-based quantum dots and Cd2+ with gut bacteria isolated from wild Salmo trutta fry.

Background: With the rapid development of nanotechnology, more and more nanoproducts are being released into the environment where they may both pose ecological risks and be toxic to living organisms. The ecotoxicological impact of quantum dots (QDs), a class of nanoparticles (NPs), on aquatic organisms is becoming an emerging issue, this due to their nano-specific properties, to the physico-chemical transformation in the environment and to the possible release of toxic metals from their structure such as Cd. Methods: In this work, (i) spectroscopic measurements of commercially available Cd-based QDs (CdSe/ZnS-COOH) were made at various pH values (5.0 and 7.0) to study their interactions (at a concentration of 4 nm) with various strains of Gram-positive and Gram-negative gut bacteria after short-term exposure and (ii) the antibacterial efficacy of QDs and Cd2+ (at a concentration 0.09-3.56 mM) against gut bacteria isolated from wild freshwater Salmo trutta fry was studied at different temperatures (15 °C and 25 °C) and pH values (5.0 and 7.0) by applying a well-established disc diffusion assay. Results: Twenty-six gut bacterial isolates from wild Salmo trutta fry were identified as Aeromonas spp., A. popoffii, A. salmonicida, A. sobria, Carnobacterium maltaromaticum, Buttiauxella sp., Listeria sp., Microbacterium sp., Shewanella putrefaciens and Serratia sp. Cd-based (CdSe/ZnS-COOH) QDs at a concentration of 4 nm were found to be stable in aqueous media (with pH 7.0) or starting to form aggregates (at pH 5.0), thus, apparently, did not release heavy metals (HMs) into the media over 48 h in conditions of light or dark and did not show antibacterial efficacy on the gut bacteria isolated from wild Salmo trutta fry after short-term (9 h and 48 h) incubations. Cd2+ was found to produce significant dose-dependent toxic effects on bacterial growth, and the size of the inhibition zones on some of the tested strains significantly correlated with temperature. The most sensitive and the most resistant to Cd2+ were the Gram-positive bacteria, for which the minimum inhibitory concentration (MIC) values of Cd2+ were 0.09-0.27 mM and 3.11-3.29 mM respectively and varied significantly between the tested temperatures (15 °C and 25 °C). The MIC values of Cd2+ for the Gram-negative bacteria (18 out of 22 strains) ranged from 0.44 to 0.71 mM and did not differ significantly between the tested temperatures. Among the selected Gram-positive and Gram-negative strains, those with the higher sensitivity towards Cd2+ also revealed relatively stronger signals of QDs photoluminescence (PL) when transferred after incubation into fresh medium without QDs. In addition, the formation of endogenous metalloporphyrins observed spectroscopically in some bacterial strains indicates certain differences in metabolic activity that may play a protective role against potential oxidative damage.

Molecular Phylogeny and Adaptive Mitochondrial DNA Evolution of Salmonids (Pisces: Salmonidae).

Salmonids are composed of anadromous and freshwater fishes, which is an important model for studying adaptive evolution. Herein, 49 salmonid complete mitochondrial genomes and those of two outgroups were used to infer a robust phylogeny for the family Salmonidae. The BI and RAxML phylogenetic trees based on 13 concatenated mitochondrial protein-coding genes showed well-supported nodes, and topologies were highly congruent. The concatenated 13 mitochondrial protein-coding genes, ND2, ND3, and ND5 genes were shown to have significantly larger dN/dS ratios in anadromous species than in freshwater species of Salmonidae, but the CYTB gene had significantly smaller dN/dS in anadromous species. The FEL analysis identified positively selected sites and negatively selected sites in each mitochondrial protein-coding gene separately. The RELAX program revealed that the ATP8 and CYTB genes supported intensified selection of the anadromous lineages. Our results demonstrated the phylogeny of Salmonidae and explored the mitochondrial DNA evolution pattern between anadromous and freshwater salmonids.

Shifting thermal regimes influence competitive feeding and aggression dynamics of brook trout (Salvelinus fontinalis) and creek chub (Semotilus atromaculatus).

The natural distributions of freshwater fish species are limited by their thermal tolerances via physiological constraints and increased interspecific competition as temperatures shift toward the thermal optima of other syntopic species. Species may mediate stress from temperature change physiologically, behaviorally, or both; but these changes may compromise competitive advantages through effects on feeding and social behavior. In the Appalachian Mountains of North America, creek chub (Semotilus atromaculatus) are found in warm-water and cold-water streams and overlap in range with brook trout (Salvelinus fontinalis) across lower thermal maxima, where they compete for food and space. As stream temperatures continue to increase due to climate change, brook trout are under increasing thermal stress which may negatively affect their ability to compete with creek chub. To examine the influences of temperature on competitive interactions between these species, we observed feeding behavior, aggression, and habitat use differences at three temperatures approaching brook trout thermal maxima (18°C, 20°C, and 22°C) among dyad pairs for all combinations of species in experimental flow-through tanks. We also examined feeding and habitat use of both species under solitary conditions. We found as temperature increased, feeding and aggression of brook trout were significantly reduced in the presence of creek chub. Creek chub pairs were more likely to occupy benthic areas and refugia while brook trout pairs used surface water more. Space use patterns significantly changed by pairing treatment. Aggression and space use shifts allowed increased exploitative and interference competition from creek chub when paired with brook trout that was not present in conspecific pairs. The decreased dominance of a top predator may lead to diverse impacts on stream community dynamics with implications for the future range restriction of brook trout and demonstrate possible mechanisms to facilitate competitive advantages of warm water generalist species under thermal stress.

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.

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.

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.

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.

Relationship between salmon egg subsidy and the distribution of an avian predator.

As a spatial subsidy, which is the phenomenon of transferring resources from a donor system to a recipient system, anadromous salmonids contribute to the supply of marine-derived nutrients to freshwater and terrestrial systems. Live salmon and salmon carcasses and eggs are utilized by various organisms and affect their abundance and distribution. However, the evaluation of the effect of salmon subsidies on the abundance and distribution of terrestrial animals is biased toward predators or scavengers that utilize spawning adults and carcasses, and few studies have focused on the effect of salmon eggs as a subsidy. To avoid underestimating the function of salmon subsidies, the response to the availability of salmon eggs in various systems should be investigated. Here, we investigated the abundance and feeding behavior of the brown dipper Cinclus pallasii, as a consumer of salmon eggs, based on the hypothesis that the availability of salmon eggs affects the diet composition and stream distribution of this small predator. In addition, to test whether changes in the abundance of brown dippers are determined by salmon spawning, their abundance was compared upstream and downstream of the check dams in three streams during the peak spawning period. Brown dippers used salmon eggs during the spawning season (53.7% of diet composition), and their abundance increased as the number of spawning redds increased. In contrast, this pattern was not observed upstream of the check dam. These results suggested that the abundance and stream distribution of brown dippers vary according to the variation in the spatiotemporal availability of salmon eggs.

Do foodborne polyethylene microparticles affect the health of rainbow trout (Oncorhynchus mykiss)?

Due to the fact that plastic pollution is a global environmental problem of modern age, studies on the impact of these synthetic materials on aquatic, and especially fish organisms, are an important part of the ecosystem and human nutrition. In our study, the toxicity of pristine polyethylene (PE) microparticles (approx. 50 µm) on rainbow trout (Oncorhynchus mykiss) was tested in three different dietary concentrations - 0.5%, 2% and 5%. After six weeks of exposure, various health indices were evaluated. Electron microscopy of the intestine revealed the disintegration of PE particles to <5 µm in size, and thus we concluded that microplastics are able to reach tissues. The haematological profile revealed changes in total red blood cells count and haematocrit (5% PE) which could be associated with spleen congestion observed histologically. The marker of lipid peroxidation was increased in gills suggesting the disruption of balance in antioxidant enzymes capacity and histopathological imaging revealed inflammation in higher PE concentrations. In addition, ammonia was decreased and calcium elevated in biochemical profile, confirming the gill damage. Electron microscopy of the gills showed lesions of lamellae and visible rings around the mucinous cell opening indicating their higher activity. Another injured was the liver tissue, as confirmed by hepatodystrophies and increased expression of pro-inflammatory genes in 2% PE. Impaired innate immunity was confirmed by an increased presence of mucinous cells and a decrease in leukocytes. Kidney damage manifested itself by higher expression of pro-inflammatory cytokines and histopathology. The damage in gills, liver and kidney together correlated with the increased antioxidant capacity of plasma. In conclusion, PE microparticles are able to affect health indices of O. mykiss. The potential problem for aquatic ecosystems and even human consumption should be considered.

Life-history strategies in salmonids: the role of physiology and its consequences.

Salmonids are some of the most widely studied species of fish worldwide. They span freshwater rivers and lakes to fjords and oceans; they include short- and long-distance anadromous migrants, as well as partially migratory and non-migratory populations; and exhibit both semelparous and iteroparous reproduction. Salmonid life-history strategies represent some of the most diverse on the planet. For this reason, salmonids provide an especially interesting model to study the drivers of these different life-history pathways. Over the past few decades, numerous studies and reviews have been published, although most have focused on ultimate considerations where expected reproductive success of different developmental or life-history strategies are compared. Those that considered proximate causes generally focused on genetics or the environment, with less consideration of physiology. Our objective was therefore to review the existing literature on the role of physiology as a proximate driver for life-history strategies in salmonids. This link is necessary to explore since physiology is at the core of biological processes influencing energy acquisition and allocation. Energy acquisition and allocation processes, in turn, can affect life histories. We find that life-history strategies are driven by a range of physiological processes, ranging from metabolism and nutritional status to endocrinology. Our review revealed that the role of these physiological processes can vary across species and individuals depending on the life-history decision(s) to be made. In addition, while findings sometimes vary by species, results appear to be consistent in species with similar life cycles. We conclude that despite much work having been conducted on the topic, the study of physiology and its role in determining life-history strategies in salmonids remains somewhat unexplored, particularly for char and trout (excluding brown trout) species. Understanding these mechanistic links is necessary if we are to understand adequately how changing environments will impact salmonid populations.

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.

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.