Exposure to hydrocarbons and chlorpyrifos alters the expression of nuclear receptors and antioxidant, detoxifying, and immune response proteins in the liver of the rainbow trout, Oncorhynchus mykiss

The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors’ regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 μg L−1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L−1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT−PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8- and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF−CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2–3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.Fil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Norte. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean) | Universidad Nacional del Comahue. Centro Regional Universitario Bariloche. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean).; ArgentinaFil: Castro, Juan Manuel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Norte. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean) | Universidad Nacional del Comahue. Centro Regional Universitario Bariloche. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean).; ArgentinaFil: Arias Darraz, Luis. Universidad Austral de Chile; ChileFil: Elías, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Bariloche; ArgentinaFil: Cárcamo, Juan Guillermo. Universidad Austral de Chile; Chile. Interdisciplinary Center for Aquaculture Research; ChileFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Norte. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean) | Universidad Nacional del Comahue. Centro Regional Universitario Bariloche. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede Junín de Los Andes-inibioma-centro de Ecología Aplicada del Neuquén (cean).; Argentin

Gills CYP1A of Oncorhynchus mykiss as a sensitive biomarker of crude oil pollution in freshwater environments

The induction of CYP1A activity (EROD) and protein expression was compared in liver and gills of rainbow trout from a stream polluted with crude oil, and through laboratory exposures to 1% and 5% of water accommodated fraction of the crude oil (WAF) for 1 and 4 days. Gills EROD increased 1.6–2.7-fold in fish from the polluted stream and during experiments, while liver EROD was induced only by 1% WAF at day 1 (1.5-fold). Contrastingly, crude oil pollution strongly induced both liver and gills CYP1A protein expression in the field (14-36-fold) and in experiments (4-25-fold). This highlights that crude oil induced CYP1A activity markedly in gills but only slightly or not at all in the liver, suggesting that differences between organ EROD activities are related to the modulation of CYP1A enzyme activity but not to the regulation at transcriptional or translational levels.Fil: Leggieri, Leonardo Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Cárcamo, Juan Guillermo. Centro Interdisciplinario para la Investigación Acuícola; Chile. Universidad Austral de Chile; ChileFil: Cerón, Gerardo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Departamento de Zoología; ArgentinaFil: Darraz, Luis Arias. Universidad Austral de Chile; ChileFil: Panebianco, Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentin

Spatial and temporal specificity of Ca2+signalling inChlamydomonas reinhardtiiin response to osmotic stress

Ca2+-dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages

Effect of Breed on Transcriptional and Protein Expression of Lipogenic Enzymes in Tail and Subcutaneous Adipose Tissue from Two Grazing Breeds of Lambs

This experiment was carried out to determine the effect of breed on mRNA and protein expression levels of lipogenic enzymes acetyl-CoA carboxylase α (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1) plus sterol regulatory element binding transcription factor 1c (SREBP1c) in the subcutaneous fat (SCF) from the back of the animal, and tail fat (TF) of both Chilota and Suffolk Down lambs grazing Calafatal. Eight Chilota and six Suffolk Down 2-month-old male lambs were allocated to graze a “Calafatal”, a typical secondary succession of Chiloé Archipelago, Chile. After 62 d, lambs were slaughtered according to Chile’s meat industry standards. Fatty acid profile, RT-qPCR, and Western blot analyses from SCF and TF samples were performed. Although the mRNA expression levels of ACC, FAS, SCD1 and SREBP1c in SCF did not differ significantly between breeds (p > 0.05), a trend to higher mRNA expression of FAS and SREBP1c in TF from Chilota lambs was observed (p = 0.06). On the other hand, FAS levels in SCF were higher in Chilota than in Suffolk Down lambs (p < 0.02), although Suffolk Down showed higher fat contents and saturated fatty acid (SFA) proportions than Chilota lambs (p < 0.01). The FAS protein expression in TF was similar in both breeds (p > 0.05). Although the fat content was higher in Suffolk Down than in Chilota lambs (p < 0.01), the SFA proportions were similar in both breeds. Finally, it can be concluded that although mRNA expression of enzymes was similar in both breeds, there were differences in some protein levels in the SCF, partially related with the fatty acid profiles, thus affecting the selection of lamb breed either for human consumption or experimental purposes

Effect of Type of Pregnancy on Transcriptional and Plasma Metabolic Response in Sheep and Its Further Effect on Progeny Lambs

The following study was performed in order to determine the effect of type of pregnancy on the transcriptional expression of genes that are engaged in angiogenesis and cell turnover/lactogenesis in the ewe mammary gland, evaluating its impact on the plasma metabolic response. In addition, an assessment of its further influence on plasma metabolic response, performance, and muscle transcriptional expression of lipogenic enzymes in progeny lambs was made. Thirteen Ile de France sheep (six twin- and seven single-bearing ewes) were allocated to graze ad libitum naturalized pasture from d 45 pre-partum to day 70 post-partum, while keeping their lambs on the same diet until day 60 after weaning. The samples were collected at different times and analyzed by qRT-PCR and plasma metabolic indicators. The data were processed using SPSS package. The results showed that twin-bearing ewes overexpressed VEGFR1 at birth, and BCL2 at birth and day 35 post-partum; however, single-bearing ewes overexpressed CAIV and IGF1 at day 35 post-partum. Similar metabolite concentrations in blood plasma were found between groups of ewes. The plasma metabolic response in lambs was similar between groups and it did not influence their performance, where a similar transcriptional expression of lipogenic enzymes in muscle was observed. Therefore, the type of pregnancy can explain the slight differences in mRNA expression that were found in angiogenesis and cell turnover/lactogenesis in mammary gland, although these differences not only did not affect the plasma metabolic response in ewes, but they also had no influence on plasma metabolic response, performance, and muscle transcriptional expression of their lambs

Effects of sequential exposure to water accommodated fraction of crude oil and chlorpyrifos on molecular and biochemical biomarkers in rainbow trout

Fish can be simultaneously or sequentially exposed to various kinds of pollutants, resulting in combined effects. Polycyclic aromatic hydrocarbons induce cytochrome P450 monooxygenase 1A (CYP1A) expression, which catalyzes the conversion of the organophosphorus insecticide chlorpyrifos (CPF) into its most active derivative, CPF-oxon. CPF-oxon inhibits CYP1A and other enzymes, including carboxylesterases (CEs) and acetylcholinesterase (AChE). We studied the effects of an in vivo exposure to crude oil water accommodated fraction (WAF) followed by an ex vivo exposure of liver tissue to CPF on the expression of Cyp1a, AhR and ARNT mRNA, CYP1A protein and on the activity of biomarker enzymes in the rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed to WAF (62 μg L−1 TPH) for 48 h. Then, liver was dissected out, sliced and exposed to 20 μg L−1 CPF ex vivo for 1 h. Liver tissue was analyzed for mRNA and protein expression and for CEs, AChE, glutathione S-transferase (GST) and CYP1A (EROD) activity. WAF induced Cyp1a mRNA and CYP1A protein expression by 10-fold and 2.5–8.3-fold, respectively, with no effect of CPF. WAF induced AhR expression significantly (4-fold) in control but not in CPF treated liver tissue. ARNT mRNA expression was significantly lowered (5-fold) by WAF. CPF significantly reduced liver EROD activity, independently of WAF pre-treatment. CEs activity was significantly inhibited in an additive manner following in vivo exposure to WAF (42%) and ex vivo exposure to CPF (19%). CPF exposure inhibited AChE activity (37%) and increased GST activity (42%).Fil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; ArgentinaFil: Leggieri, Leonardo Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; ArgentinaFil: Arias Darraz, Luis. Universidad Austral de Chile; ChileFil: Cárcamo, Juan Guillermo. Centro Interdisciplinario para la Investigación Acuícola; Chile. Universidad Austral de Chile; ChileFil: Venturino, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigaciones en Toxicología Ambiental y Agrobiotecnología del Comahue. Instituto de Biotecnología Agropecuaria del Comahue | Universidad Nacional del Comahue. Facultad de Ciencias Agrarias. Centro de Investigaciones en Toxicología Ambiental y Agrobiotecnología del Comahue. Instituto de Biotecnología Agropecuaria del Comahue; ArgentinaFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; Argentin

Cyps molecular biomarkers in rainbow trout Oncorhynchus mykiss to assess oil contamination in a patagonian stream

Cytochrome P450 (CYP) gene expressions were studied by semi-quantitative polymerase chain reaction (PCR) to assess oil pollution using liver and gills of juvenile rainbow trout Oncorhynchus mykiss sampled from La Mina stream (Bariloche, Argentina), up and downstream of a discharge of petroleum spill from an unproductive wellbore. Data was complemented with laboratory exposure to water accommodated fraction (WAF) from the oil spill. Oil exposure induced gill and liver CYP1A transcription (1.2-1.4-fold), which was used as reference biomarker. In both organs, CYP2K1 transcript was induced after 5 % WAF exposure, 24 h (1.8-2.0-fold), and it was down-regulated in more extended time (1.2-1.5-fold). CYP2M1 transcript levels were down-regulated in liver of impacted wild trout and in trout exposed to 5 % WAF during 96 h (1.5-1.7-fold). CYP2K1 and CYP2M1 are involved in the metabolism of fatty acids and estrogenic hormones, and its down-regulation would indicate metabolic alterations. CYP3A27 levels were only induced in liver of wild trout (1.6-fold), possibly related to crosstalk between aryl hydrocarbon receptor and pregnane X receptor signaling pathway. While CYP1A mRNA expression appears to be a more robust biomarker for detecting the effects of crude oil under different dose and exposure time, the expression of CYP2K1, CYP2M1 and CYP3A27 mRNAs would indicate different time exposure to oil pollution, particularly under high oil concentration in water. Hence, patterns of multiple CYP genes expression in rainbow trout gills and liver could be especially valuable for monitoring programs of crude oil pollution and remediation actions on Patagonian freshwater ecosystems.Se estudió la expresión génica de citocromos P450 (CYP) mediante reacción semicuantitativa en cadena de la polimerasa (PCR, por sus siglas en inglés), en muestras de hígado y branquias de trucha arcoíris Oncorhynchus mykiss juveniles capturadas en un arroyo que es afectado por un derrame de petróleo. El estudio se completó con exposiciones in vivo a la fracción soluble del petróleo del vertedero (WAF). El CYP1A, usado como biomarcador de referencia, fue inducido por el petróleo (1.2-1.4-veces) en branquias e hígado. En ambos órganos, el CYP2K1 fue inducido después de 24 h con 5 % WAF (1.8-2.0-veces), y subregulado en tiempos más extensos (1.2-1.5-veces). CYP2M1 fue subregulado en el hígado de truchas contaminadas y de aquellas expuestas a 96 h con 5 % (1.5-1.7-veces). Dado que CYP2K1 y CYP2M1 participan del metabolismo de ácidos grasos y de hormonas estrogénicas, la subregulación indicaría alteraciones en estos metabolismos. CYP3A27 fue inducido sólo en el hígado de las truchas silvestres afectadas por petróleo, posiblemente por efectos cruzados entre las señalizaciones de los receptores de hidrocarburos de arilo y X de pregnano. Mientras que la expresión de CYP1A mRNA pareciera ser un biomarcador más robusto para detectar los efectos del petróleo crudo bajo diferentes dosis y tiempos de exposición, las expresiones de CYP2K1, CYP2M1 y CYP3 mRNAs permitirían identificar distintos tiempos de ex-posición a la contaminación por petróleo, particularmente cuando las concentraciones de crudo sean altas. Así, los patrones múltiples de expresión de los genes CYP en branquias e hígado de las trucha arcoíris podrían ser útiles en programas de monitoreo y remediación de contaminaciones de petróleo en ecosistemas acuáticos de Patagonia.Fil: Leggieri, Leonardo Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Cerón, Geardo Abel. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arias Darraz, Luis. Universidad Austral de Chile; ChileFil: Fuentes Monasterio, Daniela. Universidad Austral de Chile; ChileFil: Cárcamo, Juan Guillermo. Universidad Austral de Chile; ChileFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentin

The insecticide chlorpyrifos modifies the expression of genes involved in the PXR and AhR pathways in the rainbow trout, Oncorhynchus mykiss

Chlorpyrifos (CPF) is an organophosphate pesticide, commonly detected in water and food. Despite CPF toxicity on aquatic species has been extensively studied, few studies analyze the effects of CPF on fish transcriptional pathways. The Pregnane X receptor (PXR) is a nuclear receptor that is activated by binding to a wide variety of ligands and regulates the transcription of enzymes involved in the metabolism and transport of many endogenous and exogenous compounds. We evaluated the mRNA expression of PXR-regulated-genes (PXR, CYP3A27, CYP2K1, ABCB1, UGT, and ABCC2) in intestine and liver of the rainbow trout, Oncorhynchus mykiss, exposed in vivo to an environmentally relevant CPF concentration. Our results demonstrate that the expression of PXR and PXR-regulated genes is increased in O. mykiss liver and intestine upon exposure to CPF. Additionally, we evaluated the impact of CPF on other cellular pathway involved in xenobiotic metabolism, the Aryl Hydrocarbon Receptor (AhR) pathway, and on the expression and activity of different biotransformation enzymes (CYP2M1, GST, FMO1, or cholinesterases (ChEs)). In contrast to PXR, the expression of AhR, and its target gene CYP1A, are reduced upon CPF exposure. Furthermore, ChE and CYP1A activities are significantly inhibited by CPF, in both the intestine and the liver. CPF activates the PXR pathway in O. mykiss in the intestine and liver, with a more profound effect in the intestine. Likewise, our results support regulatory crosstalk between PXR and AhR pathways, where the induction of PXR coincides with the downregulation of AhR-mediated CYP1A mRNA expression and activity in the intestine.Fil: de Anna, Julieta Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Arias Darraz, Luis. Universidad Austral de Chile; ChileFil: Painefilú, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Cárcamo, Juan Guillermo. Universidad Austral de Chile; ChileFil: Moura Alves, Pedro. University of Oxford; Reino UnidoFil: Venturino, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Centro de Investigaciones en Toxicología Ambiental y Agrobiotecnología del Comahue. Universidad Nacional del Comahue. Facultad de Ciencias Agrarias. Centro de Investigaciones en Toxicología Ambiental y Agrobiotecnología del Comahue; ArgentinaFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentin