Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus

Highlights

• fads and elovl are expressed from embryo, increasing from 15 days after hatching.

• fads and elovl expression is regulated by organogenesis and diet on larvae ontogeny.

• fads1, fads2 and elovl2 are mainly expressed in intestine, while elovl5 in liver.

• A. tropicus possess the enzymatic machinery to synthetize LC-PUFA from PUFA.

Abstract

Long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA), including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are essential in multiple physiological processes, especially during early development of vertebrates. LC-PUFA biosynthesis is achieved by two key families of enzymes, fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl). The present study determined the expression patterns of genes encoding desaturases (fads1 and fads2) and elongases (elovl2 and elovl5) involved in the LC-PUFA biosynthesis during early life-stages of the tropical gar Atractosteus tropicus. We further analyzed the fatty acid profiles during early development of A. tropicus to evaluate the impact of Fads and Elovl enzymatic activities. Specific oligonucleotides were designed from A. tropicus transcriptome to perform qPCR (quantitative polymerase chain reaction) on embryonic and larval stages, along with several organs (intestine, white muscle, brain, liver, heart, mesenteric adipose, kidney, gill, swim bladder, stomach, and spleen) collected from juvenile specimens. Fatty acid content of feeds and embryonic and larval stages were analyzed. Results show that fads1, fads2, elovl2 and elovl5 expression was detected from embryonic stages with expression peaks from day 15 post hatching, which could be related to transcriptional and dietary factors. Moreover, fads1, fads2 and elovl2 showed a higher expression in intestine, while elovl5 showed a higher expression in liver, suggesting that the tropical gar activates its LC-PUFA biosynthetic machinery to produce ARA, EPA and DHA to satisfy physiological demands at crucial developmental milestones during early development.

Introduction

C₁₈ polyunsaturated fatty acids (PUFA) like linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3) cannot be synthesized de novo by vertebrates, including fishes, and are thus regarded as dietary essential fatty acids (Monroig et al., 2018). C₁₈ PUFA are precursors of physiologically important long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA) including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) (Castro et al., 2016; Monroig et al., 2018). In vertebrates, LC-PUFA play a crucial role in the membrane's structure fluidity and signaling processes, which are relevant in many physiological functions such as growth, neurological functions, inflammation, immune response, and reproduction (Colombo et al., 2016; Ayisi et al., 2018).

Biosynthesis of LC-PUFA from C₁₈ PUFA is mostly performed by two key enzyme families, Fatty acyl desaturases (Fads) and Elongation of very long-chain fatty acids (Elovl). Fads introduce a new double bond (unsaturation) between the carboxyl terminus of a fatty acyl chain and a pre-existing double bond, whereas Elovl enzymes catalyze the initial, rate-limiting condensation reaction of fatty acyl chain elongation (Jakobsson et al., 2006; Guillou et al., 2010). In mammals, two Fads (Fads1 and Fads2) and three Elovl (Elovl2, Elovl4, and Elovl5) are the main enzymes involved in LC-PUFA biosynthesis (Guillou et al., 2010; Castro et al., 2016). The biosynthesis of ARA from LA and EPA from ALA, is achieved by two distinct pathways as follows: one is Δ6 desaturation (Fads2) – elongation (Elovl5) – Δ5 desaturation (Fads1) and another is elongation (Elovl5) – Δ8 desaturation (Fads2) – Δ5 desaturation (Fads1). Subsequently, DHA can be biosynthesized via the so-called “Sprecher pathway”, which comprises two consecutive elongation steps from EPA to produce tetracosapentaenoic acid (24:5n-3), followed by Δ6 desaturation (Fads2) and a chain shortening step (partial β-oxidation). An alternative pathway for DHA biosynthesis can be operated in some teleosts and consists of one single elongation from EPA to produce docosapentaenoic acid (22:5n-3), which is directly converted to DHA via the action of a Δ4 desaturase (Oboh et al., 2017).

In contrast to mammals, all Fads genes isolated from most Teleostei species, except for Elopomorpha species, are Fads2 orthologs, suggesting that all teleosts have lost Fads1 genes during evolution (Li et al., 2018; Lopes-Marques et al., 2018; Garrido et al., 2019), suggesting that their substrate specificity has diversified because of the specific evolutionary history of each species, and environmental factors including their habitat (marine vs freshwater) (Ishikawa et al., 2019), trophic level and ecology (Kabeya et al., 2017b, Li et al., 2018). Therefore, in teleost, alternative pathways for LC-PUFA biosynthesis has been characterized where the enzymes encoded by fads2 can recognize and desaturase different substrates than their non-teleost vertebrate counterparts, thus the Δ6Δ8 desaturase specific activity typically contained in vertebrate Fads2 (Castro et al., 2016; Monroig et al., 2018), where teleost Fads2 also shows Δ4 and Δ6Δ5 desaturases activities (Monroig et al., 2011; Fonseca-Madrigal et al., 2014; Morais et al., 2015; Kabeya et al., 2017; Oboh et al., 2017; Li et al., 2018; Lopes-Marques et al., 2018). Overall, functional diversification of Fads2 is a particularly common trait among teleost species inhabiting freshwater where it has been hypothesized that a deficient supply of LC-PUFA through the diet could partly compensate for the loss of fads1 (Kabeya et al., 2017a; Monroig et al., 2018). Through the functional diversification of the Fads2 family, many freshwater teleosts kept the fatty acid elongase elovl2, a gene absent in Neoteleostei genomes, a group including farmed marine finfish species. The elovl2 gene encodes an elongase that is pivotal for the conversion of 22:5n-3 into 24:5n-3 and hence, plays a key role in DHA biosynthesis via the Sprecher pathway (Sprecher, 2000). The Fads2 family in marine teleost is less diverse compared to their freshwater counterparts, also, marine teleosts have lost elovl2, this could help to explain why they require LC-PUFA in their diet to guarantee a normal growth and development, preventing deficiency symptoms (Monroig et al., 2018). This is typically achieved in aquaculture by formulating diets that include high inclusion levels of marine ingredients, particularly fish oil, increasing the production of aquaculture feeds. On the contrary, C₁₈ PUFA such as LA and ALA are provided by including vegetable oils in the diet, satisfying the essential fatty acid requirements for freshwater species since they have the enzymes to convert LA and ALA into LC-PUFA (e.g., Ferraz et al., 2019).

The tropical gar (Atractosteus tropicus) is a carnivorous freshwater species native to the southeast of Mexico and Central America that belongs to the Lepisosteidae family, classified within the Holostei group, which includes two genera, Lepisosteus with four species, and Atractosteus, with three species. From an evolutionary perspective, gars are paleontological records and a current relict of an ancient fish clade that diverged before the teleost-specific whole genome duplication (WGD or 3R) that occurred approximately 230–400 million year ago (mya) (Braasch et al., 2016; Venkatachalam et al., 2017), shows that gars and humans possess a fads1 encoding a desaturase with Δ5 preference, and fads2 encoding a desaturase with Δ6 preference as previously confirmed in L. oculatus (Lopes-Marques et al., 2018), where that species possesses Fads1 (Δ5) and Fads2 (Δ6Δ8) which are responsible for the LC-PUFA biosynthesis, demonstrating that the Lepisosteidae family conserves the desaturase machinery associated with tetrapods and that did not suffer the loss of Fads1 as found in more recently emerged fish lineages. While, as previous mentioned, teleosts only present Fads2 that shows mechanisms of bifunctionalization (Δ5Δ6). Currently wild populations of A. tropicus are being exploited although culture technology for A. tropicus developed in recent years has enabled a constant production that satisfies the demands of the regional market of southeastern Mexican states like Veracruz, Campeche, and Chiapas (Márquez-Couturier et al., 2006). Progresses have been made to understand important aspects of the A. tropicus nutrition and physiology, those include the characterization of digestive enzyme activities and the histological description of the digestive system through the species early development (Frías-Quintana et al., 2015), estimation of dietary lipid requirements (Huerta-Ortiz et al., 2018), the relative gene expression of fatty acid synthase (fas), acetyl-CoA carboxylase 1 (acc1) and carnitine palmitoyltransferase 1C (cpt1c) during larval ontogeny and across juvenile's organs (Jiménez-Martínez et al., 2019), as well as the effect of dietary lipid sources on growth, survival, cannibalism, and the relative gene expression fas, acc1 and cpt1c during early development (Jiménez-Martínez et al., 2020). However, little is known about the ability of A. tropicus to efficiently utilize vegetal oils-based diets that are more sustainable, which could help to reduce the feed production cost.

Therefore, each species possesses different capacity to biosynthesize LC-PUFA from PUFA, depending on factors such as the relative activity, availability, and affinity level of desaturases and elongases (Alhazzaa et al., 2018). However, in general terms, marine carnivorous fishes show more limited ability to synthesize LC-PUFA than fresh-water fishes and require the inclusion of C20 and C22 LC-PUFA in their diet (Yıldız et al., 2017; Alhazzaa et al., 2018), using fish oil to fulfill such fatty acid requirement. Nevertheless, fish oil is increasingly recognized as an environmentally unsustainable and economically unviable practice (Tocher, 2015; Chen et al., 2018). However, vegetables oils lack of n-3 LC-PUFA (C ≥ 20), therefore the replacement of FO by VO led the reduction in the n-3 LC-PUFA in diets (Yıldız et al., 2017). Consequently, the understanding of the physiological roles of fatty acyl desaturases and elongases in A. tropicus and assess their activity patterns during the entire life cycle is of strong interest for its ability to enable efficient and effective use of sustainable plant-based diet alternatives in aquaculture, as well to maintaining the nutritional quality of flesh (i.e., ARA, EPA, and DHA content of farmed fish) (Tocher, 2015; Yıldız et al., 2017). Here we investigated the expression levels of the fatty acyl desaturases fads1 (Δ5) and fads2 (Δ6), and fatty acyl elongases elovl2 and elovl5, involved in biosynthesis of LC-PUFA during development of the ancestral tropical gar A. tropicus. Moreover, we further determined the fatty acid profiles during the A. tropicus development to elucidate the contribution that activity of fads and elovl encoded enzymes have in the biosynthesis of the physiologically relevant LC-PUFA.