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Download at PC and HG Using Rneasy Plus Universal Kits (QIAGEN, Downloads/Salmo Salar-Annotation.Gff3.Gz) Jin et al. BMC Genomics (2018) 19:253 https://doi.org/10.1186/s12864-018-4651-8 RESEARCHARTICLE Open Access Transcriptional development of phospholipid and lipoprotein metabolism in different intestinal regions of Atlantic salmon (Salmo salar) fry Yang Jin1* , Rolf Erik Olsen1, Mari-Ann Østensen1, Gareth Benjamin Gillard3, Sven Arild Korsvoll4, Nina Santi4, Arne Bjørke Gjuvsland2, Jon Olav Vik2, Jacob Seilø Torgersen4, Simen Rød Sandve2 and Yngvar Olsen1 Abstract Background: It has been suggested that the high phospholipid (PL) requirement in Atlantic salmon (Salmo salar)fryis due to insufficient intestinal de-novo synthesis causing low lipoprotein (LP) production and reduced transport capacity of dietary lipids. However, in-depth ontogenetic analysis of intestinal PL and LP synthesis with the development of salmon has yet to be performed. Therefore, in this paper we used RNA-Seq technology to investigate the expression of genes involved in PL synthesis and LP formation throughout early developmental stages and associate insufficient expression of synthesis pathways in salmon fry with its higher dietary PL requirement. There was a special focus on the understanding homologous genes, especially those from salmonid-specific fourth vertebrate whole-genome duplication (Ss4R), and their contribution to salmonid specific features of regulation of PL metabolic pathways. Salmon fry were sampled at 0.16 g (1 day before first-feeding), 2.5 and 10 g stages of development and transcriptomic analysis was applied separately on stomach, pyloric caeca and hindgut of the fish. Results: In general, we found up-regulated pathways involved in synthesis of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and LP in pyloric caeca of salmon between 0.16 and 10 g. Thirteen differentially expressed genes (q < 0.05) in these pathways were highly up-regulated in 2.5 g salmon compared to 0.16 g, while only five more differentially expressed (q < 0.05) genes were found when the fish grew up to 10 g. Different homologous genes were found dominating in stomach, pyloric caeca and hindgut. However, the expression of dominating genes in pathways of PL and LP synthesis were much higher in pyloric caeca than stomach and hindgut. Salmon-specific homologous genes (Ss4R) had similar expression during development, while other homologs had more diverged expression. Conclusions: The up-regulation of the de-novo PtdCho and PtdEtn pathways confirm that salmon have decreasing requirement for dietary PL as the fish develops. The similar expressions between Ss4R homologous genes suggest that the functional divergence of these genes was incomplete compared to homologs derived from other genome duplication. The results of the present study have provided new information on the molecular mechanisms of phospholipid synthesis and lipoprotein formation in fish. Keywords: Atlantic salmon, Biosynthesis, Gene expression, Homologous genes, Intestinal regions, Lipoprotein, Phospholipids, RNA-Seq, Whole genome duplication * Correspondence: [email protected] 1Department of Biology, Faculty of Natural Sciences, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jin et al. BMC Genomics (2018) 19:253 Page 2 of 11 Background Many homologous genes in mammals were found be- Phospholipids (PL) are the main constituent of all bio- long to gene families controlling the same enzymatic logical cell membranes and separate the intracellular and processes but have distinct regulation in different tissues extracellular aqueous environments. In addition to pro- and developmental stages [1]. In this respect the Atlantic viding a structural scaffold for cell membranes, PL are salmon has another layer of functional genome complex- also involved in numerous biological functions, such as ity as it experienced two extra rounds of whole genome provision of metabolic energy, cell membrane transport, duplication (WGD) compared to mammals, at the base and regulation of metabolism [1]. PL are also key struc- of all teleost (Ts3R) and in a common ancestor of all sal- tural components of lipoproteins (LP), which are in- monids ~ 100–80 Mya (Ss4R) [15]. Of the Ts3R and volved in transport of dietary lipids from intestinal Ss4R gene duplicates, ~ 20 and 55% respectively are still enterocytes to liver and peripheral tissues [2, 3]. It is well retained as expressed genes in the genome [16]. This known that dietary inclusions of PL can improve growth dramatic increase in the number of homologous genes and performance in many fish species including Atlantic in salmon thus necessitates a careful annotation of PL salmon (Salmo salar)[4], Atlantic cod (Gadus morhua) synthesis and LP formation pathway genes and their [5], and rainbow trout (Oncorhynchus mykiss)[6]. Dietary tissue-specific expression regulation to improve our un- and biliary PL is mainly digested by intestinal phospholip- derstanding of salmon PL metabolism. ase A2 in fish, resulting in 1-acyl lyso-phospholipids (lyso- In this paper we annotate and characterize gene regu- PL) and free fatty acids (FFA). Subsequently, both lyso-PL lation involved in PL synthesis and LP formation in and FFA are absorbed into intestinal enterocytes and re- different intestinal regions (SM, PC and HG) during esterified into PL before being exported to the rest of the early developmental stages of salmon. Our aims are to body [7]. In addition, PL can be synthesized de-novo in (I) improve our understanding of the homologous genes, enterocytes from glycerol-3-phosphate (G-3-P) in fish [8]. especially from Ss4R, and their contribution to salmonid A minimum requirement of PL is associated with early specific features of regulation of PL metabolism path- developmental stages of fish, but so far no minimum re- ways and to (II) specifically investigate the association of quirement has been demonstrated in adult fish [8]. In insufficient expression of PL and LP synthesis pathways line with this, dietary PL has been shown to enhance in salmon fry with its higher dietary PL requirement. growth and survival in Atlantic salmon fry up to 2.5 g, but not in larger fish [4, 9]. It has been suggested that Methods the higher PL requirement is due to insufficient ability Fish, diet and sampling procedure of de-novo synthesis in the intestine leading to low LP Atlantic salmon eggs were hatched and cultivated at production and consequently reduced transport capacity AquaGen Breeding Centre (Kyrksæterøra, Norway). of dietary lipids [8]. This was supported by previous From first-feeding, the fish were fed a normal commer- histological studies in salmonids, showing lipid accu- cial diet which satisfies the nutritional requirement of mulation in intestinal enterocytes when fed PL defi- salmon, but without any additional PL supplement cient diet [9, 10]. However, these differences were not (Additional file 1). The diet was produced by EWOS AS evident in a previous transcriptomic study where gene (Bergen, Norway). Six salmon individuals were sampled expressions in PL biosynthesis pathways were un- at sizes of 0.16 g (n = 6, 1 day before first feeding), 2.5 g changed in 2.5 g salmon fed by PL-supplemented diet (n = 6, 65 days after first feeding) and 10 g (n =6, [11]. No study has examined the gene expression in 100 days after first feeding). The fish were euthanized by salmon smaller than 2.5 g. 1 g/L MS-222 (FINQUEL, Argent chemical labs, The intestinal tract of salmon consists of several re- Washington, USA) buffered with same amount of so- gions, with different functions in lipid digestion, absorp- dium bicarbonate before dissection. The belly of the tion and transport. It is generally believed that pyloric fish was dissected and immediately placed in 1 mL caeca (PC), rather than stomach (SM) or hindgut (HG), RNALater. Tissues were stored for 24 h at 4 °C for is the predominant region for lipid absorption and trans- sufficient penetration of RNALater, before being dis- port in salmon [12, 13]. Therefore, PL and LP were as- sected in sterile petri dish filled with RNALater. The sumed to be mostly synthesized in PC region. However, entire intestinal tract was carefully separated from other tissues like SM and HG could also have ability of belly, connective tissues were removed and the intes- synthesizing PL due to its structural roles in cell mem- tinal contents were squeezed. Samples of stomach branes [1]. LP has occasionally been observed in hind- (SM, section between the end of esophagus and start gut, suggesting some lipid absorption and transport of pyloric caeca), pyloric caeca (PC, section with fin- activities in the region [14]. So far no study has demon- gerlike projections) and hindgut (HG, section after strated the expression of PL metabolic pathways in SM mid intestine) were dissected under dissecting micro- and HG of fish. scope and immediately placed in in 2 mL cryo tubes Jin et al.
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