Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.

Aquaculture is currently the fastest-growing sector in animal production, with an average annual growth rate of 7.5% since 1970. In Chile, the industry is largely driven by salmonid farming, with Salmo salar (Atlantic salmon) accounting for over 65% of national production. This species has shown the most significant growth within the sector. This growth is achieved by having high-density farming, which results in high levels of stress due to overcrowding and the appearance of pathogens such as the Infectious Salmon Anemia (ISA) virus, Bacterial Kidney Disease (BKD), Caligus sea lice (Caligus rogercresseyi), and Piscirickettsiosis (SRS) caused by Piscirickettsia salmonis, among others. This study evaluated the toxicity of the two most commonly used antibiotics in the salmon industry—oxytetracycline and florfenicol—at four concentrations each, using primary liver cell cultures of Atlantic salmon (Salmo salar). Oxidative stress was assessed through enzymatic activity and gene expression of oxidative markers, including cytochrome P450, catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). Samples were analyzed at 1, 3, 6, 12, and 48 h post-exposure. These findings reveal time- and dose-dependent oxidative responses in salmon liver cells to OTC and FLO, providing critical insights into the sublethal cellular effects of antibiotics commonly used in aquaculture, which indicates the presence of a high amount of free radicals in the liver cells, indicating toxicity of both antibiotics.

The aquaculture industry in Chile, as in the rest of the world, has rapidly grown, becoming a crucial economic sector. However, diseases pose a major threat, causing significant economic losses and environmental impacts. Various antimicrobials, particularly Oxytetracycline and Florfenicol, are used to combat these diseases, which has boosted production and mitigated economic losses. However, excessive antibiotic use has led to pathogen resistance, necessitating higher doses. This overuse can cause side effects in fish, including liver damage and immunosuppression. This study aimed to determine the impact of multiple doses of florfenicol and oxytetracycline on the SHK-11 cell line of Salmo salar by analyzing the expression of genes related to innate immunity and oxidative stress by qRT-PCR in addition to the quantification of immune system proteins via dot blot. The experimental treatments were the following: cells were stimulated with different concentrations of oxytetracycline (0.25, 0.5, and 1.5 µg/mL) and florfenicol (1, 10, and 20 µg/mL) for time kinetics of 0.5, 1, 3, 6, 12, 24, and 48 h. For both cases, controls consisting of cells without antibiotics were included. The expression of the immune system genes was mostly inhibited compared to the control. However, it was observed that TLR-1 and MyD88 present a joint activation pattern at different times and concentrations for both antibiotics. Regarding the expression of CAT and GPx, transcripts were increased in the early stages of stimulation with oxytetracycline and florfenicol, followed by a subsequent decrease in gene expression. This study provides relevant information to understand the effect of antibiotics at the cellular level in one of the most important species for global aquaculture, the Atlantic salmon.

Aquaculture fish are kept for long periods in sea cages or tanks. Consequently, accumulated stress causes the fish to present serious problems with critical economic losses. Fish food has been supplemented to reduce this stress, using many components as amino acids such as tryptophan. This study aims to determine the transcriptional effect of tryptophan and cortisol on primary cell cultures of salmon head and posterior kidney. Our results indicate activation of the kynurenine pathway and serotonin activity when stimulated with tryptophan and cortisol. An amount of 95% of tryptophan is degraded by the kynurenine pathway, indicating the relevance of knowing how this pathway is activated and if stress levels associated with fish culture trigger its activation. Additionally, it is essential to know the consequence of increasing kynurenic acid “KYNA” levels in the short and long term, and even during the fish ontogeny.

Rising ocean temperatures due to climate change, combined with the intensification of anthropogenic activity, may lead to changes in the physiology and distribution of native species. Compounding climate stress, microplastic particles (MPs) enter the oceans through wastewater and the breakdown of macroplastics. Depending on their composition, they can be harmful and act as a vehicle for toxic substances, although their effects on native Antarctic and subantarctic species are unknown. Notothenioid fish are members of this group and are found inside and outside Antarctica, such as the Harpagifer, which has adapted to the cold and is particularly sensitive to thermal increases. Here, we aimed to evaluate the innate immune response in the head kidney, spleen, and foregut of two notothenoid fish, Harpagifer antarcticus and Harpagifer bispinis, exposed to elevated temperatures and PVC (polyvinyl chloride) microplastics. Adults from both species were collected on King George Island (Antarctica) and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or exposed to a temperature increase (TI) or PVC microplastics (MPs), separately or in combination (MPs + TI). MP exposures were oral (gavage) for 24 h or aqueous (in a bath) for 24 and 48 h. Using real-time qPCR, we evaluated the relative gene expression of markers involved in the innate immune response, including tlr2 (toll-like receptor 2), tlr4 (toll-like receptor 4), myd88 (myeloid differentiation factor 88), nfkb (nuclear factor kb), il6 (interleukin 6), and il8 (irterleukin 8). We found differences between treatments when H. antarcticus and H. bispinis were exposed independently to MPs or thermal increase (TI) in the experiment with a cannula, showing an up-regulation in transcripts. In contrast, a down-regulation was observed when exposed in combination to MP + TI, which looked to be tissue-dependent. However, transcripts related to innate immunity in the bath experiment increased when exposure to both stressors was combined, mostly at 48 h. These results highlight the importance of evaluating the effects of multiple stressors, both independently and in combination, and whether these species will have the capacity to adapt or survive under these conditions, especially in waters where temperature is increasing and pollution is also rising, primarily from MP-PVC, a plastic widely used in various industries and among the population.