RNA-Seq-Based Analysis of Cortisol-Induced Differential Gene Expression Associated with Piscirickettsia salmonis Infection in Rainbow Trout (Oncorhynchus mykiss) Myotubes

Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes—124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol

De novo Assembly and Analysis of Tissue-Specific Transcriptomes of the Edible Red Sea Urchin Loxechinus albus Using RNA-Seq

Edible red sea urchin (Loxechinus albus) is an endemic echinoderm species of the Chilean coasts. The worldwide demand for high-quality gonads of this species has addressed the depletion of its natural populations. Studies on this sea urchin are limited, and genomic information is almost nonexistent. Hence, generate a transcriptome is crucial information that will considerably enrich molecular data and promote future findings for the L. albus aquaculture. Here, we obtained transcriptomic data of the edible red sea urchin by Illumina platform. Total RNA was extracted from gonads, intestines, and coelomocytes of juvenile urchins, and samples were sequenced using MiSeq Illumina technology. A total of 91,119,300 paired-end reads were de novo assembled, 185,239 transcripts produced, and a reference transcriptome created with 38.8% GC content and an N50 of 1769 bp. Gene ontology analysis revealed notable differences in the expression profiles between gonads, intestines, and coelomocytes, allowing the detection of transcripts associated with specific biological processes and KEGG pathways. These data were validated using 12 candidate transcripts by real-time qPCR. This dataset will provide a valuable molecular resource for L. albus and other species of sea urchins

RNA seq analysis of the head kidney transcriptome response to handlingstress in the red cusk eel (Genypterus chilensis)

Stress is a primary contributing factor of fish disease and mortality in aquaculture. We have previously reported that the red cusk-eel (Genypterus chilensis), an important farmed marine fish, demonstrates a handling-stress response that results in increased juvenile mortality, which is mainly associated with skeletal muscle atrophy and liver steatosis. To better understand the systemic effects of stress on red cusk-eel immune-related gene expression, the present study assessed the transcriptomic head-kidney response to handling-stress. The RNA sequencing generated a total of 61,655,525 paired-end reads from control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,840 transcripts and created a reference transcriptome with a N50 of 1426 bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 upregulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant upregulation of the biological processes, like response to stress, response to biotic stimulus, and immune response. Conversely, a significant down-regulation of biological processes is associated with metabolic processes. These results were validated by RT-qPCR analysis for nine candidate genes involved in the immune response. The present data demonstrated that short term stress promotes the immune innate response in the marine teleost G. chilensis. This study is an important step towards understanding the immune adaptive response to stress in nonmodel teleost species.Fondo de Financiamiento de Centros de Investigación en Áreas Prioritarias: FONDAP INCAR 15110027 (to JAV and AM); FONDAP Center for Genome Regulation CRG 15090007 (to CM) and CONICYT, FONDECYT/Regular No. 1120261 (to HS) and 1171318 (to JAV)

Additional file 1: Table S1. of mRNA-seq reveals skeletal muscle atrophy in response to handling stress in a marine teleost, the red cusk-eel (Genypterus chilensis)

Complete list of up-regulated and down-regulated transcripts in response to handling stress. (XLSX 66 kb

Additional file 2: Table S2. of mRNA-seq reveals skeletal muscle atrophy in response to handling stress in a marine teleost, the red cusk-eel (Genypterus chilensis)

Enriched Molecular functions of up-regulated and down-regulated transcripts in response to handling stress. (XLSX 10 kb

Additional file 5: Table S5. of mRNA-seq reveals skeletal muscle atrophy in response to handling stress in a marine teleost, the red cusk-eel (Genypterus chilensis)

Primer sequences for qPCR assay, amplicon size and PCR. (XLSX 10 kb

Plasmatic levels of ALT (alanine aminotransferase), AST (aspartate aminotransferasa), and AP (alkaline phosphatase).

<p>All data are represented as means ± SEM (n = 4). Significant differences between control and stressed groups are shown as * (P < 0.05) and ** (P < 0.01).</p

Over-represented functional categories in clusters of up-regulated transcripts in response to handling stress.

<p>Over-represented functional categories in clusters of up-regulated transcripts in response to handling stress.</p

Quantitative real time PCR validation of differentially expressed transcripts.

<p>Expression of fold changes measured by RNA-seq and qPCR are indicated in the grey and black columns, respectively. <i>agtr2</i> (type-2 angiotensin ii receptor), <i>anxa2</i> (annexin a2), <i>cav1</i> (caveolin-1), <i>ctgf</i> (connective tissue growth factor precursor), <i>jun</i> (transcription factor ap-1), <i>lepr</i> (leptin receptor precursor), <i>plat</i> (tissue-type plasminogen activator precursor), <i>plxnd1</i> (plexin-b2 precursor), <i>pkd1</i> (polycystin-1 precursor), <i>s1pr1</i> (sphingosine 1-phosphate receptor 1), <i>thbs1</i> (thrombospondin-2 precursor), <i>tgfbr3</i> (transforming growth factor beta receptor type 3 precursor), <i>hmgcr</i> (3-hydroxy-3-methylglutaryl-coenzyme a reductase), <i>hmgcs1</i> (hydroxymethylglutaryl- cytoplasmic), <i>dhcr7</i> (7-dehydrocholesterol reductase), <i>nsdhl</i> (sterol-4-alpha-carboxylate 3- decarboxylating), <i>acat2</i> (acetyl-CoA acetyltransferase), <i>c14orf1</i> (ergosterol biosynthetic protein), <i>cyp51a1</i> (lanosterol 14-alpha demethylase), <i>cyb5r3</i> (nadh-cytochrome b5 reductase 3), <i>fdps</i> (farnesyl pyrophosphate synthase), <i>tecr</i> (trans- enoyl- reductase), <i>hsd17b7</i> (3-keto-steroid reductase), <i>idi1</i> (isopentenyl-diphosphate delta-isomerase 1), <i>lss</i> (lanosterol synthase), <i>mvd</i> (diphosphomevalonate decarboxylase), <i>fau</i> (40S ribosomal protein). Significant differences between the control and stressed groups are shown as * (P < 0.05) and ** (P < 0.01).</p