R. Oyarzún

Combined effects of high stocking density and Piscirickettsia salmonis treatment on the immune system, metabolism and osmoregulatory responses of the Sub-Antarctic Notothenioid fish Eleginops maclovinus

The aim of this study was to evaluate immunological, metabolic and osmoregulatory secondary stress responses in Eleginops maclovinus specimens submitted to three different stocking densities: i) low (3.1 kg m(-3)), medium (15 kg m(-3)) and high (60 kg m(-3)) during 10 days, alone or in combination with a previous treatment of a protein extract of the pathogen Piscirickettsia salmonis (0.5 μg g weight body(-1)). Plasma, liver, gill and kidney samples were obtained at the end of both experiments. Plasma cortisol and amino acid levels increased, while plasma glucose, triglyceride and lactate levels decreased at higher stocking densities. However, no effects were observed on serum Immunoglobulin type M (IgM anti P. salmonis level) values. Gill Na(+), K(+)-ATPase activity enhanced under these experimental conditions, suggesting an osmotic imbalance. Energy metabolism changes, assessed by metabolite concentrations and enzyme activities, indicated a reallocation of energetic substrates at higher stocking densities. Specimens inoculated with a protein extract of P. salmonis and maintained at different stocking densities showed primary stress response, as all groups enhanced plasma cortisol concentrations. Serum IgM levels increased after treatment with P. salmonis extract but a negative influence of high stocking density on IgM production was observed when immune system was activated. Furthermore, treatment with P. salmonis protein extract evoked deep changes in the metabolite stores in all tissues tested, indicating a mobilization of energy substrates in response to infection. The results show that stocking density induced immunological, metabolic and osmoregulatory secondary stress responses in E. maclovinus specimens and that previous treatment with P. salmonis compromise these changes.

Isolation Driven Divergence in Osmoregulation in Galaxias maculatus (Jenyns, 1848) (Actinopterygii: Osmeriformes).

Background Marine species have colonized extreme environments during evolution such as freshwater habitats. The amphidromous teleost fish, Galaxias maculatus is found mainly migrating between estuaries and rivers, but some landlocked populations have been described in lakes formed during the last deglaciation process in the Andes. In the present study we use mtDNA sequences to reconstruct the historical scenario of colonization of such a lake and evaluated the osmoregulatory shift associated to changes in habitat and life cycle between amphidromous and landlocked populations. Results Standard diversity indices including the average number of nucleotide differences (Π) and the haplotype diversity index (H) indicated that both populations were, as expected, genetically distinctive, being the landlocked population less diverse than the diadromous one. Similarly, pairwise GST and NST comparison detected statistically significant differences between both populations, while genealogy of haplotypes evidenced a recent founder effect from the diadromous stock, followed by an expansion process in the lake. To test for physiological differences, individuals of both populations were challenged with a range of salinities from 0 to 30 ppt for 8 days following a period of progressive acclimation. The results showed that the landlocked population had a surprisingly wider tolerance to salinity, as landlocked fish survival was 100% from 0 to 20 ppt, whereas diadromous fish survival was 100% only from 10 to 15 ppt. The activity of ATPase enzymes, including Na+/K+-ATPase (NKA), and H+-ATPase (HA) was measured in gills and intestine. Activity differences were detected between the populations at the lowest salinities, including differences in ATPases other than NKA and HA. Population differences in mortality are not reflected in enzyme activity differences, suggesting divergence in other processes. Conclusions These results clearly demonstrate the striking adaptive changes of G. maculatus osmoregulatory system, especially at hyposmotic environments, associated to a drastic shift in habitat and life cycle at a scale of a few thousand years.

Identification, characterization and modulation of ferritin-H in the sub-Antarctic Notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis

&NA; Ferritin is a major iron storage protein essential not only in the infectious process, but also in any circumstance generating oxidative stress. In this study, the cDNA coding sequence of ferritin‐H was obtained from the sub‐Antarctic Notothenioid fish Eleginops maclovinus through transcriptomic analysis of the head kidney. This sequence contained a 534 bp open reading frame that coded for a 177 amino acid protein with a molecular weight of 20,786.2 Da and a theoretical pI of 5.56. The protein displayed a region of iron putative response elements in the 5′UTR, two putative ferritin iron‐binding region signatures, and seven characteristic amino acids with ferroxidase functions. Phylogenetic analysis related this sequence to ferritin‐H sequences of other Antarctic Notothenioid fish, sharing 96.61% similarity. Constitutive gene expression analysis in different organs revealed increased ferritin‐H gene expression in the gills, spleen, muscle, and liver. After infection with two bacterial strains of Piscirickettsia salmonis (LF‐89 and Austral‐005), ferritin‐H was differentially expressed depending on bacterial strain and tissue. This study provides relevant information towards understanding the iron metabolism of a sub‐Antarctic Notothenioid fish. HighlightsFerritin is a major iron storage protein essential in the infectious process.Phylogenetic related this sequence to ferritin‐H sequences of other Antarctic Notothenioid fish, sharing 96.61% homology.Ferritin‐H was differentially expressed depending on bacterial strain type of P. salmonis and tissue.

Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon ( Salmo salar ) and Coho salmon ( Oncorhynchus kisutch )

Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection.

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.

Temperature modulates the immunological response of the sub-antarctic notothenioid fish Eleginops maclovinus injected with Piscirickettsia salmonis

Abstract Eleginops maclovinus is a eurythermic fish that under natural conditions lives in environments with temperatures ranging from 4 to 18 °C and can be usually captured near salmon farming areas. The aim of this study was to evaluate the effect of temperature over the innate and adaptive immune response of E. maclovinus challenged with Piscirickettsia salmonis following different treatments: C— (control injection with culture medium at 12 °C), C+ (bacterial injection at 12 °C), 18 °C c/A + B (injection with culture medium in acclimation at 18 °C), 18 °C c/A + B (bacterial injection in acclimation at 18 °C), 18 °C s/A + M (injection with culture medium without acclimation at 18 °C) and 18 °C s/A + B (bacterial injection without acclimation at 18 °C). Each injection had 100 &mgr;L of culture medium or with 100 &mgr;L at a concentration 1 × 108 of live bacteria, sampling six fish per group at 4, 8, 12, 16 and 20 days post‐injection (dpi). Expression of the mRNA related with the innate immune response gene (TLR1, TLR5, TLR8, NLRC3, NLRC5, MyD88 and IL‐1&bgr;) as well as the adaptive immune response gene (MHCI, MHCII, IgMs and IgD) were measured in spleen and head kidney. Gene expression profiles were treatment‐type and time dependent. Levels of Immunoglobulin M (IgM) increased in challenged groups with P. salmonis from day 8–20 post challenge, which suggest activation of B cells IgM + through P. salmonis epitope detection. Additionally, a rise in temperature from 12 °C (C+) to 18 °C (with/without acclimation) also resulted in antibody increment detected in serum with significant differences between “18 °C c/A + B” and “18 °C s/A + B” groups. This is the first study that evaluates the effect of temperature changes and mRNA expression related with immune system gene over time on E. maclovinus, a native wild life fish that cohabits in the salmon farming environment. HighlightsThe increase in temperature affects the gene transcription related with innate and adaptive immune response.The increase in temperature (with and without acclimatation) increased the amount of antibodies detected in serum.The temperature is modulating the immune response in Eleginops maclovinus.

Immunological response of the Sub-Antarctic Notothenioid fish Eleginops maclovinus injected with two strains of Piscirickettsia salmonis

ABSTRACT Eleginops maclovinus is an endemic fish to Chile that lives in proximity to salmonid culture centers, feeding off of uneaten pellet and salmonid feces. Occurring in the natural environment, this interaction between native and farmed fish could result in the horizontal transmission of pathogens affecting the aquaculture industry. The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis. Treatment injections (in duplicate) were as follows: control (100 &mgr;L of culture medium), wild type LF‐89 strain (100 &mgr;L, 1 × 108 live bacteria), and antibiotic resistant strain Austral‐005 (100 &mgr;L, 1 × 108 live bacteria). The fish were sampled at various time‐points during the 35‐day experimental period. The gene expression of TLRs (1, 5, and 8), NLRCs (3 and 5), C3, IL‐1&bgr;, MHCII, and IgMs were significantly modulated during the experimental period in both the spleen and gut (excepting TLR1 and TLR8 spleen expressions), with tissue‐specific expression profiles and punctual differences between the injected strains. Anti‐P. salmonis antibodies increased in E. maclovinus serum from day 14–28 for the LF‐89 strain and from day 14–35 for the Austral‐005 strain. These results suggest temporal activation of the innate and adaptive immune responses in E. maclovinus tissues when injected by distinct P. salmonis strains. The Austral‐005 strain did not always cause the greatest increases/decreases in the number of transcripts, so the magnitude of the observed immune response (mRNA) may not be related to antibiotic resistance. This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish. HighlightsEleginops maclovinus is living in proximity to salmonid culture centers feeding off of uneaten pellet and salmonid feces.The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis.The results suggest activation of the immune response in E. maclovinus injected by distinct P. salmonis strains.This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish.