Biochemical and molecular studies of the polyunsaturated fatty acid desaturation pathway in fish

Fish have an absolute dietary requirement for certain polyunsaturated fatty acids (PUFA) termed “essential fatty acids” (EFA) that include members of both the n-6 and n-3 series typified by linoleic acid, 18:2n-6, and α-linolenic acid, 18:3n-3. However, the biologically active forms of EFA are generally the C20 and C22 metabolites of 18:2n-6 and 18:3n-3, viz. 20:4n-6, 20:5n-3 and 22:6n-3. Some fish species can convert C18 PUFA to the C20 and C22 PUFA through a series of alternating desaturation and chain elongation reactions mediated by microsomal systems containing elongases and Δ6 and Δ5 fatty acid desaturases. In species that cannot perform these conversions, the C20 and C22 PUFA themselves are dietary EFA and their C18 homologues do not satisfy EFA requirements. The extent to which the foregoing statements apply quantitatively to a given fish species varies widely. Therefore, a vital area in lipid nutrition in fish is the provision of sufficient amounts of the correct EFA to satisfy the requirements for normal growth and development, requirements that can vary quantitatively during the life of the fish and are particularly important factors in larval marine fish. This paper reviews the work on defining and characterising the fatty acid desaturation and elongation pathway in fish. Biochemical studies have been advanced by the use of cell cultures which have elucidated key parts of the pathway. Thus, the presence of the so-called Sprecher shunt, where 22:6n-3 is produced from 20:5n-3 through two successive elongations and a Δ6 desaturase followed by peroxisomal chain shortening, was demonstrated in trout. Similarly, the block in the pathway in marine and/or piscivorous fish could be due to either a deficiency of C18-20 elongase or Δ5 desaturase and this varies between different marine species. Recent work has focussed on the molecular biology of the pathway with the cloning of fatty acid desaturases and elongases from a variety of fish species. Zebrafish have been used as a model species and a unique desaturase possessing both Δ6 and Δ5 activity along with an elongase with very high C18-20 activity have been cloned and characterised. Understanding this pathway is of increased importance due to the current dependence of salmonid and marine fish aquaculture on fish oil, the supply of which is becoming increasingly limited and unsustainable, necessitating the use in fish feeds of sustainable plant oils, rich in C18 PUFA, but devoid of C20 and C22 PUFA

Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the Genbank database identified several EST sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript, ZfELO, encoding a polypeptide of 291 amino acid residues was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame (ORF) shared high similarity with the elongases of C. elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish ORF conferred the ability to lengthen the chain of a range of C18, C20 and C22 polyunsaturated fatty acids, indicating that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is not only feasible, but that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-14C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexanoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar)

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFA) and, with declining fisheries, farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term, and will constrain continuing growth of aquaculture activities. A greater understanding of how fish metabolise and biosynthesise HUFA may lead to effective use of more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimising the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterisation of two genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase cDNA, SalDes, was isolated that included an open reading frame (ORF) of 1362 bp specifying a protein of 454 amino acids. The protein sequence included all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing a haem-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast, Saccharomyces cerevisiae, showed SalDes is predominantly an omega-3 Δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3n-3). The desaturase showed only low levels of Δ6 activity towards C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that includes an ORF of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo includes characteristic features of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast, SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C18 > C20 > C22. Thus, all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3 are displayed by this one polypeptide product

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and α-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18 > C20 >C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids

Performance Evaluation of Highly Admixed Tanzanian Smallholder Dairy Cattle Using SNP Derived Kinship Matrix

The main purpose of this study was to understand the type of dairy cattle that can be optimally used by smallholder farmers in various production environments such that they will maximize their yields without increasing the level of inputs. Anecdotal evidence and previous research suggests that the optimal level of taurine inheritance in crossbred animals lies between 50 and 75% when considering total productivity in tropical management clusters. We set out to assess the relationship between breed composition and productivity for various smallholder production systems in Tanzania. We surveyed 654 smallholder dairy households over a 1-year period and grouped them into production clusters. Based on supplementary feeding, milk productivity and sale as well as household wealth status four clusters were described: low-feed–low-output subsistence, medium-feed–low-output subsistence, maize germ intensive semi-commercial and feed intensive commercial management clusters. About 839 crossbred cows were genotyped at approximately 150,000 single nucleotide polymorphism (SNP) loci and their breed composition determined. Percentage dairyness (proportion of genes from international dairy breeds) was estimated through admixture analysis with Holstein, Friesian, Norwegian Red, Jersey, Guernsey, N’Dama, Gir, and Zebu as references. Four breed types were defined as RED–GUE (Norwegian Red/Friesian–Guernsey; Norwegian Red/Friesian–Jersey), RED–HOL (Norwegian Red/Friesian–Holstein), RED–Zebu (Norwegian Red/Friesian–Zebu), Zebu–RED (Zebu–Norwegian Red/Friesian) based on the combination of breeds that make up the top 76% breed composition. A fixed regression model using a genomic kinship matrix was used to analyze milk yield records. The fitted model accounted for year-month-test-date, parity, age, breed type and the production clusters as fixed effects in the model in addition to random effects of animal and permanent environment effect. Results suggested that RED–Zebu breed type with dairyness between 75 and 85% is the most appropriate for a majority of smallholder management clusters. Additionally, for farmers in the feed intensive management group, animals with a Holstein genetic background with at least 75% dairy composition were the best performing. These results indicate that matching breed type to production management group is central to maximizing productivity in smallholder systems. The findings from this study can serve as a basis to inform the development of the dairy sector in Tanzania and beyond

Validity of Verbal Autopsy Procedures for Determining Malaria Deaths in Different Epidemiological Settings in Uganda

BACKGROUND: Verbal autopsy (VA) procedures can be used to estimate cause of death in settings with inadequate vital registries. However, the sensitivity of VA for determining malaria-specific mortality may be low, and may vary with transmission intensity. We assessed the diagnostic accuracy of VA procedures as compared to hospital medical records for determining cause of death in children under five in three different malaria transmission settings in Uganda, including Tororo (high), Kampala (medium), and Kisoro (low). METHODS AND FINDINGS: Caretakers of children who died in participating hospitals were interviewed using a standardized World Health Organization questionnaire. Medical records from the child's hospitalization were also reviewed. Causes of death based on the VA questionnaires and the medical records were assigned independently by physician reviewers and then compared. A total of 719 cases were included in the final analysis, 67 in Tororo, 600 in Kampala, and 52 in Kisoro. Malaria was classified as the underlying or contributory cause of death by review of medical records in 33 deaths in Tororo, 60 in Kampala, and 0 in Kisoro. The sensitivity of VA procedures for determining malaria deaths in Tororo was 61% (95% CI 44-78%) and 50% in Kampala (95% CI 37-63%). Specificity for determining malaria deaths in Tororo and Kampala was high (>88%), but positive predictive value varied widely, from 83% in Tororo to 34% in Kampala (difference 49%, 95% CI 31-67, p<0.001). The difference between the cause-specific mortality fraction for malaria as determined by VA procedures and medical records was -11% in Tororo, +5% in Kampala, and +14% in Kisoro. CONCLUSIONS: Our results suggest that these VA methods have an acceptable level of diagnostic accuracy for determining malaria deaths at the population level in high and medium transmission areas, but not in low transmission areas

The genome landscape of indigenous African cattle

Background: The history of African indigenous cattle and their adaptation to environmental and human selection pressure is at the root of their remarkable diversity. Characterization of this diversity is an essential step towards understanding the genomic basis of productivity and adaptation to survival under African farming systems. Results: We analyze patterns of African cattle genetic variation by sequencing 48 genomes from five indigenous populations and comparing them to the genomes of 53 commercial taurine breeds. We find the highest genetic diversity among African zebu and sanga cattle. Our search for genomic regions under selection reveals signatures of selection for environmental adaptive traits. In particular, we identify signatures of selection including genes and/ or pathways controlling anemia and feeding behavior in the trypanotolerant N’Dama, coat color and horn development in Ankole, and heat tolerance and tick resistance across African cattle especially in zebu breeds. Conclusions: Our findings unravel at the genome-wide level, the unique adaptive diversity of African cattle while emphasizing the opportunities for sustainable improvement of livestock productivity on the continent

Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds

status: publishe