RESUMO
Smolting in Atlantic salmon Salmo salar is a critical life-history stage that is preparatory for downstream migration and entry to seawater that is regulated by abiotic variables including photoperiod and temperature. The present study was undertaken to determine the interaction of temperature and salinity on salinity tolerance, gill osmoregulatory proteins and cellular and endocrine stress in S. salar smolts. Fish were exposed to rapid changes in temperature (from 14 to 17, 20 and 24°C) in fresh water (FW) and seawater (SW), with and without prior acclimation and sampled after 2 and 8 days. Fish exposed simultaneously to SW and 24°C experienced 100% mortality, whereas no mortality occurred in any of the other groups. The highest temperature also resulted in poor ion regulation in SW with or without prior SW acclimation, whereas no substantial effect was observed in FW. Gill Na+ -K+ -ATPase (NKA) activity increased in SW fish compared to FW fish and decreased with high temperature in both FW and SW. Gill Nkaα1a abundance was high in FW and Nkaα1b and Na+ -K+ -2Cl- cotransporter high in SW, but all three were lower at the highest temperature. Gill Hsp70 levels were elevated in FW and SW at the highest temperature and increased with increasing temperature 2 days following direct transfer to SW. Plasma cortisol levels were elevated in SW at the highest temperature. Our results indicate that there is an important interaction of salinity and elevated temperature on osmoregulatory performance and the cellular stress response in S. salar, with an apparent threshold for osmoregulatory failure in SW above 20°C.
Assuntos
Brânquias/enzimologia , Temperatura Alta , Salmo salar/sangue , Tolerância ao Sal , Equilíbrio Hidroeletrolítico , Aclimatação/fisiologia , Animais , Sistema Endócrino , Água Doce , Proteínas de Choque Térmico HSP70/metabolismo , Osmorregulação , Salinidade , Salmão/metabolismo , Água do Mar , Sódio/sangue , ATPase Trocadora de Sódio-Potássio/metabolismo , Estresse Fisiológico , TemperaturaRESUMO
MicroRNAs are key non-coding RNA molecules that play a relevant role in the regulation of gene expression through translational repression and/or transcript cleavage during normal development and physiological adaptation processes like stress. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become the approach normally used to determine the levels of microRNAs. However, this approach needs the use of endogenous reference. An improper selection of endogenous references can result in confusing interpretation of data. The aim of this study was to identify and validate appropriate endogenous reference miRNA genes for normalizing RT-qPCR survey of miRNAs expression in four different tissues of Atlantic salmon, under handling and confinement stress conditions associated to early or primary stress response. Nine candidate reference normalizers, including microRNAs and nuclear genes, normally used in vertebrate microRNA expression studies were selected from literature, validated by RT-qPCR and analyzed by the algorithms geNorm and NormFinder. The results revealed that the ssa-miR-99-5p gene was the most stable overall and that ssa-miR-99-5p and ssa-miR-23a-5p genes were the best combination. Moreover, the suitability of ssa-miR-99-5p and ssa-miR-23a-5p as endogeneuos reference genes was demostrated by the expression analysis of ssa-miR-193-5p gene.
Assuntos
MicroRNAs/genética , Salmo salar/genética , Estresse Fisiológico/genética , Animais , Regulação da Expressão Gênica , Estudos de Associação Genética , Hidrocortisona/sangue , MicroRNAs/metabolismo , Padrões de Referência , Salmo salar/sangueRESUMO
Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.
Assuntos
Anticorpos Monoclonais/sangue , Isavirus/isolamento & purificação , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/virologia , Animais , Canadá , Chile , Ensaio de Imunoadsorção Enzimática , Europa (Continente) , Doenças dos Peixes/virologia , Isavirus/patogenicidade , América do Norte , Noruega , Infecções por Orthomyxoviridae/veterinária , Salmo salar/sangue , Salmo salar/virologia , EscóciaRESUMO
The year-round presence of ovigerous females of the parasite Caligus rogercresseyi in the fish farms of southern Chile results in a continuous source of the copepodid (infestive) stage of this louse. The short generation time in spring-summer could lead to high abundances of this copepodid, potentially leading to high infestation levels for fish. Knowing how heavy lice infestations affect Salmo salar can help determine how to time antiparasitic treatments so as to both minimize the treatment impact and reduce lice infestation levels for fish. This study aimed to describe the effects of high infestations of the copepodid stage of C. rogercresseyi on the physiology of S. salar. Two groups of S. salar were used: an infested group (75 copepodids per fish) and a control group (not infested). Sixty-five days after the first infestation, the infested fish group was re-infested at an infestation pressure of 200 copepodids per fish. Sampling was done prior to and following the second infestation, at 56 and 67 days (the latter 2 days following the second infestation). Several physiological variables were measured: cortisol (primary stress response) and glucose, proteins, amino acids, triglycerides, lactate, osmolality levels, and number and diameter of skin mucous cells (secondary stress responses). The plasma cortisol, glucose, and triglyceride levels were altered in the heavily infested fish, as was the diameter of skin mucous cells. These results suggest that heavy infestations of C. rogercresseyi lead to an acute stress response, metabolic reorganization, and increased mucus production in S. salar under heavy infestation conditions.
Assuntos
Copépodes/fisiologia , Ectoparasitoses/sangue , Doenças dos Peixes/sangue , Salmo salar/parasitologia , Estresse Fisiológico , Aminoácidos/sangue , Animais , Glicemia/análise , Proteínas Sanguíneas/metabolismo , Ectoparasitoses/metabolismo , Ectoparasitoses/veterinária , Feminino , Doenças dos Peixes/metabolismo , Proteínas de Peixes/sangue , Hidrocortisona/sangue , Ácido Láctico/sangue , Muco/metabolismo , Salmo salar/sangue , Salmo salar/metabolismo , Pele/metabolismo , Triglicerídeos/sangueRESUMO
Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression or its effects on innate immunity are unclear even in mammals. To study the possible presence of PrRP in peripheral organs, PrRP expression patterns and their effect on innate immunity were characterised in SHK-1 cells and head kidney (HK) leukocytes purified from the salmonid, Salmo salar. We detected immunoreactive cells in leukocytes from blood and HK of S. salar and found that PrRP mRNA was abundantly expressed in these cells. We have recently reported that physiological concentrations of native PRL, downstream of neuropeptide PrRP were able to induce expression of pro-inflammatory cytokines and the production of reactive oxygen species (ROS) in HK leukocytes and macrophages from S. salar and Sparus aurata. It is of interest to note that in this work we have revealed that synthetic PrRP was able to induce expression of pro-inflammatory cytokines (interleukins) IL-1ß, IL-6, IL-8, IL-12 and PRL. We also show here that PrRP increased both (ROS) production and phagocytosis. Taken together, our results demonstrate for the first time that PrRP may be a local modulator of innate immune responses in leukocytes from S. salar.
Assuntos
Proteínas de Peixes/imunologia , Hormônio Liberador de Prolactina/imunologia , Salmo salar/imunologia , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/farmacologia , Expressão Gênica , Imunidade Inata , Interleucinas/genética , Leucócitos/imunologia , Leucócitos/metabolismo , Fagocitose , Prolactina/genética , Hormônio Liberador de Prolactina/genética , Hormônio Liberador de Prolactina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Explosão Respiratória , Salmo salar/sangue , Salmo salar/genéticaRESUMO
Two systems for transporting live salmon (Salmo salar) were compared in terms of their effects on blood variables, muscle pH and rigor index: an 'open system' well-boat with recirculated sea water at 13.5 degrees C and a stocking density of 107 kg/m3 during an eight-hour journey, and a 'closed system' well-boat with water chilled from 16.7 to 2.1 degrees C and a stocking density of 243.7 kg/m3 during a seven-hour journey. Groups of 10 fish were sampled at each of four stages: in cages at the farm, in the well-boat after loading, in the well-boat after the journey and before unloading, and in the processing plant after they were pumped from the resting cages. At each sampling, the fish were stunned and bled by gill cutting. Blood samples were taken to measure lactate, osmolality, chloride, sodium, cortisol and glucose, and their muscle pH and rigor index were measured at death and three hours later. In the open system well-boat, the initial muscle pH of the fish decreased at each successive stage, and at the final stage they had a significantly lower initial muscle pH and more rapid onset of rigor than the fish transported on the closed system well-boat. At the final stage all the blood variables except glucose were significantly affected in the fish transported on both types of well-boat.