Salmo salar glucocorticoid receptors analyses of alternative splicing variants under stress conditions.

Cortisol is the main corticosteroid in teleosts, exerting multiple functions by activating glucocorticoid receptors (GR). Most teleost species have two GR genes, gr-1 and gr-2. Some teleost also presents two splice variants for gr-1; gr-1a and gr-1b. In this study, we report for first time the presence of 2 homeologous genes for gr-1 and gr-2, located on chromosomes 4q-13q (gr-1) and 5p-9q (gr-2) of the Salmo salar genome. Furthermore, our results describe gr-1 splice variants derived from chromosome 4 and 13, sharing typical teleost GR elements such as the 9 amino acid insertion in the DNA binding domain (DBD) and variations in the length of the ligand binding domain (LBD). Three splice variants were predicted for the gr-2 homeologous gene in chromosome 5, with differences of a 5 amino acid insertion in the DBD. We also identified an uncommon truncated gr-2 gene in chromosome 9 in salmon, which lacked the DBD and LBD domains. Finally, by designing specific primers for each predicted splice variant, we validated and evaluated the expression of their transcripts in S. salar subjected to stress caused by stocking density. Differences were observed in the expression of all identified mRNAs, revealing that gr-1 and gr-2 splice variants were upregulated in head kidney and gills of post-stressed fish. In conclusion, our findings suggest that from specific salmonid genomic duplication (125 MYA), two gene copies of each GR receptor were generated in S. salar. The identified splice variants could contribute to the variability of GR receptor complex modulation expression during stressful events, leading to variations in physiological responses in fish.

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.

Fuzzy-based assessment of groundwater intrinsic vulnerability of a volcanic aquifer in the Chilean Andean Valley.

A fuzzy logic approach has been proposed to face the uncertainty caused by sparse data in the assessment of the intrinsic vulnerability of a groundwater system with parametric methods in Las Trancas Valley, Andean Mountain, south-central Chile, a popular touristic place in Chile, but lacking of a centralized drinking and sewage water public systems; this situation is a potentially source of groundwater pollution. Based on DRASTIC, GOD, and EKv and the expert knowledge of the study area, the Mamdani fuzzy approach was generated and the spatial data were processed by ArcGIS. The groundwater system exhibited areas with high, medium, and low intrinsic vulnerability indices. The fuzzy approach results were compared with traditional methods results, which, in general, have shown a good spatial agreement even though significant changes were also identified in the spatial distribution of the indices. The Mamdani logic approach has shown to be a useful and practical tool to assess the intrinsic vulnerability of an aquifer under sparse data conditions.

Identification and expressional analysis of NLRC5 inflammasome gene in smolting Atlantic salmon (Salmo salar).

The NOD-like receptors (NLRs) were recently identified as an intracellular pathogen recognition receptor family in vertebrates. While the immune system participation of NLRs has been characterized and analyzed in various mammalian models, few studies have considered NLRs in teleost species. Therefore, this study analyzed the Atlantic salmon (Salmo salar) NLRC5. Structurally, Atlantic salmon NLRC5 presented leucine-rich repeat subfamily genes. Phylogenetically, NLRC5 was moderately conserved between S. salar and other species. Real-time quantitative PCR revealed NLRC5 expression in almost all analyzed organs, with greatest expressions in the head kidney, spleen, and hindgut. Furthermore, NLRC5 gene expression decreased during smolt stage. These data suggest that NLRC5 participates in the Atlantic salmon immune response and is regulated, at least partly, by the smoltification process, suggesting that there is a depression of immune system from parr at smolt stage. This is the first report on the NLRC5 gene in salmonid smolts.

Application of water quality indices and analysis of the surface water quality monitoring network in semiarid North-Central Chile.

Surface water quality has increasing importance worldwide and is particularly relevant in the semiarid North-Central Chile, where agriculture and mining activities are imposing heavy pressure on limited water resources. The current study presents the application of a water quality index in four watersheds of the 29°-33°S realm for the period 1999-2008, based on the Canadian Council of Ministers for the Environment approach and the Chilean regulation for irrigation water quality. In addition, two modifications to the index are tested and a comprehensive characterization of the existing monitoring network is performed through cluster analysis. The basins studied show fairly good water quality in the overall, specially the Limarí basin. On the other hand, the lower index values were obtained for the headwaters of Elqui, associated with the El Indio mining district. The first modification of the indicator (i.e., to consider parameters differentially according to their effect on human health or the environment) did not produce major differences with respect to the original index, given the generally good water quality. The second modification (i.e., to consider as threshold values the more restrictive figures derived from a set of regulations) yielded important differences in the indicator values. Finally, an adequate characterization of the monitoring network was obtained. The results presented spatial coherence and the information can be used as a basis for the optimization of the monitoring network if required.

A new method to map groundwater-dependent ecosystem zones in semi-arid environments: A case study in Chile.

Groundwater (GW) use has intensified in recent decades, threatening the ecological integrity of groundwater-dependent ecosystems (GDEs). The study of GDEs is limited; therefore, integrated, interdisciplinary environmental approaches that guarantee their monitoring and management amid current climate and anthropogenic changes are needed. A new geospatial method with an integrated and temporal approach was developed through a multicriteria approximation, taking into account expert opinion, remote sensing-GIS, and fieldwork to map groundwater-dependent ecosystem zones (GDEZ). A survey of experts (N = 26) was conducted to assign degrees of importance to the various geospatial parameters, and the mapping was carried out using 14 parameters. The reclassified parameters were normalized on a scale of 1 to 5 according to the degree of probability of the presence of GDE. The validation was carried out through fieldwork and statistical analysis. Then, the spatio-temporal changes amid changing GW levels were assessed using the summer season normalized difference vegetation index (NDVI). Two GDEZ maps were obtained, for 2002 and 2017, between which the high- and very-high-probability zones of GDEs decreased by 31,887 ha (~ 38%). The most sensitive temporal parameters that most influenced the spatio-temporal changes on GDEs were precipitation and land use, with rain exerting a slightly the greatest influence. It was also demonstrated that identified ecosystems decreased in area or were affected by aquifer depletion (NDVI-GW, r Pearson ≥0.74). This validated method allows spatio-temporal changes in GDEs to be mapped and analyzed at an annual scale and is transferable to other arid and semi-arid environments.

Stakeholder participation within the public environmental system in Chile: major gaps between theory and practice.

The main objective of this paper is to present a critical analysis of the stakeholder participation process within the Environmental Impact Assessment System in Chile, after ca. 14 years of being enforced. This analysis is sustained by the description and analysis of the stakeholder participation possibilities in a representative rural area of North-Central Chile. The Environmental Basis Act 19300, enacted in 1994, considers the participation of the local community in the environmental impact assessment of new projects. However, this possibility is very limited and difficult to exert, often resulting in frustration for the participants. This is due to a number of reasons, such as the imbalance of resources and knowledge among the majority of participating communities and project proponents, the complexity and administrative and legal constraints to participation, and the dominant interest of the Central Government in approving investments, specifically in energy and natural resources related projects, which generate wealth and jobs. Also, the State's rush to develop Internet-based communication and management systems has built a barrier for poor, traditional communities. This factor is clearly reflected in the case study considered. Results show that there is generally a significant lack of knowledge about institutions and participation tools. From this base, we intend to raise concern on these selected aspects that could be addressed to improve the effectiveness of the existing framework, both in Chile and in other developing countries, where immature environmental impact assessment and public management systems face similar pressures in relation to the sustainable use of their natural resources. Finally, some basic steps are proposed in order to make the community participation an effective tool for sustainable development.

Natural factors and mining activity bearings on the water quality of the Choapa basin, North Central Chile: insights on the role of mafic volcanic rocks in the buffering of the acid drainage process.

This contribution analyzes water chemical data for the Choapa basin, North Central Chile, for the period 1980-2004. The parameters considered are As, Cu Fe, pH, EC, SO4⁻², Cl⁻¹, and HCO[Formula: see text], from samples taken in nine monitoring stations throughout the basin. Results show rather moderate contents of As, Cu, and Fe, with the exception of the Cuncumén River and the Aucó creek, explained by the influence of the huge porphyry copper deposit of Los Pelambres and by the presence of mining operations, respectively. When compared against results obtained in previous researches at the neighboring Elqui river basin, which host the El Indio Au-Cu-As district, a much reduced grade of pollution is recognized for the Choapa basin. Considering the effect of acid rock drainage (ARD)-related Cu contents on the fine fraction of the sediments of both river basins, the differences recorded are even more striking. Although the Los Pelambres porphyry copper deposit, on the headwaters of the Choapa river basin, is between one and two orders of magnitude bigger than El Indio, stream water and sediments of the former exhibit significantly lower copper contents than those of the latter. A main factor which may explain these results is the smaller degree of H( + )-metasomatism on the host rocks of the Los Pelambres deposit, where mafic andesitic volcanic rocks presenting propylitic hydrothermal alteration are dominant. This fact contrast with the highly altered host rocks of El Indio district, where most of them have lost their potential to neutralize ARD.

Differential Metabolic and Transcriptional Responses of Gilthead Seabream (Sparus aurata) Administered with Cortisol or Cortisol-BSA.

Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata.

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.

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.

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.