Anatomy, immunology, digestive physiology and microbiota of the salmonid intestine: Knowns and unknowns under the impact of an expanding industrialized production.

Increased industrialized production of salmonids challenges aspects concerning available feed resources and animal welfare. The immune system plays a key component in this respect. Novel feed ingredients may trigger unwarranted immune responses again affecting the well-being of the fish. Here we review our current knowledge concerning salmon intestinal anatomy, immunity, digestive physiology and microbiota in the context of industrialized feeding regimes. We point out knowledge gaps and indicate promising novel technologies to improve salmonid intestinal health.

Skull Morphology Variation as Related to Trophic Specialization in Three Forms of Salvelinus malma (Salmonidae) from the Kamchatka River Basin.

New data on the correlation between morphology and trophic specialization of three riverine charr forms from the Kamchatka River basin (stone charr, white charr, and Dolly Varden) are presented. The paper analyzes the food preferences of the fishes according to their stomach contents and retrospectively assesses their nutrition according to the ratio of stable nitrogen isotopes. The three charr forms are compared in terms of the head shape and structure of the cartilaginous skull for the first time. The correlation between the type of their nutrition and structural features of their skull is shown. Both general and specific adaptive features of the morphology of piscivorous stone and white charrs are revealed in comparison with benthivorous Dolly Varden.

DNA methylation reprogramming, TE derepression, and postzygotic isolation of nascent animal species.

The genomic shock hypothesis stipulates that the stress associated with divergent genome admixture can cause transposable element (TE) derepression, which could act as a postzygotic isolation mechanism. TEs affect gene structure, expression patterns, and chromosome organization and may have deleterious consequences when released. For these reasons, they are silenced by heterochromatin formation, which includes DNA methylation. Here, we show that a significant proportion of TEs are differentially methylated between the "dwarf" (limnetic) and the "normal" (benthic) whitefish, two nascent species that diverged some 15,000 generations ago within the Coregonus clupeaformis species complex. Moreover, TEs are overrepresented among loci that were demethylated in hybrids, indicative of their transcriptional derepression. These results are consistent with earlier studies in this system that revealed TE transcriptional derepression causes abnormal embryonic development and death of hybrids. Hence, this supports a role of DNA methylation reprogramming and TE derepression in postzygotic isolation of nascent animal species.

Extreme premaxillary protrusion in the king-of-the-salmon, Trachipterus altivelis.

The king-of-the-salmon, Trachipterus altivelis (Lampriformes), has an unusual set of oral jaws which allow it the ability to protrude the entire upper jaw, containing the premaxilla and the maxilla bones, to extreme distances. Here, we provide a short description of the cranial anatomy and mechanism of jaw protrusion in T. altivelis using hand-drawn illustrations (by KF), supplemented by CT-scans. We then place the protrusion abilities of T. altivelis into context by comparing anatomical jaw protrusion with protrusion from other members of the Lampriformes, other unrelated species with highly protrusile jaws, and unrelated species with more stereotypical amounts of jaw protrusion. Through these comparisons we demonstrate that T. altivelis is indeed, capable of some of the most extreme premaxillary protrusion as of yet discovered, even when taking into account the extreme morphological modifications that facilitate said protrusion. That is to say, T. altivelis can protrude the premaxilla farther than one would predict from the length of the ascending process alone.

Rapid niche expansion by selection on functional genomic variation after ecosystem recovery.

It is well recognized that environmental degradation caused by human activities can result in dramatic losses of species and diversity. However, comparatively little is known about the ability of biodiversity to re-emerge following ecosystem recovery. Here, we show that a European whitefish subspecies, the gangfisch Coregonus lavaretus macrophthalmus, rapidly increased its ecologically functional diversity following the restoration of Lake Constance after anthropogenic eutrophication. In fewer than ten generations, gangfisch evolved a greater range of gill raker numbers (GRNs) to utilize a broader ecological niche. A sparse genetic architecture underlies this variation in GRN. Several co-expressed gene modules and genes showing signals of positive selection were associated with GRN and body shape. These were enriched for biological pathways related to trophic niche expansion in fishes. Our findings demonstrate the potential of functional diversity to expand following habitat restoration, given a fortuitous combination of genetic architecture, genetic diversity and selection.

Impacts of temperature, morpholine, and chronic radiation on the embryonic development of round whitefish (Prosopium cylindraceum).

During incubation, round whitefish embryos may experience fluctuating or elevated temperatures from natural (e.g., seasonal temperature changes) and/or anthropogenic sources. Anthropogenic sources like once-through cooling discharges from nuclear power plants can also expose embryos to chemicals (e.g., morpholine) and/or radiation. To examine the effects of these potential stressors on embryogenesis, round whitefish were incubated under fluctuating or constant temperatures, with morpholine or 137 Cs gamma rays. We report the percentage of prehatch and posthatch mortality, developmental rate, hatch dynamics, and morphometrics at 4 development stages. Embryos reared at constant temperatures had delayed developmental stage onset and median hatch, higher mortality at constant 8 °C, and lower mortality at ≤5 °C, compared with embryos reared under seasonal temperature regimes. Embryos incubated with ≥500 mg L-1 morpholine (>200× regulatory limits) had advanced hatch, reduced body size, and increased prehatch (100% at 1000 mg L-1 ) and posthatch (≈95% at 500 mg L-1 ) mortality compared with controls. Relative to controls, embryos irradiated with ≥0.16 mGy/d had larger body mass early in development, and all irradiated embryos had decreased posthatch mortality; the lowest dose was >300× discharge limits. Our study suggests that fluctuating or elevated temperatures and high-dose morpholine can alter development rate, hatch dynamics, and growth, and/or increase mortality compared with embryos reared at constant temperatures of ≤5 °C; conversely, low-dose irradiation had transient developmental effects but may benefit early posthatch survival. Environ Toxicol Chem 2018;37:2593-2608. © 2018 SETAC.

Ecomorphological divergence drives differential mercury bioaccumulation in polymorphic European whitefish (Coregonus lavaretus) populations of subarctic lakes.

Resource polymorphism, whereby ancestral trophic generalists undergo divergence into multiple specialist morphs, is common in salmonid fish populations inhabiting subarctic lakes. However, the extent to which such resource specialization into the three principal lake habitats (littoral, profundal, and pelagic) affects patterns of contaminant bioaccumulation remains largely unexplored. We assessed total mercury concentrations (THg) of European whitefish (Coregonus lavaretus (L.)) and their invertebrate prey in relation to potential explanatory variables across 6 subarctic lakes, of which three are inhabited by polymorphic (comprised of four morphs) and three by monomorphic populations. Among invertebrate prey, the highest THg concentrations were observed in profundal benthic macroinvertebrates, followed by pelagic zooplankton, with concentrations lowest in littoral benthic macroinvertebrates in both lake types. Broadly similar patterns were apparent in whitefish in polymorphic systems, where average age-corrected THg concentrations and bioaccumulation rates were the highest in pelagic morphs, intermediate in the profundal morph, and the lowest in the littoral morph. In monomorphic systems, age-corrected THg concentrations were generally lower, and showed pronounced lake-specific variation. In the polymorphic systems, we found significant relationships between whitefish muscle tissue THg concentration and gill raker count, resource use, lipid content and maximum length, whilst no such relationships were apparent in the monomorphic systems. Across all polymorphic lakes, the major variables explaining THg in whitefish were gill raker count and age, whereas in monomorphic systems, the factors were lake-specific. Whitefish resource polymorphism across the three main lake habitats therefore appears to have profound impacts on THg concentration and bioaccumulation rate. This highlights the importance of recognizing such intraspecific diversity in both future scientific studies and mercury monitoring programs.

Adaptation and acclimation of traits associated with swimming capacity in Lake Whitefish (coregonus clupeaformis) ecotypes.

BACKGROUND: Improved performance in a given ecological niche can occur through local adaptation, phenotypic plasticity, or a combination of these mechanisms. Evaluating the relative importance of these two mechanisms is needed to better understand the cause of intra specific polymorphism. In this study, we reared populations of Lake Whitefish (Coregonus clupeaformis) representing the'normal' (benthic form) and the 'dwarf' (derived limnetic form) ecotypes in two different conditions (control and swim-training) to test the relative importance of adaptation and acclimation in the differentiation of traits related to swimming capacity. The dwarf whitefish is a more active swimmer than the normal ecotype, and also has a higher capacity for aerobic energy production in the swimming musculature. We hypothesized that dwarf fish would show changes in morphological and physiological traits consistent with reductions in the energetic costs of swimming and maintenance metabolism. RESULTS: We found differences in traits predicted to decrease the costs of prolonged swimming and standard metabolic rate and allow for a more active lifestyle in dwarf whitefish. Dwarf whitefish evolved a more streamlined body shape, predicted to lead to a decreased drag, and a smaller brain, which may decrease their standard metabolic rate. Contrary to predictions, we also found evidence of acclimation in liver size and metabolic enzyme activities. CONCLUSION: Results support the view that local adaptation has contributed to the genetically-based divergence of traits associated with swimming activity. Presence of post-zygotic barriers limiting gene flow between these ecotype pairs may have favoured repeated local adaptation to the limnetic niches.

Morphology and ultrastructure of Brachymystax lenok tsinlingensis spermatozoa by scanning and transmission electron microscopy.

This study was conducted to investigate Brachymystax lenok tsinlingensis spermatozoa cell morphology and ultrastructure through scanning and transmission electron microscopy. Findings revealed that the spermatozoa can be differentiated into three major parts: a spherical head without an acrosome, a short mid-piece, and a long, cylindrical flagellum. The mean length of the spermatozoa was 36.11±2.84µm, with a spherical head length of 2.78±0.31µm. The mean anterior and posterior head widths were 2.20±0.42µm and 2.55±0.53µm, respectively. The nuclear fossa was positioned at the base of the nucleus that contained the anterior portion of flagellum and a centriolar complex (proximal and distal centrioles). The short mid-piece was located laterally to the nucleus and possessed just one spherical mitochondrion with a mean diameter of 0.65±0.14µm. The spermatozoa flagellum was long and cylindrical, and could be separated into two parts: a long main-piece and a short end-piece. The main piece of the flagellum had short irregular side-fins. The axoneme composed the typical '9+2' microtubular doublet structure and was enclosed by the cell membran e. This study confirmed that B. lenok tsinlingensis spermatozoa can be categorized as teleostean "Type I" spermatozoa; 'primitive' or 'ect-aquasperm type' spermatozoa. To the best of the authers knowledge, this was the first study conducted on the morphology and ultrastructure of B. lenok tsinlingensis spermatozoa.

Structure of "species flocks" in fishes: Channels of the splanchnocranium divergence in endemic lacustrine benthivorous charrs (Salvelinus, Salmonidae, Teleostei) in Lake Kronotskoye, Kamchatka.

The structure of splanchnocranium bones has been studied in four endemic benthivorous charrs (the genus Salvelinus) from Lake Kronotskoe (Kamchatka). It has been found that, according to the whole set of characters of the splanchnocranium structure, the most expressed differences are observed between specialized forms, nosed and largemouth charrs, inhabiting different biotopes of the lake. Differences between small-mouth and white charrs are less pronounced, and the species are characterized by generalized features of the structure of jaws. It can be suggested that, in addition to diverse feeding preferences within a feeding niche, an extremely high diversity of charrs in Lake Kronotskoe is due to a complex geomorphological structure of the water system. Thus, the "species flock" comprising a complex set of diverse benthivorous morphotypes has been formed and exists in Lake Kronotskoe. Charrs from Lake Kronotskoe are a representative example of sympatric speciation by way of formation of new forms differing from each other in morphological, ecological, and trophic features.

Patterns of variation and covariation in the shapes of mandibular bones of juvenile salmonids in the genus Oncorhynchus.

What is the nature of evolutionary divergence of the jaw skeleton within the genus Oncorhynchus? How can two associated bones evolve new shapes and still maintain functional integration? Here, we introduce and test a "concordance" hypothesis, in which an extraordinary matching of the evolutionary shape changes of the dentary and angular articular serves to preserve their fitting together. To test this hypothesis, we examined morphologies of the dentary and angular articular at parr (juvenile) stage, and at three levels of biological organization­between salmon and trout, between sister species within both salmon and trout, and among three types differing in life histories within one species, Oncorhynchus mykiss. The comparisons show bone shape divergences among the groups at each level; morphological divergence between salmon and trout is marked even at this relatively early life history stage. We observed substantial matching between the two mandibular bones in both pattern and amount of shape variation, and in shape covariation across species. These findings strongly support the concordance hypothesis, and reflect functional and/or developmental constraint on morphological evolution. We present evidence for developmental modularity within both bones. The locations of module boundaries were predicted from the patterns of evolutionary divergences, and for the dentary, at least, would appear to facilitate its functional association with the angular articular. The modularity results suggest that development has biased the course of evolution.

Revalidation and redescription of Brachymystax tsinlingensis Li, 1966 (Salmoniformes: Salmonidae) from China.

Brachymystax tsinlingensis Li, 1966 is revalidated and redescribed. It can be distinguished from all congeners by the following combination of characteristics: no spots on operculum; gill rakers 15-20; lateral-line scales 98-116; pyloric caeca 60-71. Unique morphological characters and genetic divergence of this species are discussed. This species has a limited distribution in several streams of the middle part of the Qinling Mountains in China. Methods for management and protection of B. tsinlingensis need to be re-evaluated.

RAD-QTL Mapping Reveals Both Genome-Level Parallelism and Different Genetic Architecture Underlying the Evolution of Body Shape in Lake Whitefish (Coregonus clupeaformis) Species Pairs.

Parallel changes in body shape may evolve in response to similar environmental conditions, but whether such parallel phenotypic changes share a common genetic basis is still debated. The goal of this study was to assess whether parallel phenotypic changes could be explained by genetic parallelism, multiple genetic routes, or both. We first provide evidence for parallelism in fish shape by using geometric morphometrics among 300 fish representing five species pairs of Lake Whitefish. Using a genetic map comprising 3438 restriction site-associated DNA sequencing single-nucleotide polymorphisms, we then identified quantitative trait loci underlying body shape traits in a backcross family reared in the laboratory. A total of 138 body shape quantitative trait loci were identified in this cross, thus revealing a highly polygenic architecture of body shape in Lake Whitefish. Third, we tested for evidence of genetic parallelism among independent wild populations using both a single-locus method (outlier analysis) and a polygenic approach (analysis of covariation among markers). The single-locus approach provided limited evidence for genetic parallelism. However, the polygenic analysis revealed genetic parallelism for three of the five lakes, which differed from the two other lakes. These results provide evidence for both genetic parallelism and multiple genetic routes underlying parallel phenotypic evolution in fish shape among populations occupying similar ecological niches.

Experimental evidence for trait utility of gill raker number in adaptive radiation of a north temperate fish.

North temperate fish in post-glacial lakes are textbook examples for rapid parallel adaptive radiation into multiple trophic specialists within individual lakes. Speciation repeatedly proceeded along the benthic-limnetic habitat axis, and benthic-limnetic sister species diverge in the number of gill rakers. Yet, the utility of different numbers of gill rakers for consuming benthic vs. limnetic food has only very rarely been experimentally demonstrated. We bred and raised families of a benthic-limnetic species pair of whitefish under common garden conditions to test whether these species (i) show heritable differentiation in feeding efficiency on zooplankton, and (ii) whether variation in feeding efficiency is predicted by variation in gill raker numbers. We used zooplankton of three different size classes to investigate prey size dependency of divergence in feeding efficiency and to investigate the effect strength of variation in the number of gill rakers. Our results show strong interspecific differences in feeding efficiency. These differences are largest when fish were tested with the smallest zooplankton. Importantly, feeding efficiency is significantly positively correlated with the number of gill rakers when using small zooplankton, also when species identity is statistically controlled for. Our results support the hypothesis that a larger number of gill rakers are of adaptive significance for feeding on zooplankton and provide one of the first experimental demonstrations of trait utility of gill raker number when fish feed on zooplankton. These results are consistent with the suggested importance of divergent selection driven feeding adaptation during adaptive radiation of fish in post-glacial lakes.

Ontogenetic shifts in morphology and resource use of cisco Coregonus artedi.

Two previously described lacustrine cisco Coregonus spp. morphs [i.e. a small (<300 mm fork length, L(F)), low-gillraker (≤44) morph and a large (≥300 mm L(F) ), high-gillraker (≥45) morph] from Great Slave Lake, NT, Canada, were found to be synonymous with cisco Coregonus artedi. Geometric body shape did not differ between the two size classes nor could they be differentiated by 24 size-corrected linear measurements, indicating that the two groups had similar phenotypes. Strong, positive correlations between all linear characters and geometric centroid size (a composite variable of fish body length, mass and age) suggested that body morphology changed with age as fish grew. Total gillraker number (N(GR)) increased with L(F) according to: N(GR) = 36.3 + 0.034L(F). Differences in gillraker number and phenotype with age and size were explained by shifts in habitat and trophic resource use. Relative abundance within 0-30, 30-60, 60-90 and >90 m depth strata differed between size classes suggesting that morphology changed when fish shifted their habitat as they grew older. Large C. artedi had lower δ(13)C and slightly higher δ(15)N, indicating greater reliance on pelagic prey resources (i.e. more or larger zooplankton, such as Mysis spp.), compared to small C. artedi, which relied slightly more on benthic prey. Gillraker shape and number have always been used as key diagnostic characters in coregonine taxonomy; based on the findings presented here, ontogenetic shifts should be accounted for in resulting classifications.

Adaptive plasticity and genetic divergence in feeding efficiency during parallel adaptive radiation of whitefish (Coregonus spp.).

Parallel phenotypic divergence in replicated adaptive radiations could either result from parallel genetic divergence in response to similar divergent selection regimes or from equivalent phenotypically plastic response to the repeated occurrence of contrasting environments. In post-glacial fish, replicated divergence in phenotypes along the benthic-limnetic habitat axis is commonly observed. Here, we use two benthic-limnetic species pairs of whitefish from two Swiss lakes, raised in a common garden design, with reciprocal food treatments in one species pair, to experimentally measure whether feeding efficiency on benthic prey has a genetic basis or whether it underlies phenotypic plasticity (or both). To do so, we offered experimental fish mosquito larvae, partially burried in sand, and measured multiple feeding efficiency variables. Our results reveal both, genetic divergence as well as phenotypically plastic divergence in feeding efficiency, with the phenotypically benthic species raised on benthic food being the most efficient forager on benthic prey. This indicates that both, divergent natural selection on genetically heritable traits and adaptive phenotypic plasticity, are likely important mechanisms driving phenotypic divergence in adaptive radiation.

Coexistence and origin of trophic ecotypes of pygmy whitefish, Prosopium coulterii, in a south-western Alaskan lake.

Ecologically, morphologically and genetically distinct populations within single taxa often coexist in postglacial lakes and have provided important model systems with which to investigate ecological and evolutionary processes such as niche partitioning and ecological speciation. Within the Salmonidae, these species complexes have been well studied, particularly within the Coregonus clupeaformis-C. laveratus (lake and European whitefish, respectively) group, but the phenomenon has been less well documented in the other whitefish genera, Prosopium and Stenodus. Here, we examined the morphology, feeding biology and genetic structure of three putative forms of the pygmy whitefish, Prosopium coulterii (Eigenmann & Eigenmann, 1892), first reported from Chignik Lake, south-western Alaska, over 40 years ago. Field collections and morphological analyses resolved a shallow water (< 5 m depth) low gill raker count form (< 15 first arch gill rakers), a deepwater (> 30 m), low gill raker form and a deepwater, high gill raker count (> 15 gill rakers) form. The two low gill raker count forms fed almost exclusively on benthic invertebrates (mostly chironomids), while the deepwater, high gill raker count form fed almost exclusively on zooplankton; differences in diet were also reflected in differences both in δ(13) C and δ(15) N stable isotopes. All three forms were characterized by the same major mitochondrial DNA clade that has been associated with persistence in, and postglacial dispersal from, a Beringian glacial refugium. Analysis of variation at nine microsatellite DNA loci indicated low, but significant differentiation among forms, especially between the two low gill raker count forms and the high gill raker count form. The extent of differentiation along phenotypic (considerable) and genetic (subtle) axes among the Chignik Lake forms is similar to that found among distinct taxa of Prosopium found in pre-glacial Bear Lake (Utah-Idaho, USA) which is probably at least ten times older than Chignik Lake. Our analyses illustrate the potential for the postglacial differentiation in traits subject to divergent natural selection across variable environments.

Species introduction promotes hybridization and introgression in Coregonus: is there sign of selection against hybrids?

Species introductions are considered one of the major drivers of biodiversity loss via ecological interactions and genetic admixture with local fauna. We examined two well-recognized fish species, native whitefish (Coregonus lavaretus) and introduced vendace (Coregonus albula), as well as their morphological hybrids in a single lake to test for selection against hybrids and backcrosses in the wild. A representative random subsample of 693 individuals (27.8%) was taken from the total catch of coregonids. This subsample was examined with the aim to select c. 50 individuals of pure whitefish (n = 52), pure vendace (n = 55) and putative hybrid (n = 19) for genetic analyses. The subsequent microsatellites and mitochondrial (mt) DNA analyses provided compelling evidence of hybridization and introgression. Of the 126 fish examined, four were found to be F(1) , 14 backcrosses to whitefish and seven backcrosses to vendace. The estimates of historical gene flow suggested higher rates from introduced vendace into native whitefish than vice versa, whereas estimates of contemporary gene flow were equal. Mitochondrial introgression was skewed, with 18 backcrosses having vendace mtDNA and only three with whitefish mtDNA. Hybrids and backcrosses had intermediate morphology and niche utilization compared with parental species. No evidence of selection against hybrids or backcrosses was apparent, as both hybrid and backcross growth rates and fecundities were high. Hybrids (F(1) ) were only detected in 2 year-classes, suggesting temporal variability in mating between vendace and whitefish. However, our data show that hybrids reached sexual maturity and reproduced actively, with backcrosses recorded from six consecutive year-classes, whereas no F(2) individuals were found. The results indicate widespread introgression, as 10.8% of coregonids were estimated to be backcrosses.

Metabolic responses to prolonged starvation, food restriction, and refeeding in the brown trout, Salmo trutta: oxidative stress and antioxidant defenses.

The effects of long-term starvation and food restriction (49 days), followed by refeeding (21 days) have been studied with respect to antioxidant defense in the liver and gills (branchial tissues) of the brown trout, Salmo trutta. Malondialdehyde levels in both tissues increased in parallel with starvation and food restriction and these values did not return to normal after the refeeding period. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in liver and gills increased during the 49 days of starvation, but glucose-6-phosphate dehydrogenase (G6PD) activities decreased. Glutathione S-transferase (GST) activity decreased in the liver at the 49th day of starvation, but increased in the branchial tissues. Some of the antioxidant enzyme activities (such as hepatic GST and branchial G6PD) returned to control values of fed fish after the refeeding period, but others (e.g. hepatic SOD and branchial GPx) did not return to normal values. In conclusion, our study indicates that total or partial food deprivation induces oxidative stress in brown trout.