Functional validation of transposable element-derived cis-regulatory elements in Atlantic salmon.

Transposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole-genome duplications (WGDs), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ∼100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. Here, we evaluated the gene-regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-CREs upstream of transcriptional start sites increased expression significantly. Our results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

Efficient transfection of Atlantic salmon primary hepatocyte cells for functional assays and gene editing.

The expansion of genomic resources for Atlantic salmon over the past half decade has enabled efficient interrogation of genetic traits by large-scale correlation of genotype to phenotype. Moving from correlation to causation will require genotype-phenotype relationships to be tested experimentally in a cost-efficient and cell context-relevant manner. To enable such future experiments, we have developed a method for the isolation and genetic manipulation of primary hepatocytes from Atlantic salmon for use in heterologous expression, reporter assay, and gene editing experiments. We chose the liver as the tissue of interest because it is the metabolic hub and many current Atlantic salmon research projects focus on understanding metabolic processes to improve traits such as the growth rate, total fat content, and omega-3 content. We find that isolated primary hepatocytes are optimally transfected with both plasmid and ribonucleoprotein using a Neon electroporator at 1,400 V, 10 ms, and 2 pulses. Transfection efficiency with plasmid and cutting efficiency with ribonucleoprotein were optimally 46% and 60%, respectively. We also demonstrate a 26 times increase in luciferase expression under the promoter of the key liver metabolic gene, elovl5b, compared to an empty vector, in line with expected liver-specific expression. Taken together, this work provides a valuable resource enabling transfection and gene editing experiments in a context-relevant and cost-effective system.

Molecular Regulation of Biosynthesis of Long Chain Polyunsaturated Fatty Acids in Atlantic Salmon.

Salmon is a rich source of health-promoting omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The LC-PUFA biosynthetic pathway in Atlantic salmon is one of the most studied compared to other teleosts. This has largely been due to the massive replacement of LC-PUFA-rich ingredients in aquafeeds with terrestrial plant oils devoid of these essential fatty acids (EFA) which ultimately pushed dietary content towards the minimal requirement of EFA. The practice would also reduce tissue content of n-3 LC-PUFA compromising the nutritional value of salmon to the human consumer. These necessitated detailed studies of endogenous biosynthetic capability as a contributor to these EFA. This review seeks to provide a comprehensive and concise overview of the current knowledge about the molecular genetics of PUFA biosynthesis in Atlantic salmon, highlighting the enzymology and nutritional regulation as well as transcriptional control networks. Furthermore, we discuss the impact of genome duplication on the complexity of salmon LC-PUFA pathway and highlight probable implications on endogenous biosynthetic capabilities. Finally, we have also compiled and made available a large RNAseq dataset from 316 salmon liver samples together with an R-script visualization resource to aid in explorative and hypothesis-driven research into salmon lipid metabolism.

Targeted mutagenesis of ∆5 and ∆6 fatty acyl desaturases induce dysregulation of lipid metabolism in Atlantic salmon (Salmo salar).

BACKGROUND: With declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1) Δ6abc/5Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c genes. Our purpose is to evaluate the genetic effect fads2 mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period. RESULTS: Both Δ6abc/5Mt and Δ6bcMt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. The Δ6abc/5Mt fish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acid de-novo synthesis, fatty acid ß-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bcMt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability. CONCLUSIONS: CRISPR-Cas9 can efficiently mutate multiple fads2 genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

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.

CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes.

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.

Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles.

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

Insecticide susceptibility of natural populations of Anopheles coluzzii and Anopheles gambiae (sensu stricto) from Okyereko irrigation site, Ghana, West Africa.

BACKGROUND: The increasing spread of insecticide resistance in malaria vectors has been well documented across sub-Saharan Africa countries. The influence of irrigation on increasing vector resistance is poorly understood, and is critical to successful and ethical implementation of food security policies. This study investigated the insecticide resistance status of An. gambiae (s.l.) mosquitoes collected from the irrigated rice area of Okyereko, a village containing about 42 hectares of irrigated field within an irrigation project plan in the Central Region of Ghana. Large amounts of insecticides, herbicides and fertilizers are commonly used in the area to boost the annual production of the rice. METHODS: Mosquito larvae were collected and adults were assayed from the F1 progeny. The resistance status, allele and genotype were characterized using WHO susceptibility testing and PCR methods respectively. RESULTS: The An. gambiae (s.l.) populations from Okyereko are highly resistant to DDT and pyrethroid insecticides, with possible involvement of metabolic mechanisms including the elevation of P450 and GST enzyme as well as P-gp activity. The population was mostly composed of An. coluzzii specimens (more than 96 %) with kdr and ace-1 frequencies of 0.9 and 0.2 %, respectively. CONCLUSION: This study brings additional information on insecticide resistance and the characterization of An. gambiae (s.l.) mosquitoes from Okyereko, which can be helpful in decision making for vector control programmes in the region.