CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.)

The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5Mt with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50–100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5Mt showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bcMt showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5Mt and Δ6bcMt demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5Mt demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.publishedVersio

Cardiac dysfunction affects eye development and vision by reducing supply of lipids in fish

Developing organisms are especially vulnerable to environmental stressors. Crude oil exposure in early life stages of fish result in multiple functional and developmental defects, including cardiac dysfunction and abnormal and smaller eyes. Phenanthrene (Phe) has a reversible impact on cardiac function, and under exposure Phe reduces cardiac contractility. Exposure to a known L-type channel blocker, nicardipine hydrochloride (Nic) also disrupts cardiac function and creates eye deformities. We aimed to investigate whether cardiac dysfunction was the major underlying mechanism of crude oil-, Phe- and Nic-induced eye malformations. We exposed Atlantic haddock (Melanogrammus aeglefinus) early embryos to Nic and crude oil (Oil) and late embryos/early larvae to Phe exposure. All three exposures resulted in cardiac abnormalities and lead to severe, eye, jaw and spinal deformities at early larval stages. At 3 days post hatching, larvae from the exposures and corresponding controls were dissected. Eyes, trunk, head and yolk sac were subjected to lipid profiling, and eyes were also subjected to transcriptomic profiling. Among most enriched pathways in the eye transcriptomes were fatty acid metabolism, calcium signaling and phototransduction. Changes in lipid profiles and the transcriptome suggested that the dysfunctional and abnormal eyes in our exposures were due to both disruption of signaling pathways and insufficient supply of essential fatty acids and other nutrients form the yolk.publishedVersio

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors

Abstract Background New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. Results Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). Conclusions In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding

Tiger Daily: April 22, 2020

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Effect of diet on molecular relationships between Atlantic cod larval muscle growth dynamics, metabolism, and antioxidant defense system

We studied molecular effects (RNAseq and qPCR) of first feeding prey types (copepods or rotifers/Artemia) on skeletal muscle myogenesis and growth dynamics (proliferation, differentiation), metabolism (glycolysis, gluconeogenesis, oxidative phosphorylation), and antioxidant defense system (production/regulation of reactive oxygen species (ROS) in cod (Gadus morhua) larval skeletal muscle. Larval somatic growth rates were significantly higher in copepod fed larvae, although shifts in gene expressions related to muscle growth dynamics between hypertrophy and hyperplasia and generation and regulation of ROS mostly occurred around 5-, 10-, and 15-mm standard length (SL) for both groups. Gene expression for cell membrane proteins (such as nox1 and igf1r) peaked at 7 mm SL in all larvae, corresponding with increased ROS expressions in cod muscle during the exponential stratified hyperplasia phase from 7 mm SL. Expression for muscle differentiation (mef2a) occurred continuously (strongest from 10 mm SL). Expressions for muscle proliferation (pcna) and hydrogen peroxide (H2O2) generation (sod1 and sod2) occurred in the 5 - 15 mm SL range, peaking at 10 mm SL in all larvae. A downregulation of sod1 and sod2 in skeletal muscle from 15 mm SL indicated the first response of the defense antioxidant system. Gene expressions related to glucose metabolism (slc2A11, pfk, fpb2, ldha) was 3 - 10 times higher in copepod-fed larvae than in rotifer/Artemia-fed larvae between 7 – 10 mm (live prey period). Copepods move faster than rotifers, and cod larvae will also gradually increase their active swimming periods, due to less viscous forces. Active swimming during the strongest muscle stratified hyperplasia phase (7 – 10 mm SL) could promote a better delivery and transport across the muscle membrane and intracellular flux through glycolysis and oxidative phosphorylation and would contribute to the observed earlier and more effective glucose metabolism in the larvae fed copepods. We suggest that active swimming is an important factor promoting cod larval muscle growth, especially during the strongest muscle hyperplasia phase between 7 and 10 mm SL. The rapid movements of copepods and better nutritional composition could play important roles in stabilizing ROS levels, promoting high swimming activities and enhancing long-term muscle growth in cod.publishedVersio

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9- mediated HDR in Atlantic salmon

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.publishedVersio

Transcriptomic analysis of dead end knockout testis reveals germ cell and gonadal somatic factors in Atlantic salmon

Sustainability challenges are currently hampering an increase in salmon production. Using sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently sterile salmon was produced by knocking out the germ cell-specific dead end (dnd). Several approaches may be applied to inhibit Dnd function, including gene knockout, knockdown or immunization. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes essential for the survival of gonadal somatic cells may be good alternative targets for sterility treatments. Our aim was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon.publishedVersio

Microbial communities associated with the parasitic copepod Lepeophtheirus salmonis.

Lepeophtheirus salmonis is a naturally occurring marine parasite of salmonid fishes in the Northern hemisphere, and a major problem in salmonid aquaculture. In addition to the direct effects on host fish, L. salmonis may act as a vector for diseases. Here, the microbial community of L. salmonis recovered from whole genome shotgun sequencing was compared between lice sampled from both the Atlantic and the Pacific, laboratory-reared and wild lice, in addition to lice displaying resistance towards chemical treatments. The analysis shows clear differences in the metagenomic composition between the Atlantic and the Pacific Ocean, whereas the resistance status of the L. salmonis or the cultivation did not have significant impact.submittedVersio

Impact of Antioxidant Feed and Growth Manipulation on the Redox Regulation of Atlantic Salmon Smolts

Accumulating evidence indicates a close relationship between oxidative stress and growth rate in fish. However, the underlying mechanisms of this relationship remain unclear. This study evaluated the combined effect of dietary antioxidants and growth hormone (GH) on the liver and the muscle redox status of Atlantic salmon. There were two sequential experimental phases (EP) termed EP1 and EP2, each lasting for 6 weeks. In EP1, Atlantic salmon were fed either low-(L, 230 mg/kg ascorbic acid (Asc), 120 mg/kg α-tocopherol (α-TOH)), or high-(H, 380 mg/kg Asc, 210 mg/kg α-TOH)vitamin diets. The vitamins were supplemented as stable forms and the feeding was continued in EP2. In EP2, half of the fish were implanted with 3 μL per g body weight of recombinant bovine GH (Posilac®, 1 mg rbGH g BW−1) suspended in sesame oil, while the other half were held in different tanks and sham-implanted with similar volumes of the sesame oil vehicle. Here, we show that increasing high levels of vitamin C and E (diet H) increased their content in muscle and liver during EP1. GH implantation decreased vitamin C and E levels in both liver and muscle but increased malondialdehyde (MDA) levels only in the liver. GH also affected many genes and pathways of antioxidant enzymes and the redox balance. Among the most consistent were the upregulation of genes coding for the NADPH oxidase family (NOXs) and downregulation of the oxidative stress response transcription factor, nuclear factor-erythroid 2-related factor 2 (nrf2), and its downstream target genes in the liver. We verified that GH increases the growth rate until the end of the trail and induces an oxidative effect in the liver and muscle of Atlantic salmon. Dietary antioxidants do lower oxidative stress but have no effect on the growth rate. The present study is intended as a starting point to understand the potential interactions between growth and redox signaling in fish.publishedVersio

Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon

Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.publishedVersio