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Saxitoxin (STX) represents a marine toxin of significant concern due to its deleterious implications for aquatic ecosystems and public food safety. As a potent paralytic agent, the role of STX in obstructing voltage-gated sodium channels (VGSCs) is well-characterized. Yet, the mechanistic details underlying its low-dose toxicity remain largely enigmatic. In the current study, zebrafish embryos and larvae were subjected to subchronic exposure of graded STX concentrations (0, 1, 10, and 100 µg/L) until the 7th day post-fertilization. A tactile stimulus-based assay was employed to evaluate potential behavioral perturbations resulting from STX exposure. Both behavioral and transcription level analyses unveiled a compromised tactile response, which was found to be associated with a notable upregulation in the mRNA of two distinct VGSC isoforms, specifically the scn8aa/ab and scn1Laa/ab transcripts, even at the minimal STX dose. Notably, exposure to this lowest STX concentration also resulted in alterations in the transcriptional patterns of pivotal genes for cholinergic and GABAergic pathways, including ache and gabra1. Furthermore, STX induced a marked decrease in the levels of the neurotransmitter GABA. Our findings underscore that prolonged low-dose STX exposure during early development can significantly compromise the tactile response behavior in zebrafish. This study reveals that chronic low-dose STX exposure of developing zebrafish alters neurotransmission pathways that converge on altered tactile behavior.
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A variety of long-term stress conditions may exist in fish cultivation, some of which are so severe that fish can no longer reestablish homeostasis. In teleost fish, the brain and gastrointestinal tract integrate signals that include the perception of stress factors regulating physiological responses, such as social stress by fish population density, where peripheral and central signals, such as peptide hormones, are the main regulators. Therefore, we proposed in this study to analyze the effect of different stock densities (SD) in the gene expression of brain neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), together with the gastrointestinal peptide hormones leptin (Lep), vasointestinal peptide (VIP), and protachykinin-1 (Prk-1) in Salmo salar post-smolt. The coding sequence of S. salar VIP and Prk-1 precursors were firstly cloned and characterized. Then, the mRNA expression of these genes, together with the NPY, Lep, and CGRP genes, were evaluated in post-smolts kept at 11 Kg/m3, 20 Kg/m3, and 40 Kg/m3. At 14 days of culture, the brain CGRP and liver leptin mRNA levels increased three and tenfold in the post-smolt salmons kept at the highest SD, respectively. The high levels of leptin were kept during all the fish culture experiments. In addition, the highest expression of intestine VIP mRNA was obtained on Day 21 in the group of 40 Kg/m3 returning to baseline on Day 40. In terms of stress biochemical parameters, cortisol levels were increased in the 20 Kg/m3 and 40 Kg/m3 groups on Day 40 and were the highest in the 20 Kg/m3 group on Day 14. This study provides new insight into the gastrointestinal signals that could be affected by chronic stress induced by high stock density in fish farming. Thus, the expression of these peptide hormones could be used as molecular markers to improve production practices in fish aquaculture.
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The feeding behavior in fish is a complex activity that relies on the ability of the brain to integrate multiple signals to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Upon stress cues including viral infection or mediators such as the proinflammatory cytokines, prostaglandins, and cortisol, both Pomc and Npy/Agrp neurons from the hypothalamus are stimulated, thus triggering a response that controls both energy storage and expenditure. However, how appetite modulators or neuro-immune cues link pathogenesis and energy homeostasis in fish remains poorly understood. Here, we provide the first evidence of a molecular linkage between inflammation and food intake in Salmon salar. We show that in vivo viral challenge with infectious pancreatic necrosis virus (IPNV) impacts food consumption by activating anorexic genes such as mc4r, crf, and pomcb and 5-HT in the brain of S. salar. At the molecular level, viral infection induces an overall reduction in lipid content in the liver, favoring the production of AA and EPA associated with the increment of elovl2 gene. In addition, infection upregulates leptin signaling and inhibits insulin signaling. These changes are accompanied by a robust inflammatory response represented by the increment of Il-1b, Il-6, Tnfa, and Pge2 as well as an increased cortisol level in vivo. Thus, we propose a model in which hypothalamic neurons respond to inflammatory cytokines and stress-related molecules and interact with appetite induction/inhibition. These findings provide evidence of crosstalk between pathogenesis-driven inflammation and hypothalamic-pituitary-adrenocortical axes in stress-induced food intake behavior in fish.
Assuntos
Infecções por Birnaviridae , Comportamento Alimentar , Hipotálamo/metabolismo , Inflamação , Metabolismo dos Lipídeos , Salmo salar/fisiologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Hipotálamo/fisiologia , Vírus da Necrose Pancreática Infecciosa , Insulina/metabolismo , Leptina/metabolismo , Salmo salar/metabolismo , Salmo salar/virologia , Transdução de SinaisRESUMO
BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.
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Comportamento Animal , Infecções por Birnaviridae/complicações , Febre/patologia , Imunidade , Vírus da Necrose Pancreática Infecciosa/fisiologia , Inflamação/patologia , Reflexo , Animais , Infecções por Birnaviridae/virologia , Regulação da Temperatura Corporal , Citocinas/metabolismo , Febre/etiologia , Peixes , Inflamação/etiologiaRESUMO
Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.
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Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunoglobulinas/metabolismo , Interferon gama/imunologia , Glicoproteínas de Membrana/metabolismo , Salmo salar/imunologia , Baço/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Biomarcadores/metabolismo , Doenças dos Peixes/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Piscirickettsia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/veterinária , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Antígeno CD83RESUMO
Teleost fish are exposed to diverse stressors in farming and wildlife conditions during their lifespan. Cortisol is the main glucocorticoid hormone involved in the regulation of their metabolic acclimation under physiological stressful conditions. In this context, increased plasma cortisol is associated with energy substrate mobilization from metabolic tissues, such as liver and skeletal muscle, to rapidly obtain energy and cope with stress. The metabolic actions of cortisol have primarily been attributed to its genomic/classic action mechanism involving the interaction with intracellular receptors, and regulation of stress-responsive genes. However, cortisol can also interact with membrane components to activate rapid signaling pathways. In this work, using the teleost fish gilthead sea bream (Sparus aurata) as a model, we evaluated the effects of membrane-initiated cortisol actions on the early modulation of glucose metabolism. For this purpose, S. aurata juveniles were intraperitoneally administrated with cortisol and with its membrane impermeable analog, cortisol-BSA. After 1 and 6 h of each treatment, plasma cortisol levels were measured, together with glucose, glycogen and lactate in plasma, liver and skeletal muscle. Transcript levels of corticosteroids receptors (gr1, gr2, and mr) and key gluconeogenesis (g6pc and pepck)- and glycolysis (pgam1 and aldo) related genes in the liver were also measured. Cortisol and cortisol-BSA administration increased plasma cortisol levels in S. aurata 1 h after administration. Plasma glucose levels enhanced 6 h after each treatment. Hepatic glycogen content decreased in the liver at 1 h of both cortisol and cortisol-BSA administration, while increased at 6 h due to cortisol but not in response to cortisol-BSA. Expression of gr1, g6pc, pgam1, and aldo were preferentially increased by cortisol-BSA in the liver. Taking all these results in consideration, we suggest that non-canonical cortisol mechanisms contribute to the regulation of the early glucose metabolism responses to stress in S. aurata.
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Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.
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Adesões Focais/efeitos dos fármacos , Hidrocortisona/farmacologia , Músculo Esquelético/efeitos dos fármacos , Oncorhynchus mykiss/genética , Transcriptoma/efeitos dos fármacos , Animais , Glicemia/análise , Proteínas de Peixes/genética , Adesões Focais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrocortisona/administração & dosagem , Hidrocortisona/sangue , Músculo Esquelético/metabolismo , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estresse Fisiológico/genética , Regulação para CimaRESUMO
Cortisol is the main glucocorticoid hormone in teleosts involved in the regulation of metabolic adjustments under both normal and stressful physiological conditions. In the skeletal muscle, cortisol modulates the energetic metabolism promoting the mobilization of glucose and other energetic substrates to overcome the stress stimulus. The effects of cortisol-mediated stress response are attributed to canonical/genomic mechanisms which involve the interaction of the hormone with its intracellular glucocorticoid receptor and, consequently, modulation of target genes. However, cortisol also can interact with membrane components, activating rapid signaling pathways with unknown contribution during the early stress response. In the present work, we evaluated the impact of membrane-initiated cortisol action over the expression of the critical modulator of energetic metabolism, pyruvate dehydrogenase kinase 2 (pdk2), in fish skeletal muscle. Juvenile rainbow trout were intraperitoneally administered with stress-related doses of cortisol and cortisol-BSA, and the expression of pdk2 was assayed by using RT-qPCR. Our results reveal that pdk2 mRNA levels increased in the skeletal muscle at one hour in both cortisol- and cortisol-BSA-treated fish. Moreover, in vitro studies revealed a biphasic response over the pdk2 regulation in myotubes mediated first through membrane-cortisol signaling pathways followed by the classic cortisol action. Finally, pdk2 up-regulation owing to cortisol and cortisol-BSA is reverted in RU486 treated myotubes, suggesting that GR signaling participates in both cortisol signaling pathways. This work suggests that non-classical cortisol pathways contribute to regulate the early metabolic response to stress in fish skeletal muscle.
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Hidrocortisona/farmacologia , Oncorhynchus mykiss/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Estresse Fisiológico/genética , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Hidrocortisona/metabolismo , Músculo Esquelético/efeitos dos fármacos , Oncorhynchus mykiss/fisiologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
Fish are ectotherm organisms that move through different thermal zones according to their physiological requirements and environmental availability, a behavior known as thermoregulation. Thermoregulation in ectothermic animals is influenced by their ability to effectively respond to thermal variations. While it is known that ectotherms are affected by thermal changes, it remains unknown how physiological and/or metabolic traits are impacted by modifications in the thermal environment. In captivity (land-based infrastructures or nets located in the open sea), fish are often restricted to spatially constant temperature conditions within the containment unit and cannot choose among different thermal conditions for thermoregulation. In order to understand how spatial variation of temperature may affect fish welfare and stress, we designed an experiment using either restricted or wide thermal ranges, looking for changes at hormonal and molecular levels. Also, thermal variability impact on fish behavior was measured. Our results showed that in Atlantic salmon (Salmo salar), a wide thermal range (ΔT 6.8°C) was associated with significant increases in monoamines hormone levels and in the expression of clock genes. Aggressive and territoriality behavior decreased, positively affecting parameters linked to welfare, such as growth and fin damage. In contrast, a restricted thermal range (ΔT 1.4°C) showed the opposite pattern in all the analyzed parameters, therefore, having detrimental effects on welfare. In conclusion, our results highlight the key role of thermal range amplitude on fish behavior and on interactions with major metabolism-regulating processes, such as hormone performance and molecular regulatory mechanisms that have positive effects on the welfare.
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Iron is a trace element, essential to support life due to its inherent ability to exchange electrons with a variety of molecules. The use of iron as a cofactor in basic metabolic pathways is essential to both pathogenic microorganisms and their hosts. During evolution, the shared requirement of micro- and macro-organisms for this important nutrient has shaped the pathogen-host relationship. Infectious pancreatic necrosis virus (IPNv) affects salmonids constituting a sanitary problem for this industry as it has an important impact on post-smolt survival. While immune modulation induced by IPNv infection has been widely characterized on Salmo salar, viral impact on iron host metabolism has not yet been elucidated. In the present work, we evaluate short-term effect of IPNv on several infected tissues from Salmo salar. We observed that IPNv displayed high tropism to headkidney, which directly correlates with a rise in oxidative stress and antiviral responses. Transcriptional profiling on headkidney showed a massive modulation of gene expression, from which biological pathways involved with iron metabolism were remarkable. Our findings suggest that IPNv infection increase oxidative stress on headkidney as a consequence of iron overload induced by a massive upregulation of genes involved in iron metabolism.
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Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/metabolismo , Fenômenos Fisiológicos da Nutrição/imunologia , Estresse Oxidativo , Viroses/veterinária , Animais , Biomarcadores , Doenças dos Peixes/patologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Ferro/metabolismo , Sobrecarga de Ferro/patologia , Transcriptoma , Carga ViralRESUMO
Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon known as behavioral fever. The cumulative evidence suggests that behavioral fever impacts positively upon lymphocyte proliferation, inflammatory cytokine expression, and other immune functions. In this study, we have explored how thermal choice during infection impacts upon underpinning molecular processes and how temperature increase is coupled to the immune response. Our results show that behavioral fever results in a widespread, plastic imprint on gene regulation, and lymphocyte proliferation. We further explored the possible contribution of histone modification and identified global associations between temperature and histone changes that suggest epigenetic remodeling as a result of behavioral fever. Together, these results highlight the critical importance of thermal choice in mobile ectotherms, particularly in response to an infection, and demonstrate the key role of epigenetic modification to orchestrate the thermocoupling of the immune response during behavioral fever.
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Regulação da Temperatura Corporal , Epigênese Genética , Peixes/fisiologia , Imunidade , Animais , Comportamento Animal , Biologia Computacional/métodos , Citocinas/metabolismo , Perfilação da Expressão Gênica , Imunidade Inata , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , TranscriptomaRESUMO
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 1426bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 up-regulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation 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 non-model teleost species.
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Enguias/genética , Enguias/imunologia , Rim/química , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Transcriptoma/genética , Transcriptoma/imunologia , Animais , Perfilação da Expressão Gênica , Cabeça/fisiologia , Rim/imunologia , Rim/metabolismo , Análise de Sequência de RNARESUMO
Sea lice infestations are a particular concern in the salmonid aquaculture industry due to damaging effects on fish growth, disease/infection susceptibility, and survival. Despite the impacts of sea lice parasitism, few studies have determined corresponding physiological thresholds, or the quantity of sea lice that can trigger measurable effects in the host immune response. The present study evaluated the mRNA expressions of immune-related genes in Salmo salar (Atlantic salmon) under infestation challenges with contrasting loads of the sea louse Caligus rogercresseyi. Specifically, two groups of S. salar were infected with either 35 (i.e. low parasitic load) or 100 (i.e. high parasitic load) copepodids per fish. At 14 days post-infestation, the mRNA levels of immune-related genes (e.g. related to oxidative stress, pro- and inflammatory responses, and the adaptive TH1/TH2 pathways) were assessed through RT-qPCR. Significant differences were found in relation to parasitic load, suggesting density-dependent effects that activated the S. salar immune system. Higher parasitic load promoted strong inflammatory and oxidative stress responses that were correlated with the TH1 immune response. This study highlights the molecular signatures for distinct parasitic loads, providing new perspectives towards fully understanding parasite-host interactions.
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Copépodes/imunologia , Ectoparasitoses/veterinária , Doenças dos Peixes/imunologia , Interações Hospedeiro-Parasita/imunologia , Salmo salar , Animais , Chile , Ectoparasitoses/imunologia , Ectoparasitoses/parasitologia , Doenças dos Peixes/parasitologia , Densidade Demográfica , Células Th1/metabolismoRESUMO
Caligus rogercresseyi, an ectoparasite affecting the Chilean salmon industry, can cause immunosuppression and physiological stress in farmed fish. Interestingly, coho salmon (Oncorhynchus kisutch) are notably resistant to infestation, whereas Atlantic salmon (Salmo salar) are phenotypically more susceptible to sea lice. However, comparative studies on immune responses to C. rogercresseyi have not been conducted. In this study, Illumina sequencing was conducted to evaluate head kidney and skin samples taken 7 and 14 days post-infestation, yielding a total of 1492 and 1522 contigs annotated to immune-related genes for Atlantic and coho salmon, respectively. Both species evidenced an upregulation of inflammatory genes. Atlantic salmon had highly upregulated TLR22 and MHCII at 14 days post-infestation, while coho salmon had highly upregulated stat5 and il1r transcripts. Fourteen transcripts related to TH1, TH2, TLR, and macrophage responses were corroborated via RT-qPCR. Statistical analyses indicated an upregulation of mmp13, cox2, il10, ccr3, tlr22a2, and tlr21 in Atlantic salmon and of ifnγ, cd83, T-bet, tlr13, and tlr19 in coho salmon. These results suggest strong differences between the Atlantic and coho salmon immune responses, where coho salmon, the more resistant species, presented a primary TH1 response. Additionally, putative roles of TLRs in salmonids against sea lice were evidenced. This study is the first comparative transcriptome analysis that reveals species-specific immune responses in salmons infected with C. rogercresseyi.
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Copépodes/fisiologia , Ectoparasitoses/veterinária , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Oncorhynchus kisutch , Salmo salar , Animais , Chile , Ectoparasitoses/genética , Ectoparasitoses/imunologia , Ectoparasitoses/parasitologia , Doenças dos Peixes/genética , Doenças dos Peixes/parasitologia , Proteínas de Peixes/metabolismo , Especificidade da Espécie , TranscriptomaRESUMO
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.
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Copépodes/genética , Perfilação da Expressão Gênica/métodos , Glutamato-Amônia Ligase/genética , Praguicidas/farmacologia , Receptores de Glutamato Metabotrópico/genética , Análise de Sequência de RNA/métodos , Animais , Células Quimiorreceptoras/efeitos dos fármacos , Copépodes/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Nitrilas/farmacologia , Organotiofosfatos/farmacologia , Piretrinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
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.
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Copépodes/metabolismo , Nitrilas/toxicidade , Praguicidas/toxicidade , Piretrinas/toxicidade , Receptores Notch/metabolismo , Animais , Copépodes/efeitos dos fármacos , Copépodes/genética , Resistência a Medicamentos/genética , Organotiofosfatos/toxicidade , Transdução de SinaisRESUMO
Despite evidence for participation in the host response to infection, the roles of many long non-coding RNAs (lncRNAs) remain unknown. Therefore, the aims of this study were to identify lncRNAs in Atlantic salmon (Salmo salar) and evaluate their transcriptomic regulation during ISA virus (ISAV) infection, an Orthomyxoviridae virus associated with high mortalities in salmonid aquaculture. Using next-generation sequencing, whole-transcriptome analysis of the Salmo salar response to ISAV infection was performed, identifying 5,636 putative lncRNAs with a mean length of 695 base pairs. The transcriptional modulation evidenced a similar number of differentially expressed lncRNAs in the gills (3,294), head-kidney (3,275), and liver (3,325) over the course of the infection. Moreover, analysis of a subset of these lncRNAs showed the following: (i) Most were similarly regulated in response to ISA virus infection; (ii) The transcript subsets were uniquely modulated in each tissue (gills, liver, and head-kidney); and (iii) A subset of lncRNAs were upregulated for each tissue and time analysed, indicating potential markers for ISAV infection. These findings represent the first discovery of widespread differential expression of lncRNAs in response to virus infection in non-model species, suggesting that lncRNAs could be involved in regulating the host response during ISAV infection.
Assuntos
Doenças dos Peixes/patologia , Imunomodulação , Isavirus/imunologia , Infecções por Orthomyxoviridae/veterinária , RNA Longo não Codificante/análise , Salmo salar , Animais , Doenças dos Peixes/virologia , Perfilação da Expressão Gênica , Brânquias/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Isavirus/patogenicidade , Rim/patologia , Fígado/patologia , Infecções por Orthomyxoviridae/patologia , RNA Longo não Codificante/genética , Análise de Sequência de DNARESUMO
Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing high mortality in farmed Atlantic salmon (Salmo salar). The collective data from the Atlantic salmon-ISAV interactions, performed "in vitro" using various salmon cell lines and "in vivo" fish infected with different ISAV isolates, have shown a strong regulation of immune related transcripts during the infection. Despite this strong defence response, the majority of fish succumb to infections with ISAV. The deficient protection of the host against ISAV is in part due to virulence factors of the virus, which allow evade the host-defence machinery. As such, the viral replication is uninhibited and viral loads quickly spread to several tissues causing massive cellular damage before the host can develop an effective cell-mediated and humoral outcome. To interrogate the correlation of the viral replication with the host defence response, we used fish that have been infected by cohabitation with ISAV-injected salmons. Whole gene expression patterns were measured with RNA-seq using RNA extracted from Head-kidney, Liver and Gills. The results show divergent mRNA abundance of functional modules related to interferon pathway, adaptive/innate immune response and cellular proliferation/differentiation. Furthermore, gene regulation in distinct tissues during the infection process was independently controlled within the each tissue and the observed mRNA expression suggests high modulation of the ISAV-segment transcription. Importantly this is the first time that strong correlations between functional modules containing significant immune process with protein-protein affinities and viral-segment transcription have been made between different tissues of ISAV-infected fish.