Herein we investigated the cellular responses of the endemic fish species Alburnus vistonicus Freyhof & Kottelat, 2007, under the variation of several physico-chemical parameters including temperature (°C), salinity (psu), dissolved oxygen (mg/L), pH and conductivity (μS/cm), which were measured in situ. Monthly fish samplings (October 2014–September 2015) were conducted in Vistonis Lake in northern Greece, a peculiar ecosystem with brackish waters in its southern part and high salinity fluctuations in its northern part. Fish gills and liver responses to the changes of the physico-chemical parameters were tested biochemically and histologically. Heat shock protein levels appeared to be correlated with salinity fluctuations, indicating the adaptation of A. vistonicus to the particular environment. The latter is also enhanced by increased Na⁺-K⁺ ATPase levels, in response to salinity increase during summer. The highest mitogen activated protein kinases phosphorylation levels were observed along with the maximum mean salinity values. A variety of histological lesions were also detected in the majority of the gill samples, without however securing salinity as the sole stress factor. A. vistonicus cellular stress responses are versatile and shifting according to the examined tissue, biomarker and season, in order for this species to adapt to its shifting habitat.

Farmed Atlantic salmon are subjected to different stressors during management routines, leading to prolonged elevated cortisol levels in the blood, a hallmark of chronic stress responses. In this study, we investigate how dexamethasone, an agonist for glucocorticoid receptors (GRs), and cortisol, an endogenous glucocorticoid, regulate gene expression in isolated A. salmon red blood cells (RBCs). Salmonid RBCs express GR genes at high levels. Transcriptional analysis revealed that dexamethasone significantly upregulated 156 genes, and led to > 100-fold induction of the genes FKBP prolyl isomerase 5 (fkbp5), the Krueppel-like factor 9 (klf9), and the DNA damage-inducible transcript 4 (ddit4). The significant upregulation of fkbp5 persisted for up to two weeks post RBC stimulation, indicating its potential as a biomarker candidate for chronic stress exposure. We compared ex vivo transcriptional responses in RBCs with responses in blood cells from A. salmon injected intraperitoneally with hydrocortisone, and found that ddit4 may be the most promising biomarker candidate for stress response in vivo. Dexamethasone and cortisol also downregulated antiviral and proteasome genes triggered by double-stranded RNA in RBCs. The results indicate that glucocorticoids have a profound effect on gene expression and putatively alter functions of A. salmon RBCs, and particularly block the antiviral response.

Piscine orthoreovirus (PRV) infection is common in aquaculture of salmonids. The three known PRV genotypes (PRV-1-3) have host species specificity and cause different diseases, but all infect and replicate in red blood cells (RBCs) in early infection phase. PRV-1 is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon (Oncorhynchus kisutch), while PRV-3 induces HSMI-like disease in farmed rainbow trout (Oncorhynchus mykiss). PRV-3 can also infect A. salmon without causing clinical disease and has been shown to cross-protect against PRV-1 infection and HSMI, while PRV-2 or inactivated adjuvanted PRV-1 vaccine only partially reduced HSMI pathologic changes. In the present work, we studied the transcriptional responses in blood cells of A. salmon two- and five-weeks post infection with PRV-1, PRV-2, PRV-3, or post injection with inactivated PRV-1 vaccine. PRV-1 and PRV-3 replicated well in A. salmon blood cells, and both induced the typical innate antiviral responses triggered by dsRNA viruses. Two weeks post infection, PRV-3 triggered stronger antiviral responses than PRV-1, despite their similar viral RNA replication levels, but after five weeks the induced responses were close to equal. PRV-2 and the InPRV-1 vaccine did not trigger the same typical antiviral responses as the replicating PRV-1 and PRV-3 genotypes, but induced genes involved in membrane trafficking and signaling pathways that may regulate physiological functions. These findings propose that the protection mediated by PRV-3 against a secondary infection by PRV-1 occur due to a potent and early activation of the same type of innate immune responses. The difference in the timing of antiviral responses may give PRV-1 an evolutionary edge, facilitating its dissemination to A. salmon heart, a critical step for HSMI development.

Fish red blood cells (RBCs) are nucleated, transcriptionally active, and key players in both gas transport and immune responses. They are the primary targets of Orthoreovirus piscis (PRV), the etiological agent of heart and skeletal muscle inflammation (HSMI), which includes three genotypes (PRV-1, PRV-2, and PRV-3), linked to circulatory disorders in farmed salmon. In Chile, PRV-3 affects the coho salmon (Oncorhynchus kisutch), but host–pathogen interactions remain poorly characterized. This study compared the interactions of PRV-3 in coho salmon and PRV-1 in Atlantic salmon (Salmo salar) using RBC infection models. RBCs were isolated from healthy juvenile salmon (n = 3) inoculated with either PRV-1 (Ct = 18.87) or PRV-3 (Ct = 21.86). Poly I:C (50 µg/mL) was used as a positive control for the antiviral response. Cells were monitored for up to 14 days post-infection (dpi). PRV-3 infection in coho salmon RBCs caused significant metabolic disruption, apoptosis from 7 dpi, and correlated with increasing viral loads. In contrast, PRV-1 infection in Atlantic salmon RBCs showed limited apoptosis and maintained cell viability. Coho salmon RBCs upregulated rig-i, mx, and pkr transcripts, indicating activation of the type I interferon pathway, whereas Atlantic salmon RBCs exhibited a more attenuated response. PRV-3 induced notable morphological changes in coho salmon RBCs, although neither PRV-3 nor PRV-1 caused hemolysis. These findings highlight species-specific differences in RBC responses to PRV infection and provide new insights into the pathogenesis of PRV-3 and PRV-1.