Cold-Driven Hemoglobin Evolution in Antarctic Notothenioid Fishes Prior to Hemoglobin Gene Loss in White-Blooded Icefishes.

Expression of multiple hemoglobin isoforms with differing physiochemical properties likely helps species adapt to different environmental and physiological conditions. Antarctic notothenioid fishes inhabit the icy Southern Ocean and display fewer hemoglobin isoforms, each with less affinity for oxygen than temperate relatives. Reduced hemoglobin multiplicity was proposed to result from relaxed selective pressure in the cold, thermally stable, and highly oxygenated Antarctic waters. These conditions also permitted the survival and diversification of white-blooded icefishes, the only vertebrates living without hemoglobin. To understand hemoglobin evolution during adaptation to freezing water, we analyzed hemoglobin genes from 36 notothenioid genome assemblies. Results showed that adaptation to frigid conditions shaped hemoglobin gene evolution by episodic diversifying selection concomitant with cold adaptation and by pervasive evolution in Antarctic notothenioids compared to temperate relatives, likely a continuing adaptation to Antarctic conditions. Analysis of hemoglobin gene expression in adult hematopoietic organs in various temperate and Antarctic species further revealed a switch in hemoglobin gene expression underlying hemoglobin multiplicity reduction in Antarctic fish, leading to a single hemoglobin isoform in adult plunderfishes and dragonfishes, the sister groups to icefishes. The predicted high hemoglobin multiplicity in Antarctic fish embryos based on transcriptomic data, however, raises questions about the molecular bases and physiological implications of diverse hemoglobin isoforms in embryos compared to adults. This analysis supports the hypothesis that the last common icefish ancestor was vulnerable to detrimental mutations affecting the single ancestral expressed alpha- and beta-globin gene pair, potentially predisposing their subsequent loss.

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.

Ocean acidification and seasonal temperature extremes combine to impair the thermal physiology of a sub-Antarctic fish.

To predict the potential impacts of climate change on marine organisms, it is critical to understand how multiple stressors constrain the physiology and distribution of species. We evaluated the effects of seasonal changes in seawater temperature and near-future ocean acidification (OA) on organismal and sub-organismal traits associated with the thermal performance of Eleginops maclovinus, a sub-Antarctic notothenioid species with economic importance to sport and artisanal fisheries in southern South America. Juveniles were exposed to mean winter and summer sea surface temperatures (4 and 10 °C) at present-day and near-future pCO2 levels (~500 and 1800 µatm). After a month, the Critical Thermal maximum and minimum (CTmax, CTmin) of fish were measured using the Critical Thermal Methodology and the aerobic scope of fish was measured based on the difference between their maximal and standard rates determined from intermittent flow respirometry. Lipid peroxidation and the antioxidant capacity were also quantified to estimate the oxidative damage potentially caused to gill and liver tissue. Although CTmax and CTmin were higher in individuals acclimated to summer versus winter temperatures, the increase in CTmax was minimal in juveniles exposed to the near-future compared to present-day pCO2 levels (there was a significant interaction between temperature and pCO2 on CTmax). The reduction in the thermal tolerance range under summer temperatures and near-future OA conditions was associated with a reduction in the aerobic scope observed at the elevated pCO2 level. Moreover, an oxidative stress condition was detected in the gill and liver tissues. Thus, chronic exposure to OA and the current summer temperatures pose limits to the thermal performance of juvenile E. maclovinus at the organismal and sub-organismal levels, making this species vulnerable to projected climate-driven warming.

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.

Proximal composition and fatty acid profile of Hemigrapsus crenulatus (H. Milne Edwards, 1837) as one of the main foods of "patagonian blenny"Eleginops maclovinus (Cuvier, 1830) / Composição proximal e perfil de ácidos graxos de Hemigrapsus crenulatus (H. Milne Edwards, 1837) como um dos principais alimentos da "blenny patagônica" Eleginops maclovinus (Cuvier, 1830)

Abstract The Patagonian blenny (Eleginops maclovinus) is species endemic to South America with physiological characteristics that would facilitate its incorporation into Chilean aquaculture. However, there is currently no specific artificial food that can be used to raise E. maclovinus. In light of this problem, this study describes the proximal composition and fatty acid profile of the crab Hemigrapsus crenulatus, one of the main foods of E. maclovinus. The purpose of the study is to serve as basic information for the development of a specific artificial diet for juveniles of this fish species. The proximal analysis of the complete body of H. crenulatus indicates that it is mainly composed of ash (35.9%), proteins (32.2%), glucides (19.8%) and minor lipids (3.6%). The fatty acid profile is 40.7% PUFAs, 29.7% MUFAs and 29.5% SAFAs, and the most abundant acids are Eicosapentaenoic (18.8%), Oleic (6.8%) and Palmitic (16.6%), respectively. H. crenulatus has highest level of proteins, lipids and PUFAs among the species of the Brachyura infraorder.

Resumo O "blenny da patagônia" (Eleginops maclovinus) é uma espécie endêmica da América do Sul, com características fisiológicas que facilitariam sua incorporação na aquicultura chilena. No entanto, atualmente não há alimento artificial específico que possa ser usado para criar E. maclovinus. Diante desse problema, este estudo descreve a composição proximal e o perfil de ácidos graxos do caranguejo Hemigrapsus crenulatus, um dos principais alimentos de E. maclovinus. O objetivo do estudo é servir como informação básica para o desenvolvimento de uma dieta artificial específica para espécies juvenis desse peixe. A análise proximal do corpo completo de H. crenulatus indica que ele é composto, principalmente, de cinzas (35,9%), proteínas (32,2%), glicídios (19,8%) e lipídios menores (3,6%). O perfil de ácidos graxos é 40,7% PUFA, 29,7% MUFA e 29,5% SAFA, e os ácidos mais abundantes são eicosapentaenoico (18,8%), oleico (6,8%) e palmítico (16,6%), respectivamente. H. crenulatus possui o mais alto nível de proteínas, lipídios e PUFAs entre as espécies da infraordem de Brachyura.

Horizontal transmission of Piscirickettsia salmonis from the wild sub-Antarctic notothenioid fish Eleginops maclovinus to rainbow trout (Oncorhynchus mykiss) under experimental conditions.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a bacterial disease that affects farmed salmonids, causing high mortalities and significant economic losses in the Chilean salmon farm industry. Given the Chilean native fish species Patagonian blenny, Eleginops maclovinus, lives in the vicinity of salmon farms, it is relevant to clarify the epidemiological role that this species could play in the transmission and/or dissemination of this pathogen. This study aimed to evaluate the bidirectional transmission of P. salmonis between the Patagonian blenny and Oncorhynchus mykiss (rainbow trout), via a cohabitation challenge model. The results of this study demonstrated the transmission of the bacteria from Patagonian blennies to rainbow trout, considering the specific mortality in cohabitant rainbow trout, reaching 46%: the necropsy of these specimens, evidencing the characteristic pathological lesions of the disease and the positive results of the qPCR analysis for P. salmonis, in the same individuals. In contrast, no mortalities of Patagonian blenny specimens were recorded in the challenged experimental groups. This study is the first report showing the horizontal transmission of P. salmonis from a native non-salmonid species, such as the Patagonian blenny, to a salmonid species, generating the disease and specific mortality in rainbow trout, using a cohabitation challenge.

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.

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.

Cellular stress responses of Eleginops maclovinus fish injected with Piscirickettsia salmonis and submitted to thermal stress.

Fluctuations in ambient temperature along with the presence of pathogenic microorganisms can induce important cellular changes that alter the homeostasis of ectothermic fish. The aim of this study was to evaluate how sudden or gradual changes in environmental temperature together with the administration of Piscirickettsia salmonis modulate the transcription of genes involved in cellular stress response in the liver of Eleginops maclovinus. Fish were subjected to the following experimental conditions in duplicate: C- 12 °C: Injection only with culture medium, C+ 12 °C: Injection with P. salmonis, AM 18 °C: Injection only with culture medium under acclimation at 18 °C, AB 18 °C: Injection with P. salmonis under acclimation at 18 °C, SM 18 °C: Injection only with culture medium and thermal shock at 18 °C and SB 18 °C: Injection with P. salmonis and thermal shock at 18 °C and sampling at 4-, 8-, 12-, 16- and 20-day post injection (dpi). The genes implied in the heat shock response (HSP70, HSC70, HSP90, and GRP78), apoptosis pathway (BAX and SMAC/Diablo), ubiquitination (E2, E3, ubiquitin, and CHIP), and 26 proteasome complex (PSMB7, PSMC1, and PSMA2) showed expression profiles dependent on time and type of injection applied. All the genes greatly increased their expression levels at day 16 and showed moderate increases at day 20, except for PSMA2 which showed a higher increase between 4- and 12-day post challenges. Our results suggest that the changes observed at the final days of the experiment are due to temperature more than P. salmonis.

Determination of acute toxicity of ammonium in juvenile Patagonian blenny (Eleginops maclovinus) / Determinação da toxicidade aguda do amônio em Babosas-da-Patagônia (Eleginops maclovinus) juvenis

Abstract Deionized ammonium (NH3) acute toxicity (LC50-96h) in Patagonian blenny juveniles (Eleginops maclovinus) was assessed. Concentrations of deionized ammonium in salt water were prepared by using 24.09 ± 2.1 g ammonium chloride (NH4Cl). Fish were exposed in triplicates to different ammonium concentrations: 0.05; 0.094; 0.175; 0.325 and 0.605 mg NH3 L-1. Additionally, a control group was included. Experimental fish were kept at a photoperiod of 16:8h. Average temperatures were 16.24 ± 1.40 °C. Oxygen concentration was 7.16 ± 0.40 mg L -1. Water pH was 7.89 ± 0.2. LC50-96 h, was estimated by using Probit statistical method (95% intervals) using EPA software (1993). Juveniles of E. maclovinus showed a LC50-96h of 0.413mg NH3 L-1 value, different from most marine species. This study presents the first record of ammonium toxicity in marine species of Chile.

Resumo A toxicidade aguda (LC50-96h) de amônia deionizada (NH3 ) em Babosas-da-Patagônia (Eleginops maclovinus) juvenis foi avaliada. As concentrações de sal de amônia deionizada em água foram preparados com 24,09 ± 2,1 g de cloreto de amônio (NH4Cl). Os peixes foram expostos em triplicata a diferentes concentrações de amônia: 0,05; 0,094; 0,175; 0,325 e 0,605 mg L-1 de NH3. Além disso, um grupo de controle foi incluído. Peixes experimentais foram mantidos em fotoperíodo de 16: 8h. As temperaturas médias foram 16,24 + 1,40 °C. A concentração de oxigênio foi 16.24 +1.40°C mg L-1 . O pH da água foi de 7,89 + 0,2. h LC50-96h, foi estimada usando o método estatístico de Probit (intervalos de 95%) usando o software EPA (1993). E. maclovinus juvenis apresentaram uma LC 50-96h de 0.413mg NH3 L-1 valor diferente da maioria das espécies marinhas. Este estudo apresenta o primeiro registro de toxicidade amónio em espécies marinhas do Chile.

High doses of Francisella noatunensis induces an immune response in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis, the etiological agent of Francisellosis, affects a large number of farmed species such as Salmo salar. This species coexists with several native species in the same ecosystem, including Eleginops maclovinus. Our objective was to evaluate the susceptibility, presence of clinical symptoms, and the ability of Eleginops maclovinus to respond to Francisella infection. For this, healthy individuals were inoculated with 1.5 × 101, 1.5 × 105, and 1.5 × 1010 bact/µL of Francisella by intraperitoneal injection, subsequently the fish were sampled on days 1, 3, 7, 14, 21, and 28 post injection (dpi). At the end of the experiment, no mortality, nor internal and external clinical signs were observed, although in the high dose anaemia was detected. Additionally, bacteria were detected in all three doses, however there was replication at day 28 only in the liver in the high dose. Analysis of gene expression by qPCR showed that the spleen generated an immune response against infection from day 1 dpi, however at day 7 dpi most of the genes suffered repressed expression; observing over expression of the genes C3, NLRC3, NLRC5, MHCI, IgM. In contrast, expression in the anterior kidney did not vary significantly during the challenge. IgM quantification showed the production of antibodies in the medium and high doses. This study provides new knowledge about Francisella infection and the long-lasting and specific immune response generated by Eleginops maclovinus. It also demonstrates its susceptibility to Francisellosis where there is a difference in the immune response according to the tissue.

Effect of ration level on growth performance, body composition, intermediary metabolism and serum parameters in juvenile Patagonian blennie Eleginops maclovinus.

Eleginops maclovinus is an endemic species of the southern cone with beneficial physiological characteristics for aquaculture. However, this species has a low growth rate under captive conditions, and the optimal feed ration together with the metabolic process is unknown. This study aimed to determine the optimum feed ration during 90 days based on growth performance, body composition, intermediary metabolism, and serum parameters. For this, fish were randomly assigned to rations of 0.5, 1, 2 and 4% of body weight. No fish mortality was registered, but all fish, developed fatty liver. The results of weight, length, growth performance (WGR, SGR), and body parameters (HSI, VSI and K) followed a similar pattern, with the lowest values observed in the 0.5% and no significant differences between rations of 1, 2 and 4%. The feed intake and feces increased with the feed ration. However, the percentage of food consumed by the fish decreased with the ration size and the feed conversion ratio was lowest in the 1% ration. Total serum proteins and calcium were lowest in the 0.5% ration and presented no differences in the rations 1, 2 and 4%, while triglyceride content was significantly different only between the rations of 0.5 and 4%. Blood cortisol levels were significantly higher in the rations of 0.5 and 1%, and decreased in rations of 2 and 4%. The lipids, fiber, and energy of the total body mass increased with the feed ration, while dry matter, proteins, and ash of the body decreased to higher feed ration. In liver, triglyceride and protein levels decreased with a larger feed ration, amino acids increased in the rations of 0.5 and 4%, while glucose levels increased in rations of 2 and 4%. Liver enzymes Glucose-6-phosphate dehydrogenase and Glutamate dehydrogenase increased their activity at a higher feed ration, while Glycogen Phosphorylase, Aspartate aminotransferase and 3-Hydroxyacyl-CoA dehydrogenase presented their highest enzymatic activity only in the 4% ration. Fructose-1,6-bisphosphate showed low enzymatic activity in rations of 2 and 4% and Glycerol-3-phosphate dehydrogenase was significantly different only between the ration of 0.5 and 1%. Finally, our results suggests that optimal feed rations for E. maclovinus corresponds to 1% since this ration size produces the highest growth and metabolism with a minimum loss of food and feces present in the environment. Additionally, we recommend to reduce the percentage of fat in the diet to avoid the development of fatty liver.

The effects of intraperitoneal administration of Francisella noatunensis subsp. noatunensis on hepatic intermediary metabolism and indicators of stress in Patagonian blennie Eleginops maclovinus.

Francisellosis is a disease produced by Francisella spp. which affects farmed fish. Eleginops maclovinus specimens can be caught close to salmon farming centers, feeding on un-consumed pellet, making the transmission of pathogens such as Francisella noatunensis possible. The aim of this study was to evaluate the effect of F. noatunensis on liver intermediary metabolism in E. maclovinus. 144 fish were injected intraperitoneally with F. noatunensis at a low dose LD (1 × 101 cells/µL), medium dose MD (1 × 105 cells/µL), high dose HD (1 × 1010 cells/µL), or with culture medium C (control), and sampled at 1, 3, 7, 14, 21 and 28 days post injection (dpi). No mortality was recorded during the experimental period, but there was a marked metabolic response in fish injected with high doses. Metabolites in plasma were lowest in the high bacterial dose. Cortisol levels were highest at day 7 in the high dose and then decreased from day 14 until the end of the study. Liver enzymes showed a similar pattern to plasma metabolites, with decreased enzymatic activity, mostly with the high bacteria dose. PK was the exception, with increased enzymatic activity in a dose-dependent manner over time. Liver metabolites were highly variable, except in the high bacterial dose where variability and total levels decreased significantly. Our results show that fish infection with F. noatunensis induces a clear stress response, especially with at the highest dose, shifting intermediary metabolism towards mobilization of energy and suggesting that E. maclovinus detects experimental infection of F. noatunensis as a stressor, which it is dependent on the bacterial dose.

Back to the future? Late Holocene marine food web structure in a warm climatic phase as a predictor of trophodynamics in a warmer South-Western Atlantic Ocean.

Stable carbon and nitrogen isotope ratios in the skeletal elements of both ancient and modern marine species from the Beagle Channel were used to compare the structure of Late Holocene and modern food webs, and predict potential changes as a result of a Sea Surface Temperature (SST) increase in the region. Complementary, ancient and modern shells of limpets and mussels were isotopically analysed to explore changes in the isotopic baseline and compare marine food webs through time after an appropriate correction for baseline shifts. Results confirmed a declining pattern of marine primary productivity during the Late Holocene in the Beagle Channel. In general, the isotopic niches overlapped largely in the ancient food web in comparison to the current marine one, with the exception of that of cormorants (Phalacrocorax sp.). Our data suggest that all the species that have undergone intense human exploitation (Arctocephalus australis, Otaria flavescens and Merluccius sp.) significantly increased their trophic levels. The most important finding of this work was the very high isotopic overlap between snoek (Thyrsites atun) and hake (Merluccius sp.) during the Late Holocene. Increasing SST as a result of global warming could favour the recolonization of the southern South-Western Atlantic Ocean by snoek from the South-Eastern Pacific Ocean, with a potential impact on the landings of the economically important Argentine and Austral hake. These findings highlight the relevance of using zooarchaeological remains for providing predictions about marine food webs changes in the near future.

Characterization of first blastomeres in Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopidae).

SummaryThere is no information about the characteristics of early cleavage in the Patagonian blennie (Eleginops maclovinus), which can be used as a diagnostic tool for embryo quality. The purpose of this investigation, therefore, was to characterize the first blastomeres of E. maclovinus morphologically. Of a 'pool' of incubated eggs at 10.7 ± 0.5°C, 100 microphotographs of blastodiscs were extracted at different incubation periods from 0.25 to 5 h after fertilization and analyzed. Blastodiscs taken at 3.5, 4.0 and 5.0 h were characterized and classified into symmetric or asymmetric groups according to their morphology. The proportions of length (L) and width (W) of each blastomere were determined to establish its symmetry. Additionally, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodisc: CB) and compared other blastodiscs (4.0 and 5.0 h). The results showed that before fertilization oocytes presented a somehow turgid aspect (maximum average diameter of 987 ± 41 µm) and after fertilization and hydration, their diameter increased to 1001.5 ± 11 µm (but not statistically significant) and presented a spherical shape. First cleavage ends after 3.5 h of development, forming two blastomeres 467 ± 45 µm length (L) and 328 ± 21 µm width (W) with a L/W ratio of 1.43 ± 0.19. The second cleavage ends after development at 4.5 h forming four blastomeres 238 ± 65 µm length and 227 ± 65 µm width with a ratio L/W of 1.06 ± 0.09. Five categories were identified during the blastomere characterization: 70% normal or symmetric; 8% with odd numbers of blastomeres; 6% unequal; 6% 'pie shaped' and 10% amorphous.

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

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 µL of culture medium or with 100 µL 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-1ß) 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.

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

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 µL of culture medium), wild type LF-89 strain (100 µL, 1 × 108 live bacteria), and antibiotic resistant strain Austral-005 (100 µL, 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-1ß, 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.

Effects of skeletal element identity, delipidation and demineralization on the analysis of stable isotope ratios of C and N in fish bone.

Stable isotope ratios of C and N in the bone tissue of three different skeletal elements (angular, cleithrum and vertebra) of three fish species from different evolutionary lineages (Clupeiformes, Atheriniformes and Notothenioidei) were determined before (δ13 Cbulk and δ15 Nbulk ) and after demineralization and delipidation (δ13 Cdml and δ15 Ndml ). One of the species had cellular bone and the other two had acellular bone. Results revealed that δ15 N and δ13 C values from different skeletal elements were interchangeable in species with acellular bone, but caution was needed in species with cellular bone, as δ15 N values varied among skeletal elements. Furthermore, δ15 Nbulk values were significantly lower than δ15 Ndml values in the three species, thus suggesting that they are not comparable. This difference is probably because δ15 Nbulk refers to total bone protein and δ15 Ndml to collagen only.

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.