Fish can show emotional fever: stress-induced hyperthermia in zebrafish

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as ‘emotional fever’. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2–4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes

RNA-Seq Reveals an Integrated Immune Response in Nucleated Erythrocytes

Background: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. Methodology/Principal Findings: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. Conclusions/Significance: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems

Divergent responses to peptidoglycans derived from different E. coli serotypes influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages

Background: Pathogen-associated molecular patterns (PAMPs) are structural components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) from bacterial cell walls. PAMP-recognition by the host results in an induction of defence-related genes and often the generation of an inflammatory response. We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. Results: Transcript profiling was assessed using function-targeted cDNA microarray hybridisation (n = 36) and results show differential responses to both PGNs that are both time and treatment dependent. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this, Gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high similarity to a generalised inflammatory priming response where multiple functional classes are related to ribosome biogenesis or cellular metabolism. Prostaglandin release was induced by both PGNs and macrophages were significantly more sensitive to PGN-O111:B4 as suggested from microarray data. Conclusion: Responses at the level of the transcriptome and the inflammatory outcome (prostaglandin synthesis) highlight the different sensitivity of the macrophage to slight differences (serotype) in peptidoglycan structure. Such divergent responses are likely to involve differential receptor sensitivity to ligands or indeed different receptor types. Such changes in biological response will likely reflect upon pathogenicity of certain serotypes and the development of disease

Molecular characterisation of the underlying mechanisms of pathogen-associated molecular pattern (PAMP) recognition in fish

La respuesta inmune innata es basada en la activación de receptores genotípicamente codificados, llamados receptores de reconocimiento de patógenos (PRR). Los PRR pueden ser proteínas solubles tal como las proteínas plasmáticas PGRPs o pueden estar anclados en las membranas celulares como los TLR. Estos receptores son capaces de reconocer a los patógenos o a sus patrones moleculares (PAMPs). La interacción PAMP-PRR provoca la activación de genes diana y promueve la producción de mediadores pro- e inflamatorios. El principal objetivo de esta tesis doctoral fue la caracterización de las respuestas de macrófagos de la trucha arcoíris Oncorhynchus mykiss, y de la dorada tratados con diferentes PAMPs y la subsecuente exploración de cambios en la expresión de genes relacionados con la respuesta inmune así como cambios globales en la respuesta transcriptómica de los macrófagos. Un objetivo especifico de este estudio fue registrar los cambios en los macrófagos activados hacia un fenotipo inflamatorio después de tratamientos con lipopolisacarido (LPS) crudo de bacterias Gram negativas, enfatizando que el peptidoglicano (PGN) es un contaminante encontrado en las preparaciones crudas de LPS. PGN es capaz de inducir la expresión de mRNA de IL-1β y IL-6 e inducir la liberación de productos inflamatorios como prostaglandinas. Los análisis de microarray fueron hechos para describir la concentración y la dependencia en el tiempo de las modulaciones transcriptómicas en los macrófagos de trucha y dorada tratados con PGN o LPS. En el caso de dorada, se diseño y valido un microarray de oligonucleótidos. Los resultados revelaron la sobre-regulación de transcritos específicos que están cercanamente relacionados con la síntesis de prostaglandinas y las vías de señalización activadas a partir de TLR. Así, el reconocimiento de PGN en peces resulta del reconocimiento de mecanismos específicos que no incluyen TLR pero si otros grupos de receptores como PGRPs o NODs. Estos mecanismos parecen ser conservados en la respuesta de defensa innata a lo largo del grupo de los vertebrados.The innate immune response is based upon the activation of a restricted number of genotypic encoded receptors, the pathogen recognition receptors (PRRs). PRRs can be soluble proteins such as plasmatic PGRPs or cell membraneanchored TLRs able to recognize pathogens or their pathogen-associated molecular patterns (PAMPs). PAMP-PRR interaction results in the activation of target genes and promotes the production of pro- and inflammatory mediators. The main goal of this dissertation was to characterise the responses of rainbow trout, Oncorhynchus mykiss, and gilthead seabream, Sparus aurata, macrophages treated with different PAMPs and to explore subsequent changes in the expression of immune related genes or global shifts in the macrophage transcriptome. A specific goal of this study was to register changes in macrophages activated toward an inflammatory phenotype after treatments with crude gram negative bacterial lipopolysaccharide (LPS) preparations, highlighting that peptidoglycan (PGN) is a contaminant within crude LPS. PGN is able to induce the mRNA expression of IL- 1β and IL-6 and release inflammatory products such as prostaglandins. Microarray analyses were made to describe concentration and time-dependent transcriptional modulations both in trout and seabream macrophages treated with PGN or LPS. In the case of sea bream, a specific oligonucleotide microarray was designed and validated for these studies. Results reveal up-regulation of specific mRNA transcripts that are closely related to prostaglandin synthesis and TLR signalling pathways. Thus PGN recognition in fish is a result of recognition mechanisms including non-TLR PRRs such as PGRPs and NODs. These mechanisms appear to be conserved throughout the vertebrate innate immune response

Evidence for the Induction of Key Components of the NOTCH Signaling Pathway via Deltamethrin and Azamethiphos Treatment in the Sea Louse Caligus rogercresseyi

The extensive use of organophosphates and pyrethroids in the aquaculture industry has negatively impacted parasite sensitivity to the delousing effects of these antiparasitics, especially among sea lice species. The NOTCH signaling pathway is a positive regulator of ABC transporter subfamily C expression and plays a key role in the generation and modulation of pesticide resistance. However, little is known about the molecular mechanisms behind pesticide resistance, partly due to the lack of genomic and molecular information on the processes involved in the resistance mechanism of sea lice. Next-generation sequencing technologies provide an opportunity for rapid and cost-effective generation of genome-scale data. The present study, through RNA-seq analysis, determined that the sea louse Caligus rogercresseyi (C. rogercresseyi) specifically responds to the delousing drugs azamethiphos and deltamethrin at the transcriptomic level by differentially activating mRNA of the NOTCH signaling pathway and of ABC genes. These results suggest that frequent antiparasitic application may increase the activity of inhibitory mRNA components, thereby promoting inhibitory NOTCH output and conditions for increased resistance to delousing drugs. Moreover, data analysis underscored that key functions of NOTCH/ABC components were regulated during distinct phases of the drug response, thus indicating resistance modifications in C. rogercresseyi resulting from the frequent use of organophosphates and pyrethroids

Evidence for the Induction of Key Components of the NOTCH Signaling Pathway via Deltamethrin and Azamethiphos Treatment in the Sea Louse Caligus rogercresseyi

The extensive use of organophosphates and pyrethroids in the aquaculture industry has negatively impacted parasite sensitivity to the delousing effects of these antiparasitics, especially among sea lice species. The NOTCH signaling pathway is a positive regulator of ABC transporter subfamily C expression and plays a key role in the generation and modulation of pesticide resistance. However, little is known about the molecular mechanisms behind pesticide resistance, partly due to the lack of genomic and molecular information on the processes involved in the resistance mechanism of sea lice. Next-generation sequencing technologies provide an opportunity for rapid and cost-effective generation of genome-scale data. The present study, through RNA-seq analysis, determined that the sea louse Caligus rogercresseyi (C. rogercresseyi) specifically responds to the delousing drugs azamethiphos and deltamethrin at the transcriptomic level by differentially activating mRNA of the NOTCH signaling pathway and of ABC genes. These results suggest that frequent antiparasitic application may increase the activity of inhibitory mRNA components, thereby promoting inhibitory NOTCH output and conditions for increased resistance to delousing drugs. Moreover, data analysis underscored that key functions of NOTCH/ABC components were regulated during distinct phases of the drug response, thus indicating resistance modifications in C. rogercresseyi resulting from the frequent use of organophosphates and pyrethroids

Divergent responses to peptidoglycans derived from different E. coli serotypes influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages

Pathogen-associated molecular patterns (PAMPs) are structural components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) from bacterial cell walls. PAMP-recognition by the host results in an induction of defence-related genes and often the generation of an inflammatory response. We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. Transcript profiling was assessed using function-targeted cDNA microarray hybridisation (n = 36) and results show differential responses to both PGNs that are both time and treatment dependent. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this, Gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high similarity to a generalised inflammatory priming response where multiple functional classes are related to ribosome biogenesis or cellular metabolism. Prostaglandin release was induced by both PGNs and macrophages were significantly more sensitive to PGN-O111:B4 as suggested from microarray data. Responses at the level of the transcriptome and the inflammatory outcome (prostaglandin synthesis) highlight the different sensitivity of the macrophage to slight differences (serotype) in peptidoglycan structure. Such divergent responses are likely to involve differential receptor sensitivity to ligands or indeed different receptor types. Such changes in biological response will likely reflect upon pathogenicity of certain serotypes and the development of disease

Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus)

DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring