Chromosome aberrations in pressure-induced triploid Atlantic salmon.

BACKGROUND: Triploid organisms have three sets of chromosomes. In Atlantic salmon, hydrostatic pressure treatment of newly fertilized eggs has been extensively used to produce triploids which are functionally sterile due to their unpaired chromosomes. These fish often perform poorly on commercial farms, sometimes without explanation. Inheritance patterns in individuals subjected to pressure treatment have not been investigated in Atlantic salmon thus far. However, work on other species suggests that this treatment can result in aberrant inheritance. We therefore studied this in Atlantic salmon by genotyping 16 polymorphic microsatellites in eyed eggs and juveniles which had been subjected to pressure-induction of triploidy. Communally reared juveniles including fish subjected to pressure-induction of triploidy and their diploid siblings were included as a control. RESULTS: No diploid offspring were detected in any of the eggs or juveniles which were subjected to hydrostatic pressure; therefore, the induction of triploidy was highly successful. Aberrant inheritance was nevertheless observed in 0.9% of the eggs and 0.9% of the juveniles that had been subjected to pressure treatment. In the communally reared fish, 0.3% of the fish subjected to pressure treatment displayed aberrant inheritance, while their diploid controls displayed 0% aberrant inheritance. Inheritance errors included two eyed eggs lacking maternal DNA across all microsatellites, and, examples in both eggs and juveniles of either the maternal or paternal allele lacking in one of the microsatellites. All individuals displaying chromosome aberrations were otherwise triploid. CONCLUSIONS: This is the first study to document aberrant inheritance in Atlantic salmon that have been subjected to pressure-induction of triploidy. Our experiments unequivocally demonstrate that even when induction of triploidy is highly successful, this treatment can cause chromosome aberrations in this species. Based upon our novel data, and earlier studies in other organisms, we hypothesize that in batches of Atlantic salmon where low to modest triploid induction rates have been reported, aberrant inheritance is likely to be higher than the rates observed here. Therefore, we tentatively suggest that this could contribute to the unexplained poor performance of triploid salmon that is occasionally reported in commercial aquaculture. These hypotheses require further investigation.

Implications for introgression: has selection for fast growth altered the size threshold for precocious male maturation in domesticated Atlantic salmon?

BACKGROUND: Mature male parr (MMP) represent an important alternative life-history strategy in Atlantic salmon populations. Previous studies indicate that the maturation size threshold for male parr varies among wild populations and is influenced by individual growth, environmental conditions, and genetics. More than ten generations of breeding have resulted in domesticated salmon displaying many genetic differences to wild salmon, including greatly increased growth rates. This may have resulted in domesticated fish with the potential to outgrow the size threshold for early maturation, or evolution of the size threshold of the trait itself. To investigate this, we performed a common-garden experiment under farming conditions using 4680 salmon from 39 families representing four wild, two wild-domesticated hybrid, and two domesticated strains. RESULTS: Domesticated salmon outgrew wild salmon 2-5-fold, and hybrids displayed intermediate growth. Overall, the numbers of MMP varied greatly among families and strains: averaging 4-12% in domesticated, 18-25% in hybrid, and 43-74% in the wild populations. However, when the influence of growth was accounted for, by dividing fish into lower and upper size modes, no difference in the incidence of MMP was detected among domesticated and wild strains in either size mode. In the lower size mode, hybrids displayed significantly lower incidences of mature males than their wild parental strains. No consistent differences in the body size of MMP, connected to domestication, was detected. CONCLUSIONS: Our data demonstrate: 1- no evidence for the evolution of the size threshold for MMP in domesticated salmon, 2- the vastly lower incidence of MMP in domesticated strains under aquaculture conditions is primarily due to their genetically increased growth rate causing them to outgrow the size threshold for early maturation, 3- the incidence of MMP is likely to overlap among domesticated and wild salmon in the natural habitat where they typically display overlapping growth, although hybrid offspring may display lower incidences of mature male parr. These results have implications for wild salmon populations that are exposed to introgression from domesticated escapees.

Ploidy elicits a whole-genome dosage effect: growth of triploid Atlantic salmon is linked to the genetic origin of the second maternal chromosome set.

BACKGROUND: The Atlantic salmon aquaculture industry is investigating the feasibility of using sterile triploids to mitigate genetic interactions with wild conspecifics, however, studies investigating diploid and triploid performance often show contrasting results. Studies have identified dosage and dosage-compensation effects for gene expression between triploid and diploid salmonids, but no study has investigated how ploidy and parent-origin effects interact on a polygenic trait in divergent lines of Atlantic salmon (i.e. slow growing wild versus fast growing domesticated phenotype). This study utilised two experiments relating to the freshwater growth of diploid and triploid groups of pure wild (0% domesticated genome), pure domesticated (100% domesticated genome), and F1 reciprocal hybrid (33%, 50% or 66% domesticated genome) salmon where triploidy was either artificially induced (experiment 1) or naturally developed/spontaneous (experiment 2). RESULTS: In both experiments, reciprocal hybrid growth was influenced by the dosage effect of the second maternal chromosome, with growth increasing as ploidy level increased in individuals with a domesticated dam (from 50% to 66% domesticated genome), and the inverse in individuals with a wild dam (from 50% to 33% domesticated genome). CONCLUSIONS: We demonstrate that the combined effect of ploidy and parent-origin on growth, a polygenic trait, is regulated in an additive pattern. Therefore, in order to maximise growth potential, the aquaculture industry should consider placing more emphasis on the breeding value of the dam than the sire when producing triploid families for commercial production.

Plasticity in response to feed availability: Does feeding regime influence the relative growth performance of domesticated, wild and hybrid Atlantic salmon Salmo salar parr?

Growth of farmed, wild and F1 hybrid Atlantic salmon parr Salmo salar was investigated under three contrasting feeding regimes in order to understand how varying levels of food availability affects relative growth. Treatments consisted of standard hatchery feeding (ad libitum), access to feed for 4 h every day, and access to feed for 24 h on three alternate days weekly. Mortality was low in all treatments, and food availability had no effect on survival of all groups. The offspring of farmed S. salar significantly outgrew the wild S. salar, while hybrids displayed intermediate growth. Furthermore, the relative growth differences between the farmed and wild S. salar did not change across feeding treatments, indicating a similar plasticity in response to feed availability. Although undertaken in a hatchery setting, these results suggest that food availability may not be the sole driver behind the observed reduced growth differences found between farmed and wild fishes under natural conditions.

Identification of quantitative genetic components of fitness variation in farmed, hybrid and native salmon in the wild.

Feral animals represent an important problem in many ecosystems due to interbreeding with wild conspecifics. Hybrid offspring from wild and domestic parents are often less adapted to local environment and ultimately, can reduce the fitness of the native population. This problem is an important concern in Norway, where each year, hundreds of thousands of farm Atlantic salmon escape from fish farms. Feral fish outnumber wild populations, leading to a possible loss of local adaptive genetic variation and erosion of genetic structure in wild populations. Studying the genetic factors underlying relative performance between wild and domesticated conspecific can help to better understand how domestication modifies the genetic background of populations, and how it may alter their ability to adapt to the natural environment. Here, based upon a large-scale release of wild, farm and wild x farm salmon crosses into a natural river system, a genome-wide quantitative trait locus (QTL) scan was performed on the offspring of 50 full-sib families, for traits related to fitness (length, weight, condition factor and survival). Six QTLs were detected as significant contributors to the phenotypic variation of the first three traits, explaining collectively between 9.8 and 14.8% of the phenotypic variation. The seventh QTL had a significant contribution to the variation in survival, and is regarded as a key factor to understand the fitness variability observed among salmon in the river. Interestingly, strong allelic correlation within one of the QTL regions in farmed salmon might reflect a recent selective sweep due to artificial selection.

Potential disease interaction reinforced: double-virus-infected escaped farmed Atlantic salmon, Salmo salar L., recaptured in a nearby river.

The role of escaped farmed salmon in spreading infectious agents from aquaculture to wild salmonid populations is largely unknown. This is a case study of potential disease interaction between escaped farmed and wild fish populations. In summer 2012, significant numbers of farmed Atlantic salmon were captured in the Hardangerfjord and in a local river. Genetic analyses of 59 of the escaped salmon and samples collected from six local salmon farms pointed out the most likely source farm, but two other farms had an overlapping genetic profile. The escapees were also analysed for three viruses that are prevalent in fish farming in Norway. Almost all the escaped salmon were infected with salmon alphavirus (SAV) and piscine reovirus (PRV). To use the infection profile to assist genetic methods in identifying the likely farm of origin, samples from the farms were also tested for these viruses. However, in the current case, all the three farms had an infection profile that was similar to that of the escapees. We have shown that double-virus-infected escaped salmon ascend a river close to the likely source farms, reinforcing the potential for spread of viruses to wild salmonids.

Genetic diversity within and among Atlantic cod (Gadus morhua) farmed in marine cages: a proof-of-concept study for the identification of escapees.

This study presents a molecular genetic characterization of Atlantic cod reared in commercial marine farms. Samples consisted of approximately 47 fish collected from nine cages located on four farms throughout Norway. In addition, 28 farmed escapees were recaptured in the sea (443 fish in total). Nine microsatellite loci and the Pan I gene were analysed, revealing a total of 181 alleles. Each sample contained 43-63% of total allelic variation. Comparing variation with published data for wild cod indicates that lower genetic variation exists within single cages than in wild populations. Significant linkage disequilibrium was observed amongst pairs of loci in all samples, suggesting a low number of contributing parental fish. Global F(ST) was 0.049, and the highest pairwise F(ST) value (pooled loci) was 0.085. For single loci, the Pan I gene was the most diagnostic, displaying a global F(ST) of 0.203. Simulations amongst the samples collected on farms revealed an overall correct self-assignment percentage of 75%, demonstrating a high probability of identifying individuals to their farm of origin. Identification of the 28 escapees revealed a single cage as the most likely source of origin for half of the escapees, whilst the remaining fish were assigned to a mixture of samples, suggesting more than one source of escapees.

Inferring Atlantic salmon post-smolt migration patterns using genetic assignment.

Understanding migratory patterns is important for predicting and mitigating unwanted consequences of environmental change or anthropogenic challenges on vulnerable species. Wild Atlantic salmon undergo challenging migrations between freshwater and marine environments, and the numbers of salmon returning to their natal rivers to reproduce have declined over several decades. Mortality from sea lice linked to fish farms within their seaward migration routes is proposed as a contributing factor to these declines. Here, we used 31 microsatellite markers to establish a genetic baseline for the main rivers in the Hardangerfjord, western Norway. Mixed stock analysis was used to assign Atlantic salmon post-smolts caught in trawls in 2013-2017 back to regional reporting units. Analyses demonstrated that individuals originating from rivers located in the inner region of the fjord arrived at the outer fjord later than individuals from middle and outer fjord rivers. Therefore, as post-smolts originating from inner rivers also have to migrate longer distances to exit the fjord, these data suggest that inner fjord populations are more likely to be at risk of mortality through aquaculture-produced sea lice, and other natural factors such as predation, than middle or outer fjord populations with earlier exit times and shorter journeys. These results will be used to calibrate models estimating mortality from sea lice on wild salmon for the regulation of the Norwegian aquaculture industry.

Major histocompatibility complex (MHC) variation and susceptibility to the sea louse Lepeophtheirus salmonis in Atlantic salmon Salmo salar.

The relationship between genetic variation in major histocompatibility complex (MHC) Class I and II genes and susceptibility to sea lice Lepeophtheirus salmonis (Krøyer) in Atlantic salmon Salmo salar (L.) was studied in cage-reared post smolts. Polymorphic repeat markers located in the 3' untranslated regions (3UTR) of the genes Sasa-UBA (MHC Class I) and Sasa-DAA (MHC Class II) were screened in 1004 fish sampled from 11 full-sibling families. This gave rise to a total of 7 and 5 alleles, and 17 and 13 genotypes respectively. Significant relationships between both Sasa-UBA-3UTR and Sasa-DAA-3UTR genotypes and abundance of lice were observed within the pooled material, within individual families, and within the pooled material with both markers combined. However, most of these associations were either weak, linked with variation in fish size among genotypes, or influenced by family background genome. Nevertheless, within one family, the Sasa-DAA-3UTR 248/278 genotype displayed a significantly higher (33%) abundance of lice compared with the Sasa-DAA-3UTR 208/258 genotype, and this difference was not influenced by fish size. Consequently, the results of this study indicate a link between MHC Class II and susceptibility to lice.

Cyclotheonamide derivatives: synthesis and thrombin inhibition. Exploration of specific structure-function issues.

Macrocyclic pentapeptide analogues (5-9) of the sponge natural product cyclotheonamide A (CtA, 3) were prepared by means of our convergent [3 + 2] synthetic protocol, in which a late-stage primary amine group is available for substitution (Maryanoff et al. Proc. Natl Acad. Sci. U.S.A. 1993, 90, 8048). These analogues, as well as CtA and cyclotheonamide B (CtB, 4), were examined for their ability to inhibit the serine protease alpha-thrombin, in comparison with suitable reference standards. We characterized Michaelis-Menten and slow-binding kinetics for the cyclotheonamide derivatives. An attempt was made to utilize the unoccupied hydrophobic S3 subsite of thrombin via analogues 5 and 6. Also, removal of the hydroxyphenyl group, which is thought to be involved in an aromatic stacking interaction with Trp60D of thrombin, was explored via analogue 9. The importance of the alpha-keto and olefin groups was examined via 7 and 8, respectively. The relationship of structure and function with the analogues proved to be less predictable than anticipated.

Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.