Influence of egg vitamin A status and egg incubation temperature on subsequent development of the early vertebral column in Atlantic salmon fry

The effect of egg vitamin A (VA) status and egg incubation temperature on the development of spinal disorders was investigated in Atlantic salmon Salmo salar fry. Atlantic salmon eggs were sorted into two groups with high VA (3·3 ± 0·1 μg retinol g⁻¹ dry mass) and low VA (2·2 ± 0·3 μg retinol g⁻¹ dry mass) status before fertilization and incubated at high (14° C) or low (8° C) temperature from 133 day degrees until the onset of feeding. High egg incubation temperatures increased the concentration of retinol in the eggs: the high VA and high temperature group displayed a significantly higher retinol concentration than the high VA and low temperature group ( P  = 0·001). After hatching, all experimental groups increased their retinol concentration. The source of the increased retinol levels was probably retinal, although astaxanthin may also be a VA precursor after hatching. Atlantic salmon fry incubated at high temperatures had increased amounts of notochord tissue. When measuring morphogenic activity in the notochord using the expression of sonic hedgehog ( shh , mRNA), however, no significant difference was found between the experimental groups. No clear effect of VA status or incubation temperature could be found on the formation of the early vertebral column although Atlantic salmon fry incubated at low temperatures had less regular constrictions of the prospective vertebral column than fry incubated at high temperatures.     

Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation

Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.     

Application of genome editing in aquatic farm animals: Atlantic salmon

Gene editing offers opportunities to solve fish farming sustainability issues that presently hampers expansion of the aquaculture industry. In for example Atlantic salmon farming, there are now two major bottlenecks limiting the expansion of the industry. One is the genetic impact of escaped farmed salmon on wild populations, which is considered the most long-term negative effect on the environment. Secondly and the utmost acute problem is the fish parasite salmon lice, which is currently causing high lethality in wild salmonids due to high concentrations of the parasite in the sea owing to sea cage salmon farming. There are also sustainability issues associated with increased use of vegetable-based ingredients as replacements for marine products in fish feed. This transition comes at the expense of the omega-3 content both in fish feed and the fish filet of the farmed fish. Reduced fish welfare represents another obstacle, and robust farmed fish is needed to avoid negative stress associated phenotypes such as cataract, bone and fin deformities, precocious maturity and higher disease susceptibility. Gene editing could solve some of these problems as genetic traits can be altered positively to reach phenotype of interest such as for example disease resistance and increased omega-3 production.

     

The vgll3 Locus Controls Age at Maturity in Wild and Domesticated Atlantic Salmon (Salmo salar L.) Males

Wild and domesticated Atlantic salmon males display large variation for sea age at sexual maturation, which varies between 1–5 years. Previous studies have uncovered a genetic predisposition for variation of age at maturity with moderate heritability, thus suggesting a polygenic or complex nature of this trait. The aim of this study was to identify associated genetic loci, genes and ultimately specific sequence variants conferring sea age at maturity in salmon. We performed a genome wide association study (GWAS) using a pool sequencing approach (20 individuals per river and phenotype) of male salmon returning to rivers as sexually mature either after one sea winter (2009) or three sea winters (2011) in six rivers in Norway. The study revealed one major selective sweep, which covered 76 significant SNPs in which 74 were found in a 370 kb region of chromosome 25. Genotyping other smolt year classes of wild and domesticated salmon confirmed this finding. Genotyping domesticated fish narrowed the haplotype region to four SNPs covering 2386 bp, containing the vgll3 gene, including two missense mutations explaining 33–36% phenotypic variation. A single locus was found to have a highly significant role in governing sea age at maturation in this species. The SNPs identified may be both used as markers to guide breeding for late maturity in salmon aquaculture and in monitoring programs of wild salmon. Interestingly, a SNP in proximity of the VGLL3 gene in humans (Homo sapiens), has previously been linked to age at puberty suggesting a conserved mechanism for timing of puberty in vertebrates.