The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

A fatty acyl desaturase (fads2) with dual Δ6 and Δ5 activities from the freshwater carnivorous striped snakehead Channa striata.

There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.

Echium oil increased the expression of a Δ4 Fads2 fatty acyl desaturase and the deposition of n-3 long-chain polyunsaturated fatty acid in comparison with linseed oil in striped snakehead (Channa striata) muscle.

Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species.

Impact of the citizen science project COLLECT on ocean literacy and well-being within a north/west African and south-east Asian context.

Plastic pollution is both a societal and environmental problem and citizen science has shown to be a useful tool to engage both the public and professionals in addressing it. However, knowledge on the educational and behavioral impacts of citizen science projects focusing on marine litter remains limited. Our preregistered study investigates the impact of the citizen science project Citizen Observation of Local Litter in coastal ECosysTems (COLLECT) on the participants' ocean literacy, pro-environmental intentions and attitudes, well-being, and nature connectedness, using a pretest-posttest design. A total of 410 secondary school students from seven countries, in Africa (Benin, Cabo Verde, Côte d'Ivoire, Ghana, Morocco, Nigeria) and Asia (Malaysia) were trained to sample plastics on sandy beaches and to analyze their collection in the classroom. Non-parametric statistical tests (n = 239 matched participants) demonstrate that the COLLECT project positively impacted ocean literacy (i.e., awareness and knowledge of marine litter, self-reported litter-reducing behaviors, attitudes towards beach litter removal). The COLLECT project also led to higher pro-environmental behavioral intentions for students in Benin and Ghana (implying a positive spillover effect) and higher well-being and nature connectedness for students in Benin. Results are interpreted in consideration of a high baseline in awareness and attitudes towards marine litter, a low internal consistency of pro-environmental attitudes, the cultural context of the participating countries, and the unique settings of the project's implementation. Our study highlights the benefits and challenges of understanding how citizen science impacts the perceptions and behaviors towards marine litter in youth from the respective regions.

Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases.

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.

Molecular cloning and ontogenic mRNA expression of fatty acid desaturase in the carnivorous striped snakehead fish (Channa striata).

There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.

Molecular cloning, phylogenetic analysis and functional characterisation of an Elovl7-like elongase from a marine crustacean, the orange mud crab (Scylla olivacea).

The capacity of crustaceans to biosynthesise long-chain polyunsaturated fatty acids has yet to be fully defined, due to the lack of evidence on the functional activities of enzymes involved in desaturation or elongation of fatty acid substrates. We report here the cloning and in vitro functional analysis of an elongase from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis placed the elovl close to the vertebrate Elovl1 and Elovl7 clade, which is distinct from the other remaining five Elovl families. The elongase was also clustered together with several elongases from crustaceans and insects. This elongase showed activities towards 16:1n-7, and at lower rate, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). To our knowledge this is the first description of a functional enzyme involved in biosynthesis of long-chained polyunsaturated fatty acids in a crustacean species. Expression of the S. olivacea elovl7-like mRNA was prominent in stomach, intestine and gill tissues, due to the need to regulate the permeability of epithelial tissue through modification of fatty acid compositions. The implication of our findings, in terms of ability of Crustacea phylum to biosynthesise polyunsaturated fatty acids is discussed.

Upregulated mRNA expression of desaturase and elongase, two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish.

BACKGROUND: Although unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed. METHODS: mRNA levels of fadsd6 and elovl5 in different ovarian follicle stages were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. For analysis of the ovarian follicular fatty acid composition, gas chromatography was used. RESULTS: Our results have shown that desaturase displayed significant upregulation in expression during the oocyte maturation stage. Expression of elongase was significantly highest in pre-vitellogenic follicles, followed by maturation stage. Fatty acid composition analysis of different ovarian follicle stages also showed that ARA level was significantly highest in pre-vitellogenic and matured follicles. DHA level was highest in both late vitellogenic and maturation stage. CONCLUSION: Collectively, our findings seem to suggest the existence of a HUFA synthesis system, which could be responsible for the synthesis of HUFA to promote oocyte maturation and possibly ovulation processes. The many advantages of zebrafish as model system to understand folliculogenesis will be useful platform to further elucidate the regulatory and mechanism aspects of ovarian HUFA synthesis.

Muscle Fatty Acid Content in Selected Freshwater Fish from Bukit Merah Reservoir, Perak, Malaysia.

One of the oldest reservoirs in Peninsular Malaysia, Bukit Merah Reservoir, is a place in which locals participate in fishing activities. Inland fisheries are important to individuals, society and the environment; whereby they generate a source of income and food security. It is essential to gauge the nutrition value of fish caught in this location as food source, especially in terms of fatty acid composition, to better demonstrate its potential towards the betterment of human health and general well-being. From an initial list of 47 fish species available in Bukit Merah Reservoir, a total of seven edible freshwater fish species were identified, namely tinfoil barb (Barbonymus schwanenfeldii), Javanese barb (Barbonymus gonionotus), hampala barb (Hampala macrolepidota), beardless barb (Cyclocheilichthys apogon), glassfish (Oxygaster anomalura), striped snakehead (Channa striata) and horseface loach (Acantopsis dialuzona), and muscle fatty acid content was analysed to determine their nutritional value. Muscle of cyprinid fish contained substantial amount of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) compared to fish from Channidae and Cobitidae families. Javanese and tinfoil barbs muscle recorded the highest levels of combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) indicating the highest nutritional value comparatively. Unexpectedly, the striped snakehead, a predatory carnivore, contained lower levels of LC-PUFA compared to detrivorous/herbivorous fishes. This further justifies that the omega-3 polyunsaturated fatty acids (PUFA) content in fish muscle varies according to their feeding habits. Even though it has been recommended that marine fish be consumed to improve health to a certain extent, there still are benefits of consuming freshwater fish, as there are several species which contain considerable amounts of beneficial omega-3 PUFA.

Identification of polyunsaturated fatty acid and diterpenoid biosynthesis pathways from draft genome of Aureispira sp. CCB-QB1.

The genus Aureispira consisting of two species, Aureispira marina and Aureispira maritima is an arachidonic acid-producing bacterium and produces secondary metabolites. In this study, we isolated a new Aureispira strain, Aureispira sp. CCB-QB1 from coastal area of Penang, Malaysia and the genome sequence of this strain was determined. The draft genome of this strain is composed of 185 contigs for 7,370,077 bases with 35.6% G+C content and contains 5911 protein-coding genes and 76 RNA genes. Linoleoyl-CoA desaturase, the key gene in arachidonic acid biosynthesis, is present in the genome. It was found that this strain uses mevalonate pathway for the synthesis of geranylgeranyl diphosphate (GGPP), which is precursor of diterpenoid, and novel pathway via futalosine for the synthesis of menaquinones. This is the first draft genome sequence of a member of the genus Aureispira.

Prolactin receptor mRNA is upregulated in discus fish (Symphysodon aequifasciata) skin during parental phase.

Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.