Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.     

Seasonal changes in hypoosmoregulatory ability in landlocked and anadromous populations of Arctic charr,Salvelinus alpinus,and Atlantic salmon,Salmo salar

Synopsis Seasonal changes in hypoosmoregulatory ability were compared in landlocked and anadromous strains of Arctic charr and Atlantic salmon. Seawater adaptability was assessed using periodic 48 h seawater challenge tests with 25. seawater. The landlocked strains of Arctic charr, two from northern Sweden and one from Southern Norway, displayed similar seasonal changes in seawater adaptability as the anadromous strain. Seawater tolerance increased during spring and remained high until the end of July — early August after which it declined. The two strains of Atlantic salmon displayed different seasonal patterns in hypoosmoregulatory ability. The anadromous strain showed a pronounced seasonal pattern with maximal seawater adaptability in early June. In contrast, seawater tolerance in the landlocked strain improved steadily during spring and remained high until late autumn. During the period of enhanced seawater tolerance, hypoosmoregulatory ability increased significantly with body size in both Arctic charr and anadromous Atlantic salmon. The minimum size at which fish were able to regulate plasma sodium following seawater transfer at a level comparable to freshwater levels (<170 mmol I^–1) differed significantly between anadromous Atlantic salmon (ca. 14 cm) and Arctic charr (ca. 22 cm). The results show that seasonal changes in hypoosmoregulatory ability are present in both Atlantic salmon and Arctic charr, and that these physiological traits are retained in the corresponding landlocked strains. However, the seasonal pattern of seawater adaptability as well as the minimum size at which seawater tolerance occurs differs between the two species.     

Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications

Although confined to fresh water, non‐parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from anadromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non‐parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non‐parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.     

Small Changes in Gene Expression of Targeted Osmoregulatory Genes When Exposing Marine and Freshwater Threespine Stickleback (Gasterosteus aculeatus) to Abrupt Salinity Transfers

Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na⁺/K⁺-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.     

SIGN EPISTASIS LIMITS EVOLUTIONARY TRADE‐OFFS AT THE CONFLUENCE OF SINGLE‐ AND MULTI‐CARBON METABOLISM IN METHYLOBACTERIUM EXTORQUENS AM1

Adaptation of one set of traits is often accompanied by attenuation of traits important in other selective environments, leading to fitness trade‐offs. The mechanisms that either promote or prevent the emergence of trade‐offs remain largely unknown, and are difficult to discern in most systems. Here, we investigate the basis of trade‐offs that emerged during experimental evolution of Methylobacterium extorquens AM1 to distinct growth substrates. After 1500 generations of adaptation to a multi‐carbon substrate, succinate (S), many lineages had lost the ability to use one‐carbon compounds such as methanol (M), generating a mixture of M⁺ and M^? evolved phenotypes. We show that trade‐offs in M^? strains consistently arise via antagonistic pleiotropy through recurrent selection for loss‐of‐function mutations to ftfL (formate‐tetrahydrofolate ligase), which improved growth on S while simultaneously eliminating growth on M. But if loss of FtfL was beneficial, why were M trade‐offs not found in all populations? We discovered that eliminating FtfL was not universally beneficial on S, as it was neutral or even deleterious in certain evolved lineages that remained M⁺. This suggests that sign epistasis with earlier arising mutations prevented the emergence of mutations that drove trade‐offs through antagonistic pleiotropy, limiting the evolution of metabolic specialists in some populations.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

Independent evolutionary origins of landlocked alewife populations and rapid parallel evolution of phenotypic traits

Alewife, Alosa pseudoharengus, populations occur in two discrete life-history variants, an anadromous form and a landlocked (freshwater resident) form. Landlocked populations display a consistent pattern of life-history divergence from anadromous populations, including earlier age at maturity, smaller adult body size, and reduced fecundity. In Connecticut (USA), dams constructed on coastal streams separate anadromous spawning runs from lake-resident landlocked populations. Here, we used sequence data from the mtDNA control region and allele frequency data from five microsatellite loci to ask whether coastal Connecticut landlocked alewife populations are independently evolved from anadromous populations or whether they share a common freshwater ancestor. We then used microsatellite data to estimate the timing of the divergence between anadromous and landlocked populations. Finally, we examined anadromous and landlocked populations for divergence in foraging morphology and used divergence time estimates to calculate the rate of evolution for foraging traits. Our results indicate that landlocked populations have evolved multiple times independently. Tests of population divergence and estimates of gene flow show that landlocked populations are genetically isolated, whereas anadromous populations exchange genes. These results support a 'phylogenetic raceme' model of landlocked alewife divergence, with anadromous populations forming an ancestral core from which landlocked populations independently diverged. Divergence time estimates suggest that landlocked populations diverged from a common anadromous ancestor no longer than 5000 years ago and perhaps as recently as 300 years ago, depending on the microsatellite mutation rate assumed. Examination of foraging traits reveals landlocked populations to have significantly narrower gapes and smaller gill raker spacings than anadromous populations, suggesting that they are adapted to foraging on smaller prey items. Estimates of evolutionary rates (in haldanes) indicate rapid evolution of foraging traits, possibly in response to changes in available resources.     

QTL mapping of freezing tolerance: links to fitness and adaptive trade‐offs

Local adaptation, defined as higher fitness of local vs. nonlocal genotypes, is commonly identified in reciprocal transplant experiments. Reciprocally adapted populations display fitness trade‐offs across environments, but little is known about the traits and genes underlying fitness trade‐offs in reciprocally adapted populations. We investigated the genetic basis and adaptive significance of freezing tolerance using locally adapted populations of Arabidopsis thaliana from Italy and Sweden. Previous reciprocal transplant studies of these populations indicated that subfreezing temperature is a major selective agent in Sweden. We used quantitative trait locus (QTL) mapping to identify the contribution of freezing tolerance to previously demonstrated local adaptation and genetic trade‐offs. First, we compared the genomic locations of freezing tolerance QTL to those for previously published QTL for survival in Sweden, and overall fitness in the field. Then, we estimated the contributions to survival and fitness across both field sites of genotypes at locally adaptive freezing tolerance QTL. In growth chamber studies, we found seven QTL for freezing tolerance, and the Swedish genotype increased freezing tolerance for five of these QTL. Three of these colocalized with locally adaptive survival QTL in Sweden and with trade‐off QTL for overall fitness. Two freezing tolerance QTL contribute to genetic trade‐offs across environments for both survival and overall fitness. A major regulator of freezing tolerance, CBF2, is implicated as a candidate gene for one of the trade‐off freezing tolerance QTL. Our study provides some of the first evidence of a trait and gene that mediate a fitness trade‐off in nature.     

Recent parallel divergence in body shape and diet source of alewife life history forms

Recent work suggests that juvenile alewives (Alosa pseudoharengus) share similar phenotypes among independently derived landlocked (freshwater resident) populations. Based on this observation, it is possible that the alewife life history forms represent a case of parallel adaptive divergence. To further evaluate this hypothesis, we describe patterns of body shape divergence between anadromous and landlocked alewife life history forms using geometric morphometrics. Our results suggest that body shape differs significantly between juveniles of the alewife forms: anadromous fish were more robust, with larger heads and deeper caudal peduncles, while landlocked fish from three independently isolated populations were more fusiform with thinner caudal peduncles and smaller heads. These differences matched population level dietary patterns, which suggest that anadromous fish consumed more littoral resources than landlocked fish. Finding consistent differences across populations of the same form supports the notion that landlocked alewives have diverged from their anadromous ancestors in a parallel manner, in response to pressures associated with being isolated in freshwater lakes. Comparing alewife phenotypes to expectations from the literature suggests that neither migration distance of the population, nor the relative availability of habitats in each lake, are likely drivers of the pattern we report. Instead, the pattern is consistent with the hypothesis that divergence between alewife forms results from the distinct effects of each form on its zooplankton prey.     

Salinity effects on osmoregulation and growth of the euryhaline flounder Paralichthys orbignyanus

The flounder, Paralichthys orbignyanus, is found in coastal and estuarine waters of the Western South Atlantic Ocean. It is being considered for aquaculture due to its high market price and wide tolerance to environmental factors such as salinity, pH, and nitrogenous compounds. The objective of this study was to characterize the ionic and osmotic regulation of P. orbignyanus over the range of its tolerated ambient salinities (0-40‰) and to evaluate the survival and growth in freshwater (0‰) and seawater (30‰) over 90 days. After 15 days of exposure to different salinities (0‰, 10‰, 20‰, 30‰ and 40‰), plasma osmolality and ionic (Na⁺, Cl⁻, K⁺ and Ca²⁺) concentrations slightly increased with salinity. The isosmotic point was estimated as 328.6 mOsm kg⁻¹ H₂O and corresponded to 10.9‰ salinity. After 90 days, survival was similar in freshwater and seawater, but osmo- and ionoregulation was significantly affected in freshwater and flounders reared in this medium showed a lower growth rate than those reared in seawater. Based on the results from this study, P. orbignyanus can be characterized as a marine/estuarine euryhaline teleost capable of hyper/hypo iono- and osmoregulation over the fluctuating salinity regime faced by this species in the environment. Furthermore, results suggest that the lower growth rate exhibited by P. orbignyanus in freshwater could be due, at least partially, to a higher energy expenditure associated to a higher branchial Na⁺, K⁺-ATPase activity in this environment.     

Early life stage salinity tolerance of wild and hatchery-reared juvenile pink salmon Oncorhynchus gorbuscha

Salinity tolerance in wild (Glendale) and hatchery (Quinsam) pink salmon Oncorhynchus gorbuscha (average mass 0·2 g) was assessed by measuring whole body [Na⁺] and [Cl^?] after 24 or 72 h exposures to fresh water (FW) and 33, 66 or 100% sea water (SW). Gill Na⁺, K⁺‐ATPase activity was measured following exposure to FW and 100% SW and increased significantly in both populations after a 24 h exposure to 100% SW. Whole body [Na⁺] and whole body [Cl^?] increased significantly in both populations after 24 h in 33, 66 and 100% SW, where whole body [Cl^?] differed significantly between Quinsam and Glendale populations. Extending the seawater exposure to 72 h resulted in no further increases in whole body [Na⁺] and whole body [Cl^?] at any salinity, but there was more variability among the responses of the two populations. Per cent whole body water (c. 81%) was maintained in all groups of fish regardless of salinity exposure or population, indicating that the increase in whole body ion levels may have been related to maintaining water balance as no mortality was observed in this study. Thus, both wild and hatchery juvenile O. gorbuscha tolerated abrupt salinity changes, which triggered an increase in gill Na⁺, K⁺‐ATPase within 24 h. These results are discussed in terms of the preparedness of emerging O. gorbuscha for the marine phase of their life cycle.     

Expression of sodium/hydrogen exchanger 3 and cation-chloride cotransporters in the kidney of Japanese eel acclimated to a wide range of salinities

Reabsorption of monovalent ions in the kidney is essential for adaptation to freshwater and seawater in teleosts. To assess a possible role of Na⁺/H⁺ exchanger 3 (NHE3) in renal osmoregulation, we first identified a partial sequence of cDNA encoding NHE3 from the Japanese eel kidney. For comparison, we also identified cDNAs encoding kidney specific Na⁺–K⁺–2Cl^? cotransporter 2 (NKCC2α) and Na⁺–Cl^? cotransporter (NCCα). In eels acclimated to a wide range of salinities from deionized freshwater to full-strength seawater, the expression of NHE3 in the kidney was the highest in eel acclimated to full-strength seawater. Meanwhile, the NCCα expression exhibited a tendency to increase as the environmental salinity decreased, whereas the NKCC2α expression was not significantly different among the experimental groups. Immunohistochemical studies showed that NHE3 was localized to the apical membrane of epithelial cells composing the second segments of the proximal renal tubule in seawater-acclimated eel. Meanwhile, the apical membranes of epithelial cells in the distal renal tubule and collecting duct showed more intense immunoreactions of NKCC2α and NCCα, respectively, in freshwater eel than in seawater eel. These findings suggest that renal monovalent-ion reabsorption is mainly mediated by NKCC2α and NCCα in freshwater eel and by NHE3 in seawater eel.     

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.     

Ontogeny of salinity tolerance and evidence for seawater-entry preparation in juvenile green sturgeon, <Emphasis Type="Italic">Acipenser medirostris</Emphasis>

We measured the ontogeny of salinity tolerance and the preparatory hypo-osmoregulatory physiological changes for seawater entry in green sturgeon (Acipenser medirostris), an anadromous species occurring along the Pacific Coast of North America. Salinity tolerance was measured every 2 weeks starting in 40-day post-hatch (dph) juveniles and was repeated until 100% survival at 34‰ was achieved. Fish were subjected to step increases in salinity (5‰ 12 h⁻¹) that culminated in a 72-h exposure to a target salinity, and treatment groups (0, 15, 20, 25, 30, 34‰; and abrupt exposure to 34‰) were adjusted as fish developed. After 100% survival was achieved (134 dph), a second experiment tested two sizes of fish for 28-day seawater (33‰) tolerance, and gill and gastrointestinal tract tissues were sampled. Their salinity tolerance increased and plasma osmolality decreased with increasing size and age, and electron microscopy revealed three types of mitochondria-rich cells: one in fresh water and two in seawater. In addition, fish held on a natural photoperiod in fresh water at 19°C showed peaks in cortisol, thyroid hormones and gill and pyloric ceca Na⁺, K⁺-ATPase activities at body sizes associated with seawater tolerance. Therefore, salinity tolerance in green sturgeon increases during ontogeny (e.g., as these juveniles may move down estuaries to the ocean) with increases in body size. Also, physiological and morphological changes associated with seawater readiness increased in freshwater-reared juveniles and peaked at their seawater-tolerant ages and body sizes. Their seawater-ready body size also matched that described for swimming performance decreases, presumably associated with downstream movements. Therefore, juvenile green sturgeon develop structures and physiological changes appropriate for seawater entry while growing in fresh water, indicating that hypo-osmoregulatory changes may proceed by multiple routes in sturgeons.     

Contemporary trait change in a classic ecological experiment: rapid decrease in alewife gill‐raker spacing following introduction to an inland lake

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The effects of sustained aerobic swimming on osmoregulatory pathways in Atlantic salmon Salmo salar smolts

Atlantic salmon Salmo salar smolts were exposed to one of the four different aerobic exercise regimens for 10 weeks followed by a 1 week final smoltification period in fresh water and a subsequent eight‐day seawater transfer period. Samples of gill and intestinal tissue were taken at each time point and gene expression was used to assess the effects of exercise training on both branchial and intestinal osmoregulatory pathways. Real‐time polymerase chain reaction (PCR) analysis revealed that exercise training up‐regulated the expression of seawater relevant genes in the gills of S. salar smolts, including Na⁺, K⁺ ATPase (nka) subunit α1b, the Na⁺, K⁺, 2 Cl^? co‐transporter (nkcc1) and cftr channel. These findings suggest that aerobic exercise stimulates expression of seawater ion transport pathways that may act to shift the seawater transfer window for S. salar smolts. Aerobic exercise also appeared to stimulate freshwater ion uptake mechanisms probably associated with an osmorespiratory compromise related to increased exercise. No differences were observed in plasma Na⁺ and Cl^? concentrations as a consequence of exercise treatment, but plasma Na⁺ was lower during the final smoltification period in all treatments. No effects of exercise were observed for intestinal nkcc2, nor the Mg²⁺ transporters slc41a2 and transient receptor protein M7 (trpm7); however, expression of both Mg²⁺ transporters was affected by salinity transfer suggesting a dynamic role in Mg²⁺ homeostasis in fishes.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.