Publicación: Characterisation of environmental stress biomarkers in Prodiamesa olivacea (Diptera) and their analysis and comparative evaluation in two chironomid species for ecotoxicity studies in natural scenarios
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2022
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Atribución-NoComercial-SinDerivadas 4.0 Internacional
info:eu-repo/semantics/openAccess
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Universidad Nacional de Educación a Distancia (España). Escuela Internacional de Doctorado. Programa de Doctorado en Ciencias
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Junto con los métodos tradicionales de investigaciones ecotoxicológicas utilizando organismos modelo, los estudios toxicológicos en organismos no modelo se están considerando un complemento perfecto para conseguir metodologías más robustas. Esta estrategia integradora nos permite descifrar la complejidad de las exposiciones en los ecosistemas naturales. El objetivo principal de este trabajo ha sido caracterizar los efectos moleculares que tres contaminantes ambientales comunes (BBP, butil bencil ftalato; BPA, bisfenol A; y BP3, benzofenona 3), así como el estrés por choque térmico provocan en poblaciones naturales de dos invertebrados acuáticos: la especie no modelo Prodiamesa olivacea (Chironomidae, Diptera) y el organismo modelo en ecotoxicología Chironomus riparius (Chironomidae, Diptera). El género de los quironómidos, un género de mosquitos no picadores, cuenta con cuatro ensayos estandarizados y aceptados internacionalmente con fines regulatorios para evaluar la toxicidad del agua y los sedimentos. En todos ellos, se utilizan especies de quironómidos y se analizan parámetros clásicos de toxicidad. Por el contrario, Prodiamesa rara vez se usa para estudios de toxicidad, pese a ser una especie interesante desde el punto de vista toxicológico ya que comparte hábitats con Chironomus pero requiere condiciones menos extremas y niveles de oxígeno más altos. Esta divergencia de requerimientos es particularmente interesante para evaluar las diferentes respuestas de ambas especies a condiciones de estrés. En esta investigación de tesis doctoral, se recolectaron larvas de cuarto estadío de ambos quironómidos en un río contaminado (Sar) de Galicia (España) y se expusieron de forma independiente a 1 μg/L BBP, BPA o BP3 durante 4 h y 24 h. Además, para evaluar el impacto del choque térmico, las larvas se expusieron a 35 °C y 39 °C durante 30', 60', 120' and 120' seguido de 2 h a temperatura ambiente. Los efectos se midieron analizando alteraciones transcripcionales y enzimáticas de diferentes biomarcadores de interés. Los resultados han permitido identificar y caracterizar por primera vez en P. olivacea nuevos biomarcadores tempranos de efecto implicados en la respuesta de estrés celular (Hsp27, Hsp60, Hsp70, Hsc70, Cdc37), el sistema endocrino (EcR, Kr-h1, JHEH), respuestas de biotransformación y estrés oxidativo (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a-14-like, Cyp6a2-like), el sistema inmune (PGRP, C-type lectin, TOLL, JAK/hopscotch) y el metabolismo energético (GAPDH). Se han descrito respuestas tóxicas diferenciales específicas de especie tras la exposición a xenobióticos y choque térmico, siendo P. olivacea el díptero que mostró una mayor sensibilidad en la mayoría de los biomarcadores moleculares estudiados. De entre los tres contaminantes de estudio, BBP fue el compuesto más tóxico para las larvas de P. olivacea ya que produjo una clara y rápida inhibición de genes relacionados con vías metabólicas esenciales. Los tres compuestos interfirieron con el sistema endocrino desencadenando un efecto antagonista de las hormonas esteroideas. El análisis transcripcional comparativo entre C. riparius y P. olivacea reveló que la exposición a xenobióticos desencadenó una temprana y aguda activación transcripcional en C. riparius, en contraposición al descenso de la actividad transcripcional observado en P. olivacea. El choque térmico activó drásticamente la respuesta de estrés celular e inhibió el sistema inmune en P. olivacea, mientras que ambas respuestas se activaron en C. riparius. Estos efectos podrían comprometer la capacidad de adaptación y supervivencia de P. olivacea y destaca la alta tolerancia de C. riparius a ambientes extremos. Las respuestas diferenciales detectadas entre P. olivacea y C. riparius proporcionan información novedosa sobre los efectos nocivos del BBP, BPA y BP3, así como del choque térmico en estos mosquitos acuáticos y destacan el potencial de P. olivacea para ser considerado un organismo centinela adecuado para estudios de ecotoxicidad en escenarios naturales.
Along with traditional ecotoxicological research using model organisms, toxicological studies in non-model organisms are being considered a complement to achieve more robust methodologies. This integrative strategy allows us to figure out the complexity of exposures in natural ecosystems. The main objective of this work has been to characterise the molecular effects that three common environmental pollutants (BBP, butyl benzyl phthalate; BPA, bisphenol A; and BP3, benzophenone 3), as well as heat stress provoke in natural populations of two aquatic invertebrates: the non-model species Prodiamesa olivacea (Chironomidae, Diptera) and model organism in ecotoxicology Chironomus riparius (Chironomidae, Diptera). The genus Chironomus has four standardised OECD tests for evaluating water and sediment toxicity, and different species can be used to assess classical toxicity parameters. In contrast, Prodiamesa is rarely used for toxicity studies, although it is an interesting toxicological species because it shares habitats with Chironomus while requiring less extreme conditions and higher oxygen levels. These different requirements are particularly interesting to assess the different responses of both species to stress conditions. In the present research, 4th instar larvae of both chironomids were collected in a contaminated river (Sar) from Galicia (Spain) and independently exposed to 1 μg/L BBP, BPA or BP3 for 4 h and 24 h. Additionally, to evaluate heat-shock impacts, larvae were exposed to 35 °C and 39 °C for 30', 60', 120' and 120' followed by 2 h at RT. The effects were measured by analysing transcriptional and enzymatic alterations of different biomarkers of interest. The results have allowed us to identify and characterise for the first time in P. olivacea new early biomarkers of effect involved in the cells stress response (Hsp27, Hsp60, Hsp70, Hsc70, Cdc37), the endocrine system (EcR, Kr-h1, JHEH), biotransformation and oxidative responses (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a-14-like, Cyp6a2-like), the immune system (PGRP, C-type lectin, TOLL, JAK/hopscotch) and the energy metabolism (GAPDH). Differential species-specific toxic responses have been described following exposure to xenobiotics and heat stress, with P. olivacea being the dipteran that showed the highest sensitivity in most of the studied biomarker genes. BBP was the most toxic to P. olivacea larvae among the three chemical tested, leading to quick and robust inhibition of genes related to critical metabolic pathways. All three compounds interfered with the endocrine system, triggering an ecdysteroid-antagonist effect. The comparative transcriptional analysis between C. riparius and P. olivacea revealed that exposure to xenobiotics resulted in strong early transcriptional activation in C. riparius, leading to downregulation in P. olivacea. Heat-shock drastically activated the cell stress response and inhibited the immune system in P. olivacea, while both responses were activated in C. riparius. These effects could compromise the adaptive capacity and survival of P. olivacea and highlights the high tolerance of C. riparius to extreme environments. The differential responses detected between P. olivacea and C. riparius provide novel information on the harmful effects of BBP, BPA, BP3 and heat-shock on these aquatic midges and highlight the potential of P. olivacea to be considered as a suitable sentinel organism for ecotoxicity studies in natural scenarios.
Along with traditional ecotoxicological research using model organisms, toxicological studies in non-model organisms are being considered a complement to achieve more robust methodologies. This integrative strategy allows us to figure out the complexity of exposures in natural ecosystems. The main objective of this work has been to characterise the molecular effects that three common environmental pollutants (BBP, butyl benzyl phthalate; BPA, bisphenol A; and BP3, benzophenone 3), as well as heat stress provoke in natural populations of two aquatic invertebrates: the non-model species Prodiamesa olivacea (Chironomidae, Diptera) and model organism in ecotoxicology Chironomus riparius (Chironomidae, Diptera). The genus Chironomus has four standardised OECD tests for evaluating water and sediment toxicity, and different species can be used to assess classical toxicity parameters. In contrast, Prodiamesa is rarely used for toxicity studies, although it is an interesting toxicological species because it shares habitats with Chironomus while requiring less extreme conditions and higher oxygen levels. These different requirements are particularly interesting to assess the different responses of both species to stress conditions. In the present research, 4th instar larvae of both chironomids were collected in a contaminated river (Sar) from Galicia (Spain) and independently exposed to 1 μg/L BBP, BPA or BP3 for 4 h and 24 h. Additionally, to evaluate heat-shock impacts, larvae were exposed to 35 °C and 39 °C for 30', 60', 120' and 120' followed by 2 h at RT. The effects were measured by analysing transcriptional and enzymatic alterations of different biomarkers of interest. The results have allowed us to identify and characterise for the first time in P. olivacea new early biomarkers of effect involved in the cells stress response (Hsp27, Hsp60, Hsp70, Hsc70, Cdc37), the endocrine system (EcR, Kr-h1, JHEH), biotransformation and oxidative responses (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a-14-like, Cyp6a2-like), the immune system (PGRP, C-type lectin, TOLL, JAK/hopscotch) and the energy metabolism (GAPDH). Differential species-specific toxic responses have been described following exposure to xenobiotics and heat stress, with P. olivacea being the dipteran that showed the highest sensitivity in most of the studied biomarker genes. BBP was the most toxic to P. olivacea larvae among the three chemical tested, leading to quick and robust inhibition of genes related to critical metabolic pathways. All three compounds interfered with the endocrine system, triggering an ecdysteroid-antagonist effect. The comparative transcriptional analysis between C. riparius and P. olivacea revealed that exposure to xenobiotics resulted in strong early transcriptional activation in C. riparius, leading to downregulation in P. olivacea. Heat-shock drastically activated the cell stress response and inhibited the immune system in P. olivacea, while both responses were activated in C. riparius. These effects could compromise the adaptive capacity and survival of P. olivacea and highlights the high tolerance of C. riparius to extreme environments. The differential responses detected between P. olivacea and C. riparius provide novel information on the harmful effects of BBP, BPA, BP3 and heat-shock on these aquatic midges and highlight the potential of P. olivacea to be considered as a suitable sentinel organism for ecotoxicity studies in natural scenarios.
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