Discovery of BbX transcription factor in the patagonian blennie: Exploring expression changes following combined bacterial and thermal stress exposure.

High-Mobility Group (HMG) proteins are involved in different processes such as transcription, replication, DNA repair, and immune response. The role of HMG proteins in the immune response of fish has been studied mainly for HMGB1, where its expression can be induced by the stimulation of viral/bacterial PAMPs and can act as a proinflammatory mediator and as a global regulator of transcription in response to temperature. However, for BbX this role remains to be discovered. In this work, we identified the BbX of E. maclovinus and evaluated the temporal expression levels after simultaneous challenge with P. salmonis and thermal stress. Phylogenetic analysis does not significantly deviate from the expected organismal relationships suggesting orthologous relationships and that BbX was present in the common ancestor of the group. BbX mRNA expression levels were very high in the intestinal tissue of E. maclovinus (foregut, midgut, and hindgut). Nevertheless, the protein levels analyzed by WB showed the highest levels of BbX protein in the liver (constitutive expression). On the other hand, the mRNA expression levels of BbX in the liver of E. maclovinus injected with P. salmonis and subjected to thermal stress showed an increase at days 16 and 20 in all treatments applied at 12 °C and 18 °C. Meanwhile, the protein levels quantified by WB showed a statistically significant increase in the HMG-Bbx at all experimental times (4, 8, 12, 16, and 20 dpi). However, at 4 dpi the HMG-Bbx protein levels were much higher than the other days evaluated. The results suggest that BbX protein may be implicated in the response mechanism to temperature and bacterial stimulation in the foregut, midgut, hindgut, and liver, according to our findings at the level of mRNA and protein. Furthermore, our WB analysis suggests an effect of P. salmonis on the expression of this protein that can be observed in condition C+ 12 °C compared to C- 12 °C. Then, there is an effect of temperature that can be evidenced in the condition AM 18 °C and SM 18 °C, compared to AB 18 °C and SB 18 °C at 4, 8, and 12 dpi. We found not differences in the levels of this protein if the thermal stress is achieved through acclimatization or shock. More research is necessary to clarify the importance of this type of HMG in the immune response and thermal tolerance in fish.

Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

Live and inactivated Piscirickettsia salmonis activated nutritional immunity in Atlantic salmon (Salmo salar).

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.

PAMPs of Piscirickettsia salmonis Trigger the Transcription of Genes Involved in Nutritional Immunity in a Salmon Macrophage-Like Cell Line.

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.

Transcriptional modulation of immune genes in gut of Sub-Antarctic notothenioid fish Eleginops maclovinus challenged with Francisella noatunensis subsp. noatunensis.

The search for functional foods that improve the immune response has traditionally been focused on lymphoid tissue and the intestinal mucosa. However, it is unknown whether there is a different immune response in different portions of the gut following exposure to a bacterial pathogen. We challenged Eleginops maclovinus intraperitoneally (i.p) with Francisella noatunensis subsp. noatunensis and measured mRNA transcripts related to innate and adaptive immune responses in different parts of the gut (foregut, midgut and hindgut). We used control (i.p only with bacterial culture medium), low dose (i.p of F. noatunensis at 1 × 101 bact/µL), medium dose (i.p of F. noatunensis at 1 × 105 bact/µL) and high dose (i.p of F. noatunensis at 1 × 1010 bact/µL) groups in our experiments. We sampled fish at days 1, 3, 7, 14, 21, and 28 post-injection. We observed tissue-specific expression of TLR1, TLR5, TLR8, MHCI, MHCII and IgM, and transcription of these immune markers was lower in foregut and higher in midgut and hindgut. We detected Francisella genetic material (DNA) in fish stimulated with a high dose from day 1-28 in foregut, midgut, and hindgut. However, we could only detect Francisella DNA in fish stimulated the medium and low dose at later timepoints in the foregut (21-28 days post injection "dpi") and hindgut (low dose from day 7-28 dpi). Our results suggest that the immune responses to bacterial pathogens occur throughout the gut, but certain segments may be more susceptible to infection because of their cellular morphology (anterior, middle and posterior).

Brain immunity response of fish Eleginops maclovinus to infection with Francisella noatunensis.

The brain's immune system is selective and hermetic in most species, including fish, favoring immune responses mediated by soluble immunomodulatory factors such as serotonin and the availability of nutrients against infectious processes. Francisella noatunensis coexist with fish such as Eleginops maclovinus, which raises questions about the susceptibility and immune response of the brain of E. maclovinus against Francisella. In this study, we inoculated fish with different doses of Francisella and took samples for 28 days. We detected bacteria in the brain of fish injected with a high concentration of Francisella at all time points. qPCR analysis of immune genes indicated a response mainly in the medium-dose and early expression of genes involved in iron metabolism. Finally, brain serotonin levels were higher than in uninfected fish in all conditions, suggesting possible immunomodulatory participation in an infectious process.

Warming and freshening activate the transcription of genes involved in the cellular stress response in Harpagifer antarcticus.

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.

Freshening effect on the osmotic response of the Antarctic spiny plunderfish Harpagifer antarcticus.

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.

Francisella noatunensis subsp. noatunensis triggers calcium metabolism gene modulation in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis is the responsible agent of Francisellosis, a bacterial disease that affects an important amount of aquatic farmed species. Eleginops maclovinus is a fish that cohabits with salmonids cages in Chile and can also act as a vector of this bacterial disease. In the present study, we evaluated calcium metabolism in the liver of E. maclovinus injected intraperitoneally with different doses of F. noatunensis subsp. noatunensis (low 1.5 × 101, medium 1.5 × 105 and high doses 1.5 × 1010 cells/µL). Fish were sampled at 1, 3, 7, 14, 21 and 28 days post injection (dpi). No mortalities nor clinical signs were observed. Plasma calcium levels were higher in the high doses group of F. noatunensis subsp. noatunensis at day 7 and 14 compared to the control group (fish injected with bacterial medium alone). Hypercalcemic factors increased at day 14 and 21 for the medium and low dose (parathyroid hormone-related protein precursor), while vitamin D3 receptor increased its expression at times 1, 3 and 7 for the low dose. On the other hand, hypocalcemic factors such as calcitonin receptor and stanniocalcin increased its expression at time 7 and 14, respectively. Calmodulin involved in calcium storage decreased its expression during all experimental days in fish subjected to high bacterial dose. Proteins involved in calcium transport, such as L-type voltage-gated calcium channel and trpv5 increased their transcription at day 1 and 14, compared to calcium sensing-receptor and plasma membrane Ca2 +- ATPase that showed peak expression at times 14 and 28. The results suggest a clear alteration of calcium metabolism, mainly in high bacterial doses. This study provides new knowledge about the calcium metabolism in fish infected with bacteria.

LPS Modulates the Expression of Iron-Related Immune Genes in Two Antarctic Notothenoids.

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.

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis.

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.

Nutritional Immunity Triggers the Modulation of Iron Metabolism Genes in the Sub-Antarctic Notothenioid Eleginops maclovinus in Response to Piscirickettsia salmonis.

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.