Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

21 páginas, 7 figuras, 3 tablas.-- Sebastian Boltaña ... et al.-- This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) licenseThis study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune responseThis study was supported by the Consolider-Ingenio Programme 2010, project CSD2007-0002 funded by the Spanish Ministry of Science and Education, Spain to SM, and FONDAP (15110027) and FONDECYT 1150585 awarded by CONICYT-Chile to Sebastian BoltañaWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).This study was supported by the Consolider-Ingenio Programme 2010, project CSD2007-0002 funded by the Spanish Ministry of Science and Education, Spain to SM, and FONDAP (15110027) and FONDECYT 1150585 awarded by CONICYT-Chile to Sebastian Boltaña.Peer reviewe

Linking stress coping styles with brain mRNA abundance of selected transcripts for Senegalese sole (Solea senegalensis) juveniles

In fish, proactive and reactive individual stress copying styles (SCS) have been used to resolve variation in molecular expression data. Stress coping styles have been previously described in several stages of Solea senegalensis by validating for the species the use of standard behavioural screening tests. The present study aimed to link behavioural SCS tests with brain transcript abundance in early Senegalese sole juveniles in order to observe the natural variation in a molecular pathway in this species. A total of 50 juveniles were subjected to three individual behavioural (Restraining, New environment and Confinement) and one group (Risk-taking) screening tests. The fish were classified in SCS categories by applying a hierarchical cluster to the variable “Total activity” (the total activity time that the fish was moving in each individual test). Three categories were defined, proactive, intermediate and reactive sole. Six transcripts were chosen and tested, one related to basic metabolism (gapdh-2), three to feeding behaviour (per1, igf-Ia, pparß) and two to the stress response (crh-BP and hsp90aa) in 30 juveniles (10 individuals per SCS category) using rt-qPCR to observe differences in the abundance of those transcripts among SCS. Four transcripts were differentially expressed (DETs) among them. The transcript gapdh-2 showed up-regulation for proactive and intermediate SCS sole while reactive individuals showed down-regulation. Target mRNAs per1, igf-Ia and pparß, showed different levels of up-regulation for proactive and reactive fish while intermediates were highly down-regulated. Surprisingly no differences in stress related transcripts were observed. Correlations were found between variation in coping styles and variation in the abundance of mRNAs involved in important biological functions in Senegalese sole. These results are the first evidence of the relationship between the behavioural individual variation and the fluctuation in brain transcripts abundance in Senegalese sole.info:eu-repo/semantics/acceptedVersio

Pesticides Drive Stochastic Changes in the Chemoreception and Neurotransmission System of Marine Ectoparasites

Scientific efforts to elucidate the mechanisms of chemical communication between organisms in marine environments are increasing. This study applied novel molecular technology to outline the effects of two xenobiotic drugs, deltamethrin (DM) and azamethiphos (AZA), on the neurotransmission system of the copepod ectoparasite Caligus rogercresseyi. Transcriptome sequencing and bioinformatics analyses were conducted to evaluate treatment effects on the glutamatergic synaptic pathway of the parasite, which is closely related to chemoreception and neurotransmission. After drug treatment with DM or AZA, stochastic mRNA expression patterns of glutamatergic synapse pathway components were observed. Both DM and AZA promoted a down-regulation of the glutamate-ammonia ligase, and DM activated a metabotropic glutamate receptor that is a suggested inhibitor of neurotransmission. Furthermore, the delousing drugs drove complex rearrangements in the distribution of mapped reads for specific metabotropic glutamate receptor domains. This study introduces a novel methodological approach that produces high-quality results from transcriptomic data. Using this approach, DM and AZA were found to alter the expression of numerous mRNAs tightly linked to the glutamatergic signaling pathway. These data suggest possible new targets for xenobiotic drugs that play key roles in the delousing effects of antiparasitics in sea lice

Developments in genomics relevant to disease control in aquaculture

21 pagesThe use of genomic technologies in aquaculture has experienced a rapid expansion over the past decade where functional genomics exemplified by microarray technology has experienced a more comprehensive development than actual genome sequencing. The significant increase in genomic resources driven by large-scale expressed sequence tag (EST) sequencing projects and more recently high throughput sequencing technologies has driven this development. Health management and prevention of disease are major issues in aquaculture, and genomics has contributed to a number of key areas in disease research including basic research aimed toward mRNA transcript discovery relevant to the immune response in diverse phylogenetic groups, evaluation of vaccine efficacy and immunostimulant diets, identification of the underlying mechanisms of disease resistance and the development of prognostic markersN

Fishing a hypothetical membrane receptor in the gilthead sea bream (Sparus aurata): role in acute stress

Trabajo presentado en el XII Conference of the Iberian Association for Comparative Endocrinology (AIEC), celebrado en Faro (Portugal), del 26 al 28 de septiembre de 2019.Cortisol is the most important hormone involved in the regulation of neuroendocrine stress response in teleosts. Effects of this hormone are mediated through its intracellular receptors, although membrane components are also involved, with unclear roles during the stress response. In this work, the contribution of membrane-initiated cortisol actions on stress-related parameters and metabolism-related genes in gilthead sea bream (Sparus aurata) were evaluated. Two in vivo experiments were performed. In the first, fish were administered with vehicle, BSA, cortisol and cortisol-BSA (membrane impermeable analogue) dissolved in saline (PBS 1X). In the second, the same treatments were replicated but using coconut oil as a vehicle. Fish were sampled after one and six hours (first experiment) and after three days (second experiment). Plasma cortisol, glucose and lactate levels were measured, and hepatic transcript levels of key genes involved in glucose metabolism, like glucose-6-phosphatase (g6pc), phosphoglycerate mutase 1 (pgam1), among others, were also analyzed. Fish implanted with each version of cortisol in both in vivo experiments reached plasma levels typically observed under an acute stress in S. aurata. Cortisol and cortisol-BSA increased glucose and lactate plasma levels after six hours of treatment. However, cortisol, but not cortisol-BSA, maintained increased plasma glucose levels after three days of treatment. Analysis by qPCR showed that expression of g6pc increased after one hour of cortisol and cortisol-BSA administration. However, cortisol, but not cortisol-BSA, maintained the g6pc up-regulation after three days. Our results suggested that membrane-initiated cortisol actions contributed to the regulation of early metabolic adaptations in S. aurata submitted to an emulated acute stress situation.This work was funded by Spanish Ministry of Science, Innovation and Universities - MICINN (AGL2016-76069-C2-1-R) awarded to JMM. The authors belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigación (MICINN, Spanish Government).Peer reviewe

The involvement of cholesterol in sepsis and tolerance to lipopolysaccharide highlighted by the transcriptome analysis of zebrafish (Danio rerio)

13 pages, 2 tables, 4 figuresSeptic shock is the most common cause of death in intensive care units due to an aggressive inflammatory response that leads to multiple organ failure. However, a lipopolysaccharide (LPS) tolerance phenomenon (a nonreaction to LPS), is also often described. Neither the inflammatory response nor the tolerance is completely understood. In this work, both of these responses were analyzed using microarrays in zebrafish. Fish that were 4 or 6 days postfertilization (dpf) and received a lethal dose (LD) of LPS exhibited 100% mortality in a few days. Their transcriptome profile, even at 4 dpf, resembled the profile in humans with severe sepsis. Moreover, we selected 4-dpf fish to set up a tolerance protocol: fish treated with a nonlethal concentration of Escherichia coli LPS exhibited complete protection against the LD of LPS. Most of the main inflammatory molecules described in mammals were represented in the zebrafish microarray experiments. Additionally and focusing on this tolerance response, the use of cyclodextrins may mobilize cholesterol reservoirs to decrease mortality after a LD dose of LPS. Therefore, it is possible that the use of the whole animal could provide some clues to enhance the understanding of the inflammatory/tolerance response and to guide drug discovery.This work was supported by grants CSD2007-00002 Aquagenomics of the Consolider-Ingenio 2010 program and AGL2011-28921-C03 IMTRA-VAC from the Spanish Ministerio de Ciencia e Innovación (MICINN) and grant 201230E057 Proyecto Intramural Especial, PIE from Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC). This work was also partially supported by grant 289202 FishForPharma from the European Union.Peer reviewe

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)