Experimental Design
All procedures performed in this study were conducted following the Mogi das Cruzes University Institutional Animal Care and Use Ethics Committee (approval number: #008/2017). In total, 150 cobia juveniles (90.85 ± 11.75 g, mean ± SD) were acquired by Redemar Alevinos (Ilha Bela, SP, Brazil), acclimated during 15 days in a 10,000 L tank (temperature average of 28 ± 0.4 ºC; dissolved oxygen 6.57 ± 0.7 mg L− 1; and total ammonia nitrogen – TAN ˂0.05 mg L− 1, mean ± SD), and fed twice a day until apparent satiation with a fish commercial diet (Guabipirá, Guabi Nutrição e Saúde Animal S.A., SP, Brazil). After the acclimatization period, the animals were transferred to three 1,000 L tanks (50 animals per tank, triplicate) and were fed twice a day for seven days with the same commercial diet. At the end of the seventh day, the animals were fasted for 24 h. After the fasting period, it was carried out an uninterrupted single feed event (ad libitum) for 10 min. Ten min before and after 30 min, 1, 2, 4, 8, 12, and 24 h of the feed event, three fish from each tank (a total of 9 individuals) were anesthetized, euthanized, and aliquots of liver were immediately frozen in liquid nitrogen, and then transferred to the ultra-freezer (-80 ºC) until the analysis.
Diet and liver fatty acid profile
The total lipids of the diet and liver were extracted using a chloroform/methanol/water mix (2:1:0.5) (modified from Folch et al. 1957). The lipid extracts were methylated using 5% HCl methanol prepared by dissolving 10% vol acetyl chloride in methanol according to Christie (2003). The fatty acid analysis was performed using a gas chromatograph (GC, model 3900, CA, USA) coupled with a flame ionization detector (FID) and a CP-8410 autosampler. The FAME analysis was carried out using a capillary column (CP-Wax 52 CB, 0.25-μm thick, 0.25-mm inner diameter, and 30-m length), and hydrogen was used as a carrier gas at a linear velocity of 22 cm/s. The column was programmed to start at 170 °C for 1 min followed by a 2.5 °C/min ramp to 240 °C and a final hold time of 5 min. The injector and FID temperature were kept at 250 and 260 °C, respectively. The FAMEs were identified by comparing their retention times to those obtained with commercial standards (Supelco, 37 components; Sigma-Aldrich; mixture Me93, Larodan, and Qualimix; PUFA fish M, menhaden Oil, Larodan). The fatty acid profile of the diet used in the trial is presented in Table 1.
Table 1 Fatty acid composition (% of total fatty acids) of the experimental diet
Fatty acids
|
|
14:0
|
2.23 ± 0.08
|
16:0
|
22.46 ± 0.24
|
18:0
|
7.30 ± 0.13
|
ΣSFA
|
31.99 ± 0.40
|
16:1n-7
|
6.16 ± 0.21
|
18:1n-9
|
26.95 ± 0.52
|
18:1n-7
|
3.46 ± 0.48
|
ΣMUFA
|
37.88 ± 0.61
|
18:3n-3
|
1.40 ± 0.05
|
20:5n-3
|
3.45 ± 0.32
|
22:5n-3
|
1.22 ± 0.11
|
22:6n-3
|
6.66 ± 0.29
|
Σn-3 PUFA
|
12.74 ± 0.34
|
18:2n-6
|
14.45 ± 0.41
|
20:4n-6
|
1.49 ± 0.15
|
22:4n-6
|
0.58 ± 0.03
|
22:5n-6
|
0.43 ± 0.16
|
Σn-6 PUFA
|
16.96 ± 0.45
|
ΣPUFA
|
30.13 ± 0.33
|
ΣLC-PUFA
|
13.84 ± 0.50
|
Σn-3/Σ n-6
|
0.75 ± 0.04
|
Others
|
0.43 ± 0.03
|
ΣSFA, ΣMUFA, ΣPUFA, n-3 ΣPUFA, n-6 ΣPUFA are the sum of saturated, monounsaturated, n-3 polyunsaturated, polyunsaturated and n-6 polyunsaturated fatty acids respectively
Rna Isolation, Library Preparation, And Sequencing
Total RNA from the liver was extracted using an RNeasy Lipid Tissue kit (Qiagen, Germantown, MD, USA) following the manufacturer's instructions. The quantity of extracted RNA was assessed by Nanodrop™ Spectrophotometer (Thermo Fisher Scientific, USA), and the quality was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Germany). The RIN values of the RNA used were all above 7.5.
RNA from the liver of three animals of each tank was used in the library construction using the TruSeq RNA Sample Preparation kit (Illumina Inc., USA) according to the manufacturer’s specifications. Transcriptome sequencing was performed with Illumina Nextseq® platform (Illumina Inc., San Diego, CA, USA) using a paired-end sequencing strategy (2×75 bp).
De novo assembly, functional annotation, and differential expression
The detailed methodology, statistics, and summary of the de novo transcriptome assembled of cobia liver tissue were previously described by Barbosa et al. (2021). Due to the massive dataset generated by the transcriptome sequencing, the differential expression and quantitative real time PCR (qRT-PCR) validation were restricted to three comparisons (30 min vs. 8 h, 30 min vs. 24 h, and 8 h vs. 24 h) based on the main differences observed in the liver fatty acid profile. Raw libraries were submitted to quality control checking, using FastQC (Andrews 2010) and Fastp was used to remove low-quality (Q < 30) reads, adapters, and other contaminant sequences. To remove rRNA reads, the high-quality reads were aligned to sequences in the SILVA ribosomal RNA (rRNA) gene database (http://www.arbsilva.de/) using SortMeRNA (Kopylova et al., 2012). The National Center for Biotechnology Information (NCBI) Univec database (https://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/) was used to remove contaminants from libraries. Quality-filtered reads were then mapped to R. canadum transcriptome available and described by Barbosa et al. (2021), using HISAT2 (Kim et al., 2019) with default parameters. StringTie (Pertea et al. 2015) was then used to assemble the mapped reads into transcripts, using the de novo transcriptome reconstruction method, allowing the identification of all transcripts present in each sample (including currently annotated genes, as well as newly identified elements and isomorphs). StringTie Merge was next used to combine redundant transcription structures, providing a non-redundant reference transcriptome, with unique identifiers. Cufflinks (Trapnell et al. 2012) were next used to estimate expression values (FPKM) for each element in the StringTie-generated reference transcriptome. Transcriptome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) software, version 3.0 (Simão et al. 2015). Finally, the reads and the merged assembly were also used to calculate expression levels and the effect size and statistical significance of observed differences using Cuffdiff (Trapnell et al. 2010). Heatmaps based on expression patterns were built with Complex Heatmap package (Gu et al. 2016) in a custom R script.
We applied Eukaryotic Non-Model Transcriptome Annotation Pipeline (EnTAP) (Hart et al. 2020) on the query transcripts (blastx, e-value ≤ e− 5) for homology against the National Center for Biotechnology Information non-redundant protein database (NCBI nr), NCBI proteins reference database (RefSeq), the curated Swiss-Prot database from UniProt Knowledgebase (UniProtKB), and the EggNOG proteins database. The EggNOG hits also helped to assign the biological function to the genes, identifying GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) terms. EnTAP functional annotation process was carried out using a Dugong container environment (Menegidio et al. 2018) in BioPortainer Workbench (Menegidio et al. 2019).
Kegg terms were annotated and organized in Pathway Level 2 classes, from KEGG Pathway Maps page (https://www.genome.jp/kegg-bin/show_brite?&htext=br08901.keg&option=-a&panel=collapse). Kegg Automatic Annotation Server (KAAS, https://www.genome.jp/kegg/kaas/) was used to annotate the transcript sequences in KEGG Orthologs (KO) terms, with the bi-directional best hit (BBH) method. KOs were utilized as input in the KEGG Mapper-Search&Color Pathway (https://www.genome.jp/kegg/tool/map_pathway2.html) to map the differentially expressed transcripts based on their modulation; Up-regulated elements were assigned as green, while Down-regulated ones received the color red. Barramundi was set as the background species because it is closely related to the cobia contained in KEGG.
Validation Of Degs
Total RNA from liver samples was purified using Kit Pure Link RNA (12183018A Invitrogen™) according to the manufacturer's instructions. To eliminate the potential contamination by genomic DNA, samples were treated with DNase using Turbo DNAfree (AM1907 Invitrogen ™), for this, previously the RNA samples were diluted to 100 ng/uL and after treatment were stored at -80 ºC. 500 ng of total RNA were used as a template to synthesize cDNA using SuperScript III Reverse Transcriptase (18080093 Invitrogen™) and random primers were used according to the manufacturer's instructions. Negative controls were also performed, without reverse transcriptase and the cDNA was stored at -20 ºC.
It was selected a total of 10 differentially expressed unigenes (DEGs) from the liver to verify the reliability of the RNA-Seq data. The primers were previously designed using the primer design software tools: IDT Oligo Analyzer (www.idtdna.com/calc/analyzer). The elongation factor 1 alpha (ef1a) and β-actn were used as endogenous reference genes. The oligonucleotides used as primers and the parameters of efficiency are described in Table 2. All primers were synthesized by Thermofisher (Invitrogen™).
Table 2
Nucleotide sequence of primers used for quantitative real-time PCR (qRT-PCR)
Gene | Primer Sequence 5’ − 3’ | Amplicon Size (bp) |
acads_fw | GATGAGGTGATGGCGACTTATC | 94 |
acads_ rv | GTACCAGTCATAGGCACACTTT |
acsl_fw | CTCAATCTTCTCTGGTGCTATGT | 146 |
acsl_rv | CTGCAGAGGCCTTGGATAAA |
cpt1_fw | GTCTCACCGCACGAGTATTT | 101 |
cpt1_rv | TTCGGTCCTGTTGCTGATG |
fas_fw | TGTGGAGCCAACCATCTTTATC | 97 |
fas _rv | GGAGGTCTGTATCAAGGGTAGA |
pdk_fw | GGCTGTTCACCTACACATACTC | 123 |
pdk_rv | CTTGAAAGTAGCGGGCATAGA |
scd_fw | CAGTCAGTGTAGTCAGTGGTTAG | 99 |
scd_rv | TAGGGAACAGGAAGCAGAGA |
elovl6-like_fw | GGTCTGACTCCACTTTGAGAAG | 96 |
elovl6-like_rv | AAGACACAGAAGGAGCCAATAG |
elovl6_fw | TGAGTGACCACAGCACTAAC | 105 |
elovl6_rv | TCATGTTCTGTGGCCTGTATG |
mcm3_fw | GACGATGAGTCGGGCTTTATT | 102 |
mcm3_rv | CAGAGGGACTACCTGGACTT |
hspa1S_fw | TCTTGCTAACCCACTCCAATTA | 75 |
hspa1S_rv | GAGGGTGGGAGGTAAGAATAAG |
β-act_fw | AGCCATGGAAGATGAAATCG | 190 |
β-act_rv | TCTCTTGCTCTGGGCTTCAT |
ef1α_fw | AGTGCGGAGGAATCGACAAG | 188 |
ef1α_rv | TGCTGGTCTCGAACTTCCAC |
fw: forward and rv: reverse
Statistical analysis
Data related to the liver fatty acid profile is presented as mean ± SD (Table 3). The comparisons between hepatic fatty acid composition of different sample times were performed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD test using the R package, with a significance level of 5% (P < 0.05).