- Atlantic Salmon ( Salmo salar. - In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. - In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. - Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3 ’ mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes.. - We also found DEGs related to lipid metabolism in the QuantSeq method. - In the TruSeq method, a missense mutation was detected in DEGs in different flesh color traits.. - The number of DEGs found in the TruSeq libraries was much higher than the QuantSeq. - Our findings suggest that Grp could inhibit the feeding behavior of low color-banded fish, resulting in the dietary carotenoid shortage. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - This makes flesh color a key criterion in the salmon farming industry [1]. - In the wild the muscle pigmentation of some salmonids is the result of the accumulation of carotenoids, primarily astaxanthin (Ax), derived from the consumption of algae which are Ax main producers, and/or crustaceans which fed on the algae [2]. - Due to its hydrophobicity, Ax is not solubilized in the aqueous environment of the fish gastrointestinal system [3]. - Changing the dietary fatty acid composition could influence the solubility of Ax in the lipid phase [3, 6].. - In this study, we compared Illumina ’ s TruSeq kit, the standard RNA-Seq (refered to as TruSeq) and Lexogen’s QuantSeq kit, the 3’ mRNA-Seq method (QuantSeq) for an Atlantic salmon flesh color investigation. - The QuantSeq reads covered only the last exon at 3’ end, while TruSeq reads covered all exons of the gene with only a slight decline in the exon at the 5’ end.. - A represents the coverage of mapped reads from every library along with transcripts from 5 ’ to 3 ’ ends in the whole Atlantic salmon genome. - clear clustering in the PCA plot (Fig. - In the TruSeq data, 191 DEGs were iden- tified while only 50 DEGs were identified from the QuantSeq data when the two subsample groups were. - 5 The differential gene expression in the two libraries: (A) Venn diagram of DEGs from TruSeq and QuantSeq libraries (B) the variation of expression within biological replicates of the 11 DEGs shared in the QuantSeq and TruSeq data. - Interestingly, the high variation in expression of these DEGs showed a similar pattern in the TruSeq and QuantSeq datasets.. - The fact that these 11 DEGs showed the same pattern with the two library methods used instil more confi- dence in the validity of these DEGs. - In the QuantSeq library, we detected 50 DEGs (Sup- plementary 2a) related to metal ion transport, oxidation- reduction process, immune responses and lipid metabol- ism. - DEGs detected in the TruSeq library (Supplementary 2b) that were different to those found in the Quantseq library, were also linked with metal ion transport, oxidation-reduction process and immune responses. - five that were DEGs only in the QuantSeq data, 5 that were DEGs only in the TruSeq data and 5 that were DEGs in both datasets. - From the five DEGs detected in the QuantSeq data, two genes, fabp1 and lyama, were validated as DEGs using RT-qPCR, and three out of five TruSeq data DEGs were validated as DEGs by RT-qPCR (P <. - Among 191 DEGs identified in the Truseq library, there were 5 DEGs with nine missense mutations and 2 DEGs with three missense mutations found in HN and LB fish,. - In 13 carotenoid-related genes in the HN fish, missense mutations were identified. - In the LB fish group, a missense mutation on transmembrane protease serine 9-like (tmprss9) (Fig. - Three missense mutations, in- cluding Ser267Gly, Ala740Asp, and Arg974Gln on duox2 gene and three other missense mutations involv- ing Leu55Phe, Ala213Thr, and Glu239Gly on duoxa1 were present in the same four of the five low color- banded fish and absent in the other phenotype (Fig. - During that summer where the temperature was for 117 days above 18 o C, flesh color was reduced initially in the front dorsal and then in the back central region. - In the present study, we sam- pled fish from a single cage in June 2017. - In the context of iron ion transport, fertn-m was up- regulated in HN fish when compared to LB fish in our study. - Subset of DEGs of interest identified in the transcriptiomes from the two library preprations.. - (B) Expression pattern of seven genes identified in the TruSeq library: cyp450 gene family cyp2k1, cyp3a27 and cyp1b1, involved in oxidation-reduction process. - fertn-m function in Atlantic salmon was well described in previous studies [44]. - enriched in the hindgut of low color fish. - catb and catl are cysteine proteases in- volved in the self-defense mechanism against fish patho- gens and the host immune defense in vertebrates [53, 54]. - the next five genes showed significant differences only in the QuantSeq library analysis, and only two of those (fabp1 and lyama) were significantly different in RT-qPCR. - the last five genes showed significant differences only in the TruSeq library, and only three out of these five genes (ovcm2, noxo1 and fclt3) were significantly different using the RT-qPCR assay. - alb2 is one of the most abundant proteins in the plasma, considered as the long-chain fatty acid trans- porter when it binds together to form complexes that circulate in the plasma [59, 60]. - We have found that alb is upregulated in the gut of HN fish and propose that this upregulation may increase the Ax absorption and transport from the gut into the blood system and then deposition in the muscle or liver. - HDL is known to have a major role in carrying fatty acids and caroten- oids in Atlantic salmon [65, 66]. - The CYP450 enzyme family, together with important functions in the oxidation-reduction process, has also been known to induce stress response as a result of a variation in temperature [67]. - In the latter study, the CYP450 2J19 gene (cyp2j19), which is considered as a carotenoid ketolase and mediates red coloration in birds, was also upregulated in the pyloric caeca of red flesh phenotype Chinook salmon [70], although in the present study, cyp2j19 was not a DEG. - This finding suggests that cyp2j19 might not be associated with the red coloration in the Atlantic salmon. - Aside from functions in the gastrointes- tinal tract, some neuroendocrine peptides also impact on short-term food satiation and signal the brain to in- hibit feeding [73]. - In the gut-brain axis, neuroendo- crine peptides are secreted by the stimulation of food in- take available in the gastrointestinal tract [73, 74]. - grp was previously shown to stimulate gastric acid release in the fish gut [73] while inhibiting gastric emptying and mediating satiety in fish [74]. - We proposee that grp may func- tion as an anorexigenic signal in the gut-brain axis dur- ing prolonged thermal stress. - This finding might apply to other ver- tebrates and be consistent with our result in the upregulation of grp in LB fish. - Accordingly, prolonged starvation is also the main rea- son for flesh discoloration, as fish likely lost a large amount of uptaken carotenoids to compensate for the amount used by physiological activities in the fish body.. - also known as sterolins) are known to participate in the physiological pathways involving dietary choles- terol and non-cholesterol sterols [79]. - It has been shown that 2 % increase in dietary cholesterol improved Ax absorption and deposition as well as Ax levels in the plasma of Atlantic salmon [82]. - Hence, in light of the aforementioned findings, additional choles- terol in Atlantic salmon diets could potentially result in increased Ax levels in the plasma, which would then be absorbed by the muscle, leading to a more intense flesh coloration. - With regard to to ATP-binding cassette, sub family, Zoric [83] reported that a missense mutation in abcg2-1a, resulting in the substitution of Asparagine with Serine in amino acid position 230 (Asn230Ser), is predicted to be associated with flesh color in Atlantic salmon because it is more prevalent in the pale than in the dark flesh phenotype. - Therefore, in our study, it is possible that Pro21Thr missense mutation on abcg8 plays an important role to- gether with abcg5 in the control of secreting carotenoid- binding cholesterols and absorption of dietary caroten- oids and mobilization of carotenoids in Atlantic salmon.. - In term of SNPs on DEGs identified in TruSeq, the six mutations on duox2-l and duoxa1-l that we detected in LB fish in the current study might potentially lead to the mismatched complex, which then affects their function of generating reactive oxygen species (ROS). - In juvenile rainbow trout, dietary Ax supposedly enhanced the antioxidant defense system, which plays a role in the inactivation of ROS [91]. - 0.05) even though they were picked as DEGs and expressed highly in the Quant- Seq and TruSeq data.. - In only QuantSeq with a low depth of sequencing, in the comparisons of 39 samples from 4 color phenotypes, there was a high variation in the number of normalized read counts within each group. - A recent study of genetic and phenotypic correla- tions indicated that improvement in growth did not end in any flesh color variation in Atlantic salmon [35]. - In the standard whole transcript method, mRNAs was fragmented before converted to cDNA. - specifically apolipoproteins, serum albumin and fatty acid-binding protein genes which are proposed to play important functions in the absorption, transport and de- position of carotenoids. - Moreover, we speculate that grp, which is highly expressed in low banded fish, could in- hibit their feeding behavior, leading to the dietary carot- enoid shortage, resulting in the flesh discoloration in Atlantic salmon. - RNA samples used in the library preparation were chosen based on the follow- ing criteria: A260/280 ratio within 2.0-2.2, A260/A230 ra- tios >. - Therefore, in the comparison of the effectiveness of QuantSeq and TruSeq libraries, the same ten randomly chosen extracted RNA samples which were used for QuantSeq library were se- lected from two groups: five of extremely HN and five of extremely LB for repeated sequencing using Illumina li- brary preparation. - The datasets supporting the conclusions of this article are available in the BioProject Accession number PRJNA706530, ID 706,530 - BioProject - NCBI (nih.gov).. - Astaxanthin deposition in fillets of Atlantic salmon Salmo salar L. - Ghrelin is involved in voluntary anorexia in Atlantic salmon raised at elevated sea temperatures. - Environmental drivers of Atlantic salmon behaviour in sea-cages: A review. - Response of Atlantic salmon Salmo salar to temperature and dissolved oxygen extremes established using animal-borne environmental sensors. - Ghrelin is involved in voluntary anorexia in Atlantic salmon raised at elevated sea temperatures.. - Appetite, metabolism and growth regulation in Atlantic salmon (Salmo salar L.) exposed to hypoxia at elevated seawater temperature. - Transcriptional responses to temperature and low oxygen stress in Atlantic salmon studied with next-generation sequencing technology. - Assessing the Pyloric Caeca and Distal Gut Microbiota Correlation with Flesh Color in Atlantic Salmon (Salmo salar L., 1758). - Multigenic Delineation of Lower Jaw Deformity in Triploid Atlantic Salmon (Salmo salar L. - Comparison of library construction kits for mRNA sequencing in the Illumina platform.. - A comparison of Illumina and Ion Torrent sequencing platforms in the context of differential gene expression. - Heterogeneous astaxanthin distribution in the fillet of Atlantic salmon post- smolt at elevated temperature is not affected by dietary fatty acid composition, metabolic conversion of astaxanthin to idoxanthin, or oxidative stress. - Understanding flesh colour variation in Atlantic salmon: molecular mechanisms and genetic effect. - University of the Sunshine Coast (USC). - characterisation of multiple ferritin isoforms in the Atlantic salmon (Salmo salar). - Lack of a role for iron in the lyme disease pathogen. - Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage. - Two ferritin subunits of Atlantic salmon (Salmo salar): cloning of the liver cDNAs and antibody preparation. - Genetic markers of the immune response of Atlantic salmon (Salmo salar) to infectious salmon anemia virus (ISAV).. - The effect of nonylphenol on gene expression in Atlantic salmon smolts. - Atlantic Salmon (Salmo salar L., 1758) Gut Microbiota Profile Correlates with Flesh Pigmentation: Cause or Effect? Marine Biotechnology. - A comparative study on innate immune parameters in the epidermal mucus of various fish species.. - A direct role for serum albumin in the cellular uptake of long-chain fatty acids. - Blood appearance, metabolic transformation and plasma transport proteins of 14 C-astaxanthin in Atlantic salmon (Salmo salar L. - Red carotenoid coloration in the zebra finch Is controlled by a cytochrome P450 gene cluster. - Red and White Chinook Salmon (Oncorhynchus tshawytscha): Differences in the Transcriptome Profile of Muscle, Liver, and Pylorus. - Gastrin-releasing peptide receptors in the central nervous system: role in brain function and as a drug target. - Gastrin-releasing peptide receptor signaling in the integration of stress and memory.. - Role of bombesin-related peptides in the mediation or integration of the stress response. - Transcriptional regulation of cholesterol and bile acid metabolism after dietary soyabean meal treatment in Atlantic salmon (Salmo salar L. - Effects of various dietary factors on astaxanthin absorption in Atlantic salmon (Salmo salar). - Characterization of genes and gene products influencing carotenoid metabolism in Atlantic salmon
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