Gene co-expression patterns in Atlantic salmon adipose tissue provide a molecular link among seasonal changes, energy balance and age at maturity.

Sexual maturation in many fishes requires a major physiological change that involves a rapid transition between energy storage and usage. In Atlantic salmon, this transition for the initiation of maturation is tightly controlled by seasonality and requires a high-energy status. Lipid metabolism is at the heart of this transition since lipids are the main energy storing molecules. The balance between lipogenesis (lipid accumulation) and lipolysis (lipid use) determines energy status transitions. A genomic region containing a transcription co-factor of the Hippo pathway, vgll3, is the main determinant of maturation timing in Atlantic salmon. Interestingly, vgll3 acts as an inhibitor of adipogenesis in mice and its genotypes are potentially associated with seasonal heterochrony in lipid storage and usage in juvenile Atlantic salmon. Here, we explored changes in expression of more than 300 genes directly involved in the processes of adipogenesis, lipogenesis and lipolysis, as well as the Hippo pathway in the adipose tissue of immature and mature Atlantic salmon with distinct vgll3 genotypes. We found molecular evidence consistent with a scenario in which immature males with different vgll3 genotypes exhibit contrasting seasonal dynamics in their lipid profiles. We also identified components of the Hippo signalling pathway as potential major drivers of vgll3 genotype-specific differences in adipose tissue gene expression. This study demonstrates the importance of adipose gene expression patterns for directly linking environmental changes with energy balance and age at maturity through genetic factors bridging lipid metabolism, seasonality and sexual maturation.

Sex-specific overdominance at the maturation vgll3 gene for reproductive fitness in wild Atlantic salmon.

Linking reproductive fitness with adaptive traits at the genomic level can shed light on the mechanisms that produce and maintain sex-specific selection. Here, we construct a multigenerational pedigree to investigate sex-specific selection on a maturation gene, vgll3, in a wild Atlantic salmon population. The vgll3 locus is responsible for ~40% of the variation in maturation (sea age at first reproduction). Genetic parentage analysis was conducted on 18,265 juveniles (parr) and 685 adults collected at the same spawning ground over eight consecutive years. A high proportion of females (26%) were iteroparous and reproduced two to four times in their lifetime. A smaller proportion of males (9%) spawned at least twice in their lifetime. Sex-specific patterns of reproductive fitness were related to vgll3 genotype. Females showed a pattern of overdominance where vgll3*EL genotypes had three-fold more total offspring than homozygous females. In contrast, males demonstrated that late-maturing vgll3*LL individuals had two-fold more offspring than either vgll3*EE or vgll3*EL males. Taken together, these data suggest that balancing selection in females contributes to the maintenance of variation at this locus via increased fitness of iteroparous vgll3*EL females. This study demonstrates the utility of multigenerational pedigrees for uncovering complex patterns of reproduction, sex-specific selection and the maintenance of genetic variation.

Genotype-specific variation in seasonal body condition at a large-effect maturation locus.

Organisms use resource allocation strategies to survive seasonal environmental changes and life-history stage transitions. Earlier studies found a transcription cofactor, vgll3, associating with maturation timing that inhibits adipogenesis in mice and affects body condition in juvenile salmon. Owing to a lack of temporal studies examining seasonality effects on phenotypes such as vgll3 genotype, body condition, maturation and different life stages, we investigated the influence of different larval and juvenile temperatures, vgll3 genotype and interactions with body condition and maturation rate. We reared Atlantic salmon for 2 years in four larval-juvenile phase temperature groups until the occurrence of mature males. We found no effect of larval temperature on the measured phenotypes or maturation rate. However, we observed an increased maturation rate in individuals of the warm juvenile temperature treatment and differences in body condition associated with vgll3 genotype. Early maturation genotype individuals had a less variable body condition across seasons compared with late maturation genotype individuals. This result suggests a vgll3 influence on resource allocation strategies; possibly linked with the early maturation process, with early maturation genotype individuals having a higher maturation rate and a higher body condition in the spring.

Sulphonamide inhibition studies of the ß-carbonic anhydrase GsaCAß present in the salmon platyhelminth parasite Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Monogenean platyhelminth Gyrodactylus salaris, a fish parasite, GsaCAß, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAß inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9-139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAß inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with KIs in the range of 16.9-24.8 µM. Although no potent GsaCAß-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the inhibition profile of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.

A pituitary gene network linking vgll3 to regulators of sexual maturation in male Atlantic salmon.

Age at maturity is a key life history trait and a significant contributor to life history strategy variation. The maturation process is influenced by genetic and environmental factors, but specific causes of variation in maturation timing remain elusive. In many species, the increase in the regulatory gonadotropin-releasing hormone 1 (GnRH1) marks the onset of puberty. Atlantic salmon, however, lacks the gnrh1 gene, suggesting gnrh3 and/or other regulatory factors are involved in the maturation process. Earlier research in Atlantic salmon has found a strong association between alternative alleles of vgll3 and maturation timing. Recently we reported strong induction of gonadotropin genes (fshb and lhb) in the pituitary of Atlantic salmon homozygous for the early maturation allele (E) of vgll3. The induction of gonadotropins was accompanied by increased expression of their direct upstream regulators, c-jun and sf1 (nr5a1b) but the regulatory connection between vgll3 and these regulators has never been investigated in any organism. In this study, we investigated the potential regulatory connection between vgll3 genotypes and these regulators through a stepwise approach of identifying a gene regulatory network (GRN) containing c-jun and sf1, and transcription factor motif enrichment analysis. We found a GRN containing c-jun with predicted upstream regulators, e2f1, egr1, foxj1 and klf4, to be differentially expressed in the pituitary. Finally, we suggest a vgll3 and Hippo pathway -dependent model for transcriptional regulation of c-jun and sf1 in the pituitary, which may have broader implications across vertebrates.

The effect of temperature and dietary energy content on female maturation and egg nutritional content in Atlantic salmon.

The environment experienced by a female influences reproductive traits in many species of fish. Environmental factors such as temperature and diet are not only important mediators of female maturation and reproduction but also of egg traits and offspring fitness through maternal provisioning. In this study, we use 3-year-old tank-reared Atlantic salmon from two Finnish populations to investigate the effect of temperature and diet on maturation and egg traits. We show that a temperature difference of 2°C is sufficient to delay maturation in female Atlantic salmon whereas a 22% reduction in dietary energy content had no effect on maturation. Diet did not influence the body size, condition or fecundity of the mature females or the size or protein content of the eggs. However, a higher energy diet increased egg lipid content. Neither female body size nor condition were associated with egg size or fat/protein composition. Our results indicate that female salmon that have a poorer diet in terms of energy content may have a reproductive disadvantage due to the lower energy provisioning of eggs. This disadvantage has the potential to translate into fitness consequences for their offspring.

Genetic coupling of life-history and aerobic performance in Atlantic salmon.

A better understanding of the genetic and phenotypic architecture underlying life-history variation is a longstanding aim in biology. Theories suggest energy metabolism determines life-history variation by modulating resource acquisition and allocation trade-offs, but the genetic underpinnings of the relationship and its dependence on ecological conditions have rarely been demonstrated. The strong genetic determination of age-at-maturity by two unlinked genomic regions (vgll3 and six6) makes Atlantic salmon (Salmo salar) an ideal model to address these questions. Using more than 250 juveniles in common garden conditions, we quantified the covariation between metabolic phenotypes-standard and maximum metabolic rates (SMR and MMR), and aerobic scope (AS)-and the life-history genomic regions, and tested if food availability modulates the relationships. We found that the early maturation genotype in vgll3 was associated with higher MMR and consequently AS. Additionally, MMR exhibited physiological epistasis; it was decreased when late maturation genotypes co-occurred in both genomic regions. Contrary to our expectation, the life-history genotypes had no effects on SMR. Furthermore, food availability had no effect on the genetic covariation, suggesting a lack of genotype-by-environment interactions. Our results provide insights on the key organismal processes that link energy use at the juvenile stage to age-at-maturity, indicating potential mechanisms by which metabolism and life-history can coevolve.

Life-history genotype explains variation in migration activity in Atlantic salmon (Salmo salar).

One of the most well-known life-history continuums is the fast-slow axis, where 'fast' individuals mature earlier than 'slow' individuals. 'Fast' individuals are predicted to be more active than 'slow' individuals because high activity is required to maintain a fast life-history strategy. Recent meta-analyses revealed mixed evidence for such integration. Here, we test whether known life-history genotypes differ in activity expression by using Atlantic salmon (Salmo salar) as a model. In salmon, variation in Vgll3, a transcription cofactor, explains approximately 40% of variation in maturation timing. We predicted that the allele related to early maturation (vgll3*E) would be associated with higher activity. We used an automated surveillance system to follow approximately 1900 juveniles including both migrants and non-migrants (i.e. smolt and parr fish, respectively) in semi-natural conditions over 31 days (approx. 580 000 activity measurements). In migrants, but not in non-migrants, vgll3 explained variation in activity according to our prediction in a sex-dependent manner. Specifically, in females the vgll3*E allele was related to increasing activity, whereas in males the vgll3*L allele (later maturation allele) was related to increasing activity. These sex-dependent effects might be a mechanism maintaining within-population genetic life-history variation.

Refining the genomic location of single nucleotide polymorphism variation affecting Atlantic salmon maturation timing at a key large-effect locus.

Efforts to understand the genetic underpinnings of phenotypic variation are becoming more and more frequent in molecular ecology. Such efforts often lead to the identification of candidate regions showing signals of association and/or selection. These regions may contain multiple genes and therefore validation of which genes are actually responsible for the signal is required. In Atlantic salmon (Salmo salar), a large-effect locus for maturation timing, an ecologically important trait, occurs in a genomic region including two genes, vgll3 and akap11, but data for clearly determining which of the genes (or both) contribute to the association have been lacking. Here, we take advantage of natural recombination events detected between the two candidate genes in a salmon broodstock to reduce linkage disequilibrium at the locus, thus enabling delineation of the influence of variation at these two genes on early maturation. By rearing 5,895 males to maturation age, of which 81% had recombinant vgll3/akap11 allelic combinations, we found that vgll3 single nucleotide polymorphism (SNP) variation was strongly associated with early maturation, whereas there was little or no association between akap11 SNP variation and early maturation. These findings provide strong evidence supporting vgll3 as the primary candidate gene in the chromosome 25 locus for influencing early maturation. This will help guide future research for understanding the genetic processes controlling early maturation. This also exemplifies the utility of natural recombinants to more precisely map causal variation underlying ecologically important phenotypic diversity.

Dissecting the loci underlying maturation timing in Atlantic salmon using haplotype and multi-SNP based association methods.

Characterizing the role of different mutational effect sizes in the evolution of fitness-related traits has been a major goal in evolutionary biology for a century. Such characterization in a diversity of systems, both model and non-model, will help to understand the genetic processes underlying fitness variation. However, well-characterized genetic architectures of such traits in wild populations remain uncommon. In this study, we used haplotype-based and multi-SNP Bayesian association methods with sequencing data for 313 individuals from wild populations to test the mutational composition of known candidate regions for sea age at maturation in Atlantic salmon (Salmo salar). We detected an association at five loci out of 116 candidates previously identified in an aquaculture strain with maturation timing in wild Atlantic salmon. We found that at four of these five loci, variation explained by the locus was predominantly driven by a single SNP suggesting the genetic architecture of this trait includes multiple loci with simple, non-clustered alleles and a locus with potentially more complex alleles. This highlights the diversity of genetic architectures that can exist for fitness-related traits. Furthermore, this study provides a useful multi-SNP framework for future work using sequencing data to characterize genetic variation underlying phenotypes in wild populations.

Strong regulatory effects of vgll3 genotype on reproductive axis gene expression in juvenile male Atlantic salmon.

Age at maturity is a major contributor to the diversity of life history strategies in organisms. The process of maturation is influenced by both genetics and the environment, and includes changes in levels of sex hormones and behavior, but the specific factors leading to variation in maturation timing are poorly understood. gnrh1 regulates the transcription of gonadotropin genes at pubertal onset in many species, but this gene is lacking in certain teleost species including Atlantic salmon (Salmo salar), which raises the possibility of the involvement of other important regulatory factors during this process. Earlier research has reported a strong association of alternative alleles of the vgll3 gene with maturation timing in Atlantic salmon, suggesting it as a potential candidate regulating reproductive axis genes. Here, we investigated the expression of reproductive axis genes in one-year-old Atlantic salmon males with immature gonads and different vgll3 genotypes during the spawning period. We detected strong vgll3 genotype-dependent differential expression of reproductive axis genes (such as fshb, lhb, amh and igf3) tested in the pituitary, and testis. In addition, we observed differential expression of jun (ap1) and nr5a1b (sf1), potential upstream regulators of gonadotropins in the pituitary, as well as axin2, id3, insl3, itch, ptgs2a and ptger4b, the downstream targets of amh and igf3 in the testis. Hereby, we provide evidence of strong vgll3 genotype-dependent transcriptional regulation of reproductive axis genes prior to sexual maturation and suggest alternative models for distinct actions of vgll3 genotypes on the related molecular processes.

Cloning, purification, kinetic and anion inhibition studies of a recombinant ß-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAß has a significant catalytic activity for the physiological reaction, CO2 + H2O ⇋ HCO3- + H+ with a kcat of 1.1 × 105 s-1 and a kcat/Km of 7.58 × 106 M-1 × s-1. This activity was inhibited by acetazolamide (KI of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAß at millimolar concentrations, but sulfamide (KI of 81 µM), N,N-diethyldithiocarbamate (KI of 67 µM) and sulphamic acid (KI of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAß is subsequently proposed as a new drug target for which effective inhibitors can be designed.

Linking vgll3 genotype and aggressive behaviour in juvenile Atlantic salmon (Salmo salar).

We tested the possibility that vgll3, a gene linked with maturation age in Atlantic salmon (Salmo salar), may be associated with behaviour by measuring aggressiveness and feeding activity in 380 juveniles with different vgll3 genotypes. Contrary to our prediction, individuals with the genotype associated with later maturation (vgll3*LL) were significantly more aggressive than individuals with the genotype associated with earlier maturation (vgll3*EE). Individuals with higher aggression were also significantly lighter in colour and had higher feeding activity. Although higher aggression was associated with higher feeding activity, there was no association between feeding activity and vgll3 genotype. Increased aggression of vgll3*LL individuals was independent of their sex and size, and genotypes did not differ in their condition factor. These results imply that aggressive behaviour may have an energetic cost impairing growth and condition, especially when food cannot be monopolized. This may have implications for individual fitness and aquaculture practices.

Major population splits coincide with episodes of rapid climate change in a forest-dependent bird.

Climate change influences population demography by altering patterns of gene flow and reproductive isolation. Direct mutation rates offer the possibility for accurate dating on the within-species level but are currently only available for a handful of vertebrate species. Here, we use the first directly estimated mutation rate in birds to study the evolutionary history of pied flycatchers (Ficedula hypoleuca). Using a combination of demographic inference and species distribution modelling, we show that all major population splits in this forest-dependent system occurred during periods of increased climate instability and rapid global temperature change. We show that the divergent Spanish subspecies originated during the Eemian-Weichselian transition 115-104 thousand years ago (kya), and not during the last glacial maximum (26.5-19 kya), as previously suggested. The magnitude and rates of climate change during the glacial-interglacial transitions that preceded population splits in pied flycatchers were similar to, or exceeded, those predicted to occur in the course of the current, human-induced climate crisis. As such, our results provide a timely reminder of the strong impact that episodes of climate instability and rapid temperature changes can have on species' evolutionary trajectories, with important implications for the natural world in the Anthropocene.

Heterogeneous genetic basis of age at maturity in salmonid fishes.

Understanding the genetic basis of repeated evolution of the same phenotype across taxa is a fundamental aim in evolutionary biology and has applications in conservation and management. However, the extent to which interspecific life-history trait polymorphisms share evolutionary pathways remains underexplored. Here, we address this gap by studying the genetic basis of a key life-history trait, age at maturity, in four species of Pacific salmonids (genus Oncorhynchus) that exhibit intra- and interspecific variation in this trait-Chinook Salmon, Coho Salmon, Sockeye Salmon, and Steelhead Trout. We tested for associations in all four species between age at maturity and two genome regions, six6 and vgll3, that are strongly associated with the same trait in Atlantic Salmon (Salmo salar). We also conducted a genome-wide association analysis in Steelhead to assess whether additional regions were associated with this trait. We found the genetic basis of age at maturity to be heterogeneous across salmonid species. Significant associations between six6 and age at maturity were observed in two of the four species, Sockeye and Steelhead, with the association in Steelhead being particularly strong in both sexes (p = 4.46 × 10-9 after adjusting for genomic inflation). However, no significant associations were detected between age at maturity and the vgll3 genome region in any of the species, despite its strong association with the same trait in Atlantic Salmon. We discuss possible explanations for the heterogeneous nature of the genetic architecture of this key life-history trait, as well as the implications of our findings for conservation and management.

Polygenic and major-locus contributions to sexual maturation timing in Atlantic salmon.

Sexual maturation timing is a life-history trait central to the balance between mortality and reproduction. Maturation may be triggered when an underlying compound trait, called liability, exceeds a threshold. In many different species and especially fishes, this liability is approximated by growth and body condition. However, environmental vs. genetic contributions either directly or via growth and body condition to maturation timing remain unclear. Uncertainty exists also because the maturation process can reverse this causality and itself affect growth and body condition. In addition, disentangling the contributions of polygenic and major loci can be important. In many fishes, males mature before females, enabling the study of associations between male maturation and maturation-unbiased female liability traits. Using 40 Atlantic salmon families, longitudinal common-garden experimentation, and quantitative genetic analyses, we disentangled environmental from polygenic and major locus (vgll3) effects on male maturation, and sex-specific growth and condition. We detected polygenic heritabilities for maturation, growth, and body condition, and vgll3 effects on maturation and body condition but not on growth. Longitudinal patterns for sex-specific phenotypic liability, and for genetic variances and correlations between sexes suggested that early growth and condition indeed positively affected maturation initiation. However, towards spawning time, causality appeared reversed for males whereby maturation affected growth negatively and condition positively via both the environmental and genetic effects. Altogether, the results indicate that growth and condition are useful traits to study liability for maturation initiation, but only until maturation alters their expression, and that vgll3 contributes to maturation initiation via condition.

Population genomics reveals repeated signals of adaptive divergence in the Atlantic salmon of north-eastern Europe.

Our ability to examine genetic variation across entire genomes has enabled many studies searching for the genetic basis of local adaptation. These studies have identified numerous loci as candidates for differential local selection; however, relatively few have examined the overlap among candidate loci identified from independent studies of the same species in different geographic areas or evolutionary lineages. We used an allelotyping approach with a 220K SNP array to characterize the population genetic structure of Atlantic salmon in north-eastern Europe and ask whether the same genomic segments emerged as outliers among populations in different geographic regions. Genome-wide data recapitulated the phylogeographic structure previously inferred from mtDNA and microsatellite markers. Independent analyses of three genetically and geographically distinct groups of populations repeatedly inferred the same 17 haploblocks to contain loci under differential local selection. The most strongly supported of these replicated haploblocks had known strong associations with life-history variation or immune response in Atlantic salmon. Our results are consistent with these genomic segments harbouring large-effect loci which have a major role in Atlantic salmon diversification and are ideal targets for validation studies.

Sex-dependent dominance at a single locus maintains variation in age at maturity in salmon.

Males and females share many traits that have a common genetic basis; however, selection on these traits often differs between the sexes, leading to sexual conflict. Under such sexual antagonism, theory predicts the evolution of genetic architectures that resolve this sexual conflict. Yet, despite intense theoretical and empirical interest, the specific loci underlying sexually antagonistic phenotypes have rarely been identified, limiting our understanding of how sexual conflict impacts genome evolution and the maintenance of genetic diversity. Here we identify a large effect locus controlling age at maturity in Atlantic salmon (Salmo salar), an important fitness trait in which selection favours earlier maturation in males than females, and show it is a clear example of sex-dependent dominance that reduces intralocus sexual conflict and maintains adaptive variation in wild populations. Using high-density single nucleotide polymorphism data across 57 wild populations and whole genome re-sequencing, we find that the vestigial-like family member 3 gene (VGLL3) exhibits sex-dependent dominance in salmon, promoting earlier and later maturation in males and females, respectively. VGLL3, an adiposity regulator associated with size and age at maturity in humans, explained 39% of phenotypic variation, an unexpectedly large proportion for what is usually considered a highly polygenic trait. Such large effects are predicted under balancing selection from either sexually antagonistic or spatially varying selection. Our results provide the first empirical example of dominance reversal allowing greater optimization of phenotypes within each sex, contributing to the resolution of sexual conflict in a major and widespread evolutionary trade-off between age and size at maturity. They also provide key empirical evidence for how variation in reproductive strategies can be maintained over large geographical scales. We anticipate these findings will have a substantial impact on population management in a range of harvested species where trends towards earlier maturation have been observed.

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

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

Captive-bred Atlantic salmon released into the wild have fewer offspring than wild-bred fish and decrease population productivity.

The release of captive-bred animals into the wild is commonly practised to restore or supplement wild populations but comes with a suite of ecological and genetic consequences. Vast numbers of hatchery-reared fish are released annually, ostensibly to restore/enhance wild populations or provide greater angling returns. While previous studies have shown that captive-bred fish perform poorly in the wild relative to wild-bred conspecifics, few have measured individual lifetime reproductive success (LRS) and how this affects population productivity. Here, we analyse data on Atlantic salmon from an intensely studied catchment into which varying numbers of captive-bred fish have escaped/been released and potentially bred over several decades. Using a molecular pedigree, we demonstrate that, on average, the LRS of captive-bred individuals was only 36% that of wild-bred individuals. A significant LRS difference remained after excluding individuals that left no surviving offspring, some of which might have simply failed to spawn, consistent with transgenerational effects on offspring survival. The annual productivity of the mixed population (wild-bred plus captive-bred) was lower in years where captive-bred fish comprised a greater fraction of potential spawners. These results bolster previous empirical and theoretical findings that intentional stocking, or non-intentional escapees, threaten, rather than enhance, recipient natural populations.