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Nutritional immunity regulates the homeostasis of micronutrients such as iron, manganese, and zinc at the systemic and cellular levels, preventing the invading microorganisms from gaining access and thereby limiting their growth. Therefore, the objective of this study was to evaluate the activation of nutritional immunity in specimens of Atlantic salmon (Salmo salar) that are intraperitoneally stimulated with both live and inactivated Piscirickettsia salmonis. The study used liver tissue and blood/plasma samples on days 3, 7, and 14 post-injections (dpi) for the analysis. Genetic material (DNA) of P. salmonis was detected in the liver tissue of fish stimulated with both live and inactivated P. salmonis at 14 dpi. Additionally, the hematocrit percentage decreased at 3 and 7 dpi in fish stimulated with live P. salmonis, unchanged in fish challenged with inactivated P. salmonis. On the other hand, plasma iron content decreased during the experimental course in fish stimulated with both live and inactivated P. salmonis, although this decrease was statistically significant only at 3 dpi. Regarding the immune-nutritional markers such as tfr1, dmt1, and ireg1 were modulated in the two experimental conditions, compared to zip8, ft-h, and hamp, which were down-regulated in fish stimulated with live and inactivated P. salmonis during the course experimental. Finally, the intracellular iron content in the liver increased at 7 and 14 dpi in fish stimulated with live and inactivated P. salmonis, while the zinc content decreased at 14 dpi under both experimental conditions. However, stimulation with live and inactivated P. salmonis did not alter the manganese content in the fish. The results suggest that nutritional immunity does not distinguish between live and inactivated P. salmonis and elicits a similar immune response. Probably, this immune mechanism would be self-activated with the detection of PAMPs, instead of a sequestration and/or competition of micronutrients by the living microorganism.

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The innate immune system can limit the growth of invading pathogens by depleting micronutrients at a cellular and tissue level. However, it is not known whether nutrient depletion mechanisms discriminate between living pathogens (which require nutrients) and pathogen-associated molecular patterns (PAMPs) (which do not). We stimulated SHK-1 cells with different PAMPs (outer membrane vesicles of Piscirickettsia salmonis "OMVs", protein extract of P. salmonis "TP" and lipopolysaccharides of P. salmonis "LPS") isolated from P. salmonis and evaluated transcriptional changes in nutritional immunity associated genes. Our experimental treatments were: Control (SHK-1 stimulated with bacterial culture medium), OMVs (SHK-1 stimulated with 1µg of outer membrane vesicles), TP (SHK-1 stimulated with 1µg of total protein extract) and LPS (SHK-1 stimulated with 1µg of lipopolysaccharides). Cells were sampled at 15-, 30-, 60- and 120-minutes post-stimulation. We detected increased transcription of zip8, zip14, irp1, irp2 and tfr1 in all three experimental conditions and increased transcription of dmt1 in cells stimulated with OMVs and TP, but not LPS. Additionally, we observed generally increased transcription of ireg-1, il-6, hamp, irp1, ft-h and ft-m in all three experimental conditions, but we also detected decreased transcription of these markers in cells stimulated with TP and LPS at specific time points. Our results demonstrate that SHK-1 cells stimulated with P. salmonis PAMPs increase transcription of markers involved in the transport, uptake, storage and regulation of micronutrients such as iron, manganese and zinc.

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Thermal and saline variations of the Southern Ocean are important signs of climate change which can alter the physiological responses of stenotic species residing at high latitudes. Our study aimed to evaluate the cellular stress response (CSR) of Harpagifer antarcticus subjected to increased ambient temperature and decreased salinity. The fish were distributed in different thermal (2, 5, 8, 11, and 14 °C) and saline (23, 28, and 33 psu) combinations for 10 days. We used qPCR analysis to evaluate the transcription of genes involved in the thermal shock response (HSP70, HSC70, HSP90, and GRP78), ubiquitination (E2, E3, ubiquitin, and CHIP), 26S proteasome complex (PSMA2, PSMB7, and PSMC1), and apoptosis (SMAC/Diablo and BAX) in the liver and gill. The expression profiles were tissue-specific and mainly dependent on temperature rather than salinity in the gill; meanwhile, in the liver, both conditions modulated the expression of these genes. Transcription of markers involved in the heat shock response was much higher in the liver than in the gill and was higher when salinity decreased and the temperature increased. Similarly, the genes involved in the ubiquitination pathway, 26S complex of the proteasome, and the apoptotic pathway showed the same pattern, being mainly induced in the liver rather than in the gill. This is the first study to show that this Antarctic fish can induce the cellular stress response in their tissues when subjected to these thermal/saline combinations.

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Global warming is having a significant impact around the world, modifying environmental conditions in many areas, including in zones that have been thermally stable for thousands of years, such as Antarctica. Stenothermal sedentary intertidal fish species may suffer due to warming, notably if this causes water freshening from increased freshwater inputs. Acute decreases in salinity, from 33 down to 5, were used to assess osmotic responses to environmental salinity fluctuations in Antarctic spiny plunderfish Harpagifer antarcticus, in particular to evaluate if H. antarcticus is able to cope with freshening and to describe osmoregulatory responses at different levels (haematological variables, muscle water content, gene expression, NKA activity). H. antarcticus were acclimated to a range of salinities (33 as control, 20, 15, 10 and 5) for 1 week. At 5, plasma osmolality and calcium concentration were both at their lowest, while plasma cortisol and percentage muscle water content were at their highest. At the same salinity, gill and intestine Na+ -K+ -ATPase (NKA) activities were at their lowest and highest, respectively. In kidney, NKA activity was highest at intermediate salinities (15 and 10). The salinity-dependent NKA mRNA expression patterns differed depending on the tissue. Marked changes were also observed in the expression of genes coding membrane proteins associated with ion and water transport, such as NKCC2, CFTR and AQP8, and in the expression of mRNA for the regulatory hormone prolactin (PRL) and its receptor (PRLr). Our results demonstrate that freshening causes osmotic imbalances in H. antarcticus, apparently due to reduced capacity of both transport and regulatory mechanisms of key organs to maintain homeostasis. This has implications for fish species that have evolved in stable environmental conditions in the Antarctic, now threatened by climate change.

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The non-specific immunity can induce iron deprivation as a defense mechanism against potential bacterial pathogens, but little information is available as to its role in Antarctic fish. In this study the response of iron metabolism related genes was evaluated in liver and head kidney of the Antarctic notothenoids Notothenia coriiceps and Notothenia rossii 7 days after lipopolysaccharide (LPS) injection. Average plasma Fe2+ concentration was unaffected by treatment in any of the species. The gene expression response to LPS varied between tissues and species, being stronger in N. coriiceps and more prominent in the head kidney than liver. The reaction to LPS was marked by increased individual variability in most genes analyzed, even when the change in expression was not statistically significant, suggesting different individual sensitivity and coping responses in these wild fish. We found that iron related genes had an attenuated and homogenous response to LPS but there was no detectable relationship between plasma Fe2+ and gene expression. However, overall in both tissues and species LPS exposure set a multilevel response that concur to promote intracellular accumulation of iron, an indication that Antarctic Notothenoids use innate nutritional immunity as a resistance mechanism against pathogens.

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Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.

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Iron deprivation is a nutritional immunity mechanism through which fish can limit the amount of iron available to invading bacteria. The aim of this study was to evaluate the modulation of iron metabolism genes in the liver and brain of sub-Antarctic notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis. The specimens were inoculated with two P. salmonis strains: LF-89 (ATCC® VR-1361™) and Austral-005 (antibiotic resistant). Hepatic and brain samples were collected at intervals over a period of 35 days. Gene expression (by RT-qPCR) of proteins involved in iron storage, transport, and binding were statistically modulated in infected fish when compared with control counterparts. Specifically, the expression profiles of the transferrin and hemopexin genes in the liver, as well as the expression profiles of ferritin-M, ferritin-L, and transferrin in the brain, were similar for both experimental groups. Nevertheless, the remaining genes such as ferritin-H, ceruloplasmin, hepcidin, and haptoglobin presented tissue-specific expression profiles that varied in relation to the injected bacterial strain and sampling time-point. These results suggest that nutritional immunity could be an important immune defense mechanism for E. maclovinus against P. salmonis injection. This study provides relevant information for understanding iron metabolism of a sub-Antarctic notothenioid fish.