Persona: Muñiz González, Ana Belén
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Muñiz González
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Publicación Chironomus riparius molecular response to polystyrene primary microplastics(ELSEVIER, 0023-04-10) Kalman, Judit; Muñiz González, Ana Belén; García, María Ángeles; Martínez Guitarte, José LuisMicroplastics are emerging as a central concern for the environment. They can be found worldwide, produced for the industry and because of plastic debris degradation. The microplastics are smaller than 5 mm, but they can also range in micro and nanometer scales, present in soil and aquatic ecosystems. Furthermore, they can have different chemical compositions, including additives with putative toxicity. Chironomus riparius is a dipteran with aquatic larvae used in toxicology tests. As a benthic organism, it can be exposed to microplastics in the water and the sediments, being able to ingest some of them depending on the size and shape. However, it is still poor knowledge of the effects that microplastics have on invertebrates, especially at the molecular level. We have analyzed the impact that 5–5.9 μm spheres of polystyrene have on the metabolism of C. riparius, studying the transcriptional activity of eighty genes, twenty-eight described here for the first time. The genes covered the endocrine response, the detoxification mechanisms, the stress response, the DNA repairing mechanisms, hypoxia, oxidative stress, apoptosis, immunity, cholesterol metabolism, energy metabolism, the circadian rhythm, signaling, and regulation of piRNAs. The results showed that at 24 h, the stress response was the most affected, while at 48 h, the endocrine response and detoxification were slightly affected. Finally, at 72 h, endocrine response, detoxification mechanisms, and lipid metabolism genes were altered. Overall, the data suggest an acute response involving stress genes downregulation, while the later response seems to move to metabolic alterations, with changes in hormonal regulation and metabolism. It could be because micrometer microplastics are confounded with food, decreasing the availability of resources for larval development. The present work shows a dynamic impact of polystyrene microspheres and provides new putative biomarkers to analyze several mechanisms involved in the cellular and physiological response to toxicants.Publicación Effects of wildfire ashes on aquatic invertebrates: First molecular approach on Chironomus riparius larvae(Elsevier, 2022-11-03) Muñiz González, Ana Belén; Campos, Isabel; Re, Ana; Martínez Guitarte, José Luis; Nelson AbrantesThe wildfire magnification in recent years has raised increasing concern about their adverse impacts on the environment. Wildfires are recognized as an important source of diffuse pollution for the nearby aquatic systems being potentially toxic to aquatic life. Albeit previous studies with wildfire runoff/ashes observed effects in aquatic organisms, to date, different severity origins of ashes and their impact at the sub-organismal level on aquatic biota have not been assessed. In this work, the molecular response of Chironomus riparius exposed to wildfire with low (LS) and high (HS) severity ashes from burnt Pine plantations was evaluated by employing an array of 42 genes related to crucial metabolic pathways by Real time-PCR. IV instar larvae were exposed for 72 h to aqueous extract of ashes (12.5 %, 25 %, 50 %, 75 % and 100 %) prepared from LS and HS ashes. Mn, Zn, and Pb were the metals found at highest concentration in both ash extracts, for HS notable Cd, Mn and Cr presence. From the 42 genes studied only 4 were not altered (22 genes modulated their response by LS and 38 genes in the case of HS) showing the opposite response at 100% with downregulated by LS and upregulated by HS. The 12.5 %, 25 %, 100 % HS and 25 % LS were the main modulators, confirmed by the integrative biomarkers response (IBR). Remarkable genotoxicity was generated by ashes even activating the apoptosis response, and endocrine disruption observed could modify the development. Moreover, detoxification and stress response were strongly activated, limiting the organism's future response to external aggressions. The employment of this novelty approach with molecular tools act as early alarm signal preventing greater damages. Overall, wildfire ashes showed to be a significant environmental disruptor to C. riparius even at lower concentration and the short exposure time employed, emphasizing the strong impact of wildfires on aquatic systems.Publicación Molecular biomarkers as tool for early warning by chlorpyrifos exposure on Alpine chironomids(Elsevier, 2021-08-26) Muñiz González, Ana Belén; Paoli, Francesca; Martínez Guitarte, José Luis; Lencioni, ValeriaPesticides used in agriculture can be transported at a medium-high distance due to the drift effect, reaching even remote areas as mountain regions, glaciers, and snow cover. With the melting process, pesticides enter freshwater glacier ecosystems, becoming a threat to wildlife fauna, mainly dominated by Diptera Chironomidae. Chlorpyrifos (CPF), as one of the most commonly used pesticides in alpine vineyards and apple orchards, is frequently detected in icemelt waters. We selected as target species, larvae of the cold stenothermal chironomid Diamesa zernyi, collected in two glacier-fed streams (Presena and Amola) in the Italian Alps. Firstly, a de novo transcriptome was obtained, and secondly, a gene array was designed to study the molecular response of a wild population of D. zernyi exposed to three sub-lethal CPF concentrations corresponding to 1/100 LC10 (0.011 μg/ L), 1/10 LC10 (0.11 μg/L), and LC10 (1.1 μg/L), for 24 h. The sub-organismal response was evaluated by Real-Time Polymerase Chain Reaction (RT-PCR), employing 40 genes related to essential metabolic routes as future candidates for biomarkers in wildlife chironomids. After 24 h, the endocrine system (E75, E93, EcR, and Met), detoxification response (GSTO3, GSTS1), and stress response (hsp75, hsp83, HYOU1) were altered. CPF seems to act as an endocrine disruptor and could lead to defective larval development, disrupted cellular homeostasis through heat shock proteins (HSPs) alteration (defective protein folding and mitochondrial functions), as well as oxidative damage (confirmed by increased GST expression). For the first time, molecular studies detected early alarm signals in wildlife in glacier environments. Our findings confirm the high environmental risk of CPF affecting aquatic insect metabolism and raise the level of concern about this pesticide in high altitude water bodies, generally considered pristine. Furthermore, this study emphasizes the incipient need to use non-model organisms for the evaluation of natural ecosystems. We also highlight the demand for research into new molecular biomarkers, and the importance of including molecular approaches in toxicology evaluations to detect the early adverse effects of pollutants.Publicación Suborganismal responses of the aquatic midge Chironomus riparius to polyethylene microplastics(Elsevier, 2021-08-20) Muñiz González, Ana Belén; Silva, Carlos J.M.; Patricio Silva, Ana L.; Campos, Diana; Pestana, João L.T.; Martínez Guitarte, José LuisFreshwater riverbeds are a major repository of microplastics (MPs) from inland activities. Benthic macroinvertebrates that live in close contact with sediments seem to ingest a considerable amount of such plastic particles. The effects of MPs on life-history traits are relatively well-known, but the suborganismal mechanisms underlying such effects remain unclear. This study addressed the potential effects of low-density polyethylene (LDPE) MPs Freshwater riverbeds are a major repository of microplastics (MPs) from inland activities. Benthic macroinvertebrates that live in close contact with sediments seem to ingest a considerable amount of such plastic particles. The effects of MPs on life-history traits are relatively well-known, but the suborganismal mechanisms underlying such effects remain unclear. This study addressed the potential effects of low-density polyethylene (LDPE) MPs on Chironomus riparius larvae at cellular and molecular levels. Fourth instar C. riparius larvae were exposed to 0.025 and 2.5 g/kg LDPE of dry sediment (sizes: <32 and 32–45 μm; with irregular shape) under laboratory conditions for 48 h. These short-term exposures to environmental concentrations of LDPE MPs induced changes in the energy reserves (mostly by decreasing carbohydrates and increasing lipids), increased antioxidant and detoxification responses (tGSH, CAT, and GST), and induced increases in the activity of AChE (related to neurotransmission). In addition, at the gene level, exposure to MPs modified mRNA levels of InR, Dis, EcR, Dronc, Met (endocrine system), Def (immune system), PARP, ATM, NLK, and Decay (DNA repair), generating important alterations in the C. riparius development and response to unfavorable situations. This study provides new evidence of the effects of LDPE MPs at the suborganismal level, filling the gap in knowledge regarding the mechanisms underlying the toxicity of MPs and spotlighting gene expression analyses as early indicators of MP toxicity in C. riparius which were confirmed by Integrated biomarker response analyses highlighting the gene expression as sensible and useful endpoints for LPDE pollution in freshwaters. These results, coupled with previous investigations on responses at the organismal level, emphasizes the potential adverse effects of LDPE MPs on C. riparius, which may compromise freshwater benthic communities, considering its ecological role within these habitats. on Chironomus riparius larvae at cellular and molecular levels. Fourth instar C. riparius larvae were exposed to 0.025 and 2.5 g/kg LDPE of dry sediment (sizes: <32 and 32–45 μm; with irregular shape) under laboratory conditions for 48 h. These short-term exposures to environmental concentrations of LDPE MPs induced changes in the energy reserves (mostly by decreasing carbohydrates and increasing lipids), increased antioxidant and detoxification responses (tGSH, CAT, and GST), and induced increases in the activity of AChE (related to neurotrans-mission). In addition, at the gene level, exposure to MPs modified mRNA levels of InR, Dis, EcR, Dronc, Met (endocrine system), Def (immune system), PARP, ATM, NLK, and Decay (DNA repair), generating important alterations in the C. riparius development and response to unfavorable situations. This study provides new evidence of the effects of LDPE MPs at the suborganismal level, filling the gap in knowledge regarding the mechanisms underlying the toxicity of MPs and spotlighting gene expression analyses as early indicators of MP toxicity in C. riparius which were confirmed by Integrated biomarker response analyses highlighting the gene expression as sensible and useful endpoints for LPDE pollution in freshwaters. These results, coupled with previous investigations on responses at the organismal level, emphasizes the potential adverse effects of LDPE MPs on C. riparius, which may compromise freshwater benthic communities, considering its ecological role within these habitats.Publicación Environmentally Relevant Concentrations of the Insecticide Fipronil Modulated Molecular Response in Chironomus riparius(Wiley, 2023-11-29) da Silva Pinto, Thandy Junio; Martínez Guitarte, José Luis; Dias, Mariana Amaral; Montagner, Cassiana Carolina; Gaeta Espindola, Evaldo Luiz; Muñiz González, Ana BelénPesticides employed worldwide for crop protection easily reach aquatic systems, which act as the main reservoirs, and become a risk factor for aquatic fauna. Fipronil is a broad‐spectrum insecticide acting on the insect nervous system; however, other effects and systems unrelated to this mechanism could be affected in non‐target organisms. Thus, the present study aimed to assess the impact of fipronil on the suborganismal response (gene expression and enzymatic activity) of Chironomus riparius larvae as a model organism in ecotoxicology. To this end, short‐term toxicity tests were carried out with fourth‐instar larvae exposed to 0.001, 0.01, and 0.1 μg L−1 of fipronil for 24 and 96 h. Messenger RNA levels of 42 genes related to diverse metabolic pathways were analyzed by real‐time polymerase chain reaction, complemented with catalase (CAT), glutathione S‐transferase (GST), and acetylcholinesterase (AChE) activities. Few effects were observed at 24 h; however, after longer exposure (96 h), genes involved in the endocrine, detoxification, stress, and immune response pathways were altered. Moreover, fipronil at 96 h increased CAT and GST activity at 0.01 μg L−1 and AChE at the highest concentrations. The results demonstrate that even low environmentally relevant fipronil concentrations can modulate the molecular response of several cellular pathways in C. riparius after short‐term exposure. These results bring new information about the underlying response of fipronil and its mode of action on a key aquatic invertebrate. Despite no effects on mortality, strong modulation at the suborganismal level emphasizes the advantage of biomarkers as early damage responses and the harmful impact of this pesticide on freshwater organisms. Environ Toxicol Chem 2024;43:405–417. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Publicación Endosulfan exposure alters transcription of genes involved in the detoxification and stress responses in Physella acuta(Springer Nature, 2020) Alonso Trujillo, María; Muñiz González, Ana Belén; Martínez Guitarte, José Luis; Springer NatureEndosulfan is a persistent pesticide that has been in use for more than five decades. During this time, it has contaminated soil, air, and water reservoirs worldwide. It is extremely toxic and harmful to beneficial non-target invertebrates, aquatic life, and even humans upon consumption, which is one of the many dangers of this pesticide since it biomagnifies in the food chain. The effects of three endosulfan concentrations (1, 10, and 100 μg/L) on the freshwater snail Physella acuta, an invasive cosmopolitan species, were examined over a week-long exposure period. Alterations in the expression of ten genes related to stress and xenobiotic detoxification were measured against the endogenous controls rpL10 and GAPDH by Real-Time polymerase chain reaction. Four genes are described here for the first time in this species, namely Hsp60, Grp78, GSTk1, and GSTm1. The rest of genes were Hsp90, sHsp16.6, cyp2u1, cyp3a7, cyp4f22, and MRP1. cyp2u1, sHsp16.6, and Grp78 expression were all altered by endosulfan. These results suggest a low pesticide concentration activates the acute response in P. acuta by affecting detoxification and stress responses and alter endoplasmic reticulum function and lipid metabolism. Furthermore, the newly identified genes extend the number of processes and cellular locations that can be analyzed in this organism.Publicación New insights about the toxicity of 2,4-D: Gene expression analysis reveals modulation on several subcellular responses in Chironomus riparius(Elsevier, 2024-09) da Silva Pinto, Thandy Junio; Martínez Guitarte, José Luis; Amaral Dias, Mariana; Montagner, Cassiana Carolina; Gaeta Espindola, Evaldo Luiz; Muñiz González, Ana Belén; ElsevierHerbicides are the main class of pesticides applied in crops and are capable of polluting the surrounding freshwater system; thus, understanding their impact on non-target species, whose mechanism of action is not described, helps to elucidate the real risks of these pollutants to the environment. 2,4-dichlorophenoxyacetic acid (2,4-D) is frequently detected in water and, due to its persistence, poses a risk to wildlife. In this way, the present work aimed to describe the implication of exposure to concentrations of 2,4-D already reported in aquatic environments in several physiological mechanisms of C. riparius at molecular and biochemical levels. To achieve this, bioassays were conducted with fourth instar larvae exposed to three concentrations of 2,4-D (0.1, 1.0, and 7.5 μg L 1). Larvae were collected after 24 and 96 h of exposure, and the expression of 42 genes, related to six subcellular mechanisms, was assessed by Real-Time PCR (RT-PCR). Besides, the activity of the enzymes catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE) was determined. The main metabolic route altered after exposure to 2,4-D was the endocrine system (mainly related to 20-hydroxyecdysone and juvenile hormone), confirming its endocrine disruptor potential. Four of the eleven stress response genes studied were down-regulated, and later exposure modulated DNA-repair genes suggesting genotoxic capacity. Moreover, only one gene from each detoxification phase was modulated at short exposure to 1.0 μg L 1. The molecular responses were not dose-dependent, and some early responses were not preserved after 96 h, indicating a transient response to the herbicide. Exposure to 2,4-D did not alter the activity of CAT, GST, and AChE enzymes. The responses described in this study reveal new mechanistic pathways of toxicity for 2,4-D in non-target organisms and highlight potential ecological consequences for chironomids in aquatic systems at the edges of agricultural fields.Publicación Impact of Global Warming on Kryal Fauna: Thermal Tolerance Response of Diamesa steinboecki (Goetghebuer, 1933; Chironomidae)(MDPI, 2023-05-26) Muñiz González, Ana Belén; Martínez Guitarte, José Luis; Lencioni, Valeria; MDPI; https://orcid.org/0000-0002-4341-9923The ice fly Diamesa steinboecki Goetghebuer, 1933 (Diptera: Chironomidae: Diamesinae) is exclusive to glacier-fed streams in the East Palaearctic region and is threatened by extinction due to global warming and glacier retreat. To date, no data are available on its thermal tolerance or ability to develop a heat shock response (HSR) or involve other biomarkers when exposed to higherthan-natural temperatures (i.e., >4–6 ◦C). Our study aimed to investigate the warmth resistance of IV-instar larvae of D. steinboecki in terms of (1) ability to survive heat shock and (2) gene expression of four genes known to be involved in the detoxification/stress response (cytochrome p450 (Cyp450), heat shock protein 70 (hsp70), hsp70 with intron and heat shock protein cognate 70 (hsc70)). Larvae were exposed to short-term shocks for 1 h at increasing temperatures (26, 28, 30, 32, 34, 36, 38, and 40 ◦C) to estimate the lethal temperature, obtaining high values (LT10 = 38.1 ◦C, LT50 = 39.2 ◦C, LT99 = 40.3 ◦C), suggesting a strong heat resistance up to 38 ◦C and a very rapid decline in survival thereafter. Moreover, gene expression analysis by real-time PCR was performed on larvae from the control (at 2 ◦C) and larvae found alive after the previous treatment at 26, 28, 30, 32, 34, 36, and 38 ◦C. Modulation of the expression was observed only for hsc70 and hsp70 genes. Specifically, hsc70 resulted in constitutive overexpression, even at 26 ◦C when all larvae were found alive without evidence of suffering. By contrast, hsp70 showed up and downregulation according to the specific temperature, suggesting the activation of an HSR at 28 ◦C, when some larvae were found alive but suffering (almost paralyzed). The results suggest that, based on LTs, D. steinboecki is more thermally tolerant than other Diamesa species (e.g., D. tonsa) from cold freshwaters, but, as in these, hsp70 and hsc70 are involved in surviving short-term heat shock. This makes the ice fly from the Alps different from Belgica antarctica and other cold-adapted organisms living in extremely cold habitats that, constantly exposed to cold, have lost the ability to develop an HSR. Further research is needed to investigate the response to prolonged exposure to temperatures higher that the natural one, giving new insights into the biological response to climate change of alpine species threatened by extinction.Publicación Combined effects of benzophenone-3 and temperature on gene expression and enzymatic activity in the aquatic larvae Chironomus riparius(Elsevier, 2020) Muñiz González, Ana Belén; Martínez Guitarte, José Luis; Elsevier; https://orcid.org/0000-0002-7722-864XClimate change and pollution are two of the main environmental problems living organisms currently face. Temperature canmodify a toxicant's effects and the organism's response to it. Globalwarming is expected to increase the temperature of freshwater ecosystems. In thiswork,we analyzed the effect of a mild temperature increase on the acute response of the aquatic larvae Chironomus riparius to the ultraviolet filter benzophenone-3 (BP3). This substance is commonly used in sunscreens and other commercial products and can reach the environment in different ways.We exposed larvae to BP3 at 18.5 or 23 °C for 8 or 24 h and analyzed the acute response at the molecular level. By quantitative real-time polymerase chain reaction (q-PCR), we studied altered messenger RNA (mRNA) levels of genes related to the endocrine system (EcR, InR and Met), detoxification mechanisms (Cyp4d2, Cyp6b7, GST d6, GST o1 and MRP-1) and stress response (Hsp22, Hsp27, Hsp70, HYOU and Gp93). Moreover, enzyme activity was evaluated, with a focus on glutathione-S-transferase (GST), phenoloxidase (PO) and acetylcholinesterase (AChE). Results showed that temperature affected the acute response of this organism by modifying the expression of EcR, Cyp6b7, GST d6, GST o1, MRP-1, Hsp22, Hsp27 and Hsp70 genes. These results suggest that even mild temperature change can affect the response of this organism to BP3 influencing short-term progress of the population. Although longer exposures are required to determine the ability of C. riparius to manage the pollutants in this novel environmental conditions, in order to know the possible mechanisms of detoxification or adaptation that may develop. This research represents a first step in the analysis of multi-stressPublicación Effects of single exposure and binary mixtures of ultraviolet filters octocrylene and 2-ethylhexyl 4-(dimethylamino) benzoate on gene expression in the freshwater insect Chironomus riparius(Springer Nature, 2018-10-22) Muñiz González, Ana Belén; Martínez Guitarte, José Luis; Springer NatureUltraviolet filters are used extensively in the production of many personal care and industrial products. These products can inadvertently pollute the environment through recreational activities. They have been associated with endocrine disruption in vertebrates but their effects in invertebrates are poorly understood. Chironomus riparius is a species of the dipteran order, with aquatic larvae that are frequently used in toxicity tests. Previously, we showed that octocrylene (OC) and 2-ethylhexyl 4-(dimethylamino) benzoate (ODPABA) differentially affected the mRNA levels of the ecdysone receptor and Hsp70 genes. For a better understanding of their mode of action, transcriptional activity by real-time PCR was analyzed in fourth instar larvae exposed to OC, OD-PABA, or a binary mixture of both. We studied 16 genes related to the endocrine system, stress, the immune system, and biotransformation mechanisms to elucidate the putative interactions between these compounds. No response was observed for the genes involved in biotransformation, suggesting that enzymes other than cytochromes P450 and glutathione-S-transferases (GSTs) could get involved in transformation of these compounds. Similarly, no response was observed for endocrine-related genes while the stress gene HYOU1 was inhibited by ODPABA, suggesting an effect in response to hypoxia. In addition, no significant interactions were observed following exposure to a binary mixture of these compounds. Overall, the results suggest a weak, acute response in different metabolic pathways and a lack of interaction between the compounds. Finally, new genes are identified in this organism, opening the possibility to analyze new cellular pathways as targets of toxicants.