Environmentally Relevant Concentrations of the Insecticide Fipronil Modulated Molecular Response in Chironomus riparius.

Pesticides 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.

History of exposure to copper influences transgenerational gene expression responses in Daphnia magna.

The establishment of transgenerational effects following chemical exposure is a powerful phenomenon, capable of modulating ecosystem health beyond exposure periods. This study assessed the transgenerational effects occurring due to copper exposure in the invertebrate D. magna at the transcriptional level, while evaluating the role of exposure history on such responses. Thus, daphnids acclimated for several generations in a copper vs. clean medium were then exposed for one generation (F0) to this metal, and monitored for the following non-exposed generations (F1, F2 and F3). Organisms differing in exposure histories showed remarkably different transcriptional profiles at the F0, with naïve organisms being more profoundly affected. These trends were confirmed for F3 treatments, which presented different transcriptional patterns for genes involved in detoxification, oxidative stress, DNA damage repair, circadian clock functioning and epigenetic regulation. Furthermore, regardless of exposure history, a great number of histone modifier genes were always found transcriptionally altered, thus suggesting the involvement of histone modifications in the response of Daphnia to metal exposure. Lastly, remarkably distinct transgenerational transcriptional responses were found between naïve and non-naïve organisms, thereby highlighting the influence of exposure history on gene expression and confirming the capacity of metals to determine transgenerational transcriptional effects across non-exposed generations.

Prevention of neurotoxicity and cognitive impairment induced by zinc nanoparticles by oral administration of saffron extract.

The accumulation of relatively higher dose of zinc oxide nanoparticles in brain was reported to produce neurotoxicity. Indeed, nanoparticles have a high ability to penetrate biological membranes and be uptaken by cells, which may cause cell disorders and physiological dysfunctions. The aim of the current study was to evaluate, whether oral administration of saffron extract, in rats, can protect from neurotoxicity and behavioural disturbances induced by chronic administration of ZnO-NPs. Daily oral administration of ZnO-NPs was performed for 21 consecutive days to induce oxidative stress-like situation. Then after the saffron extract was concomitantly administrated in several rat groups to overcome the nanotoxicological effect induced by ZnO-NPs. In the frontal cortex, the hippocampus and the cerebellum, ZnO-NPs induced a H2 O2 -oxydative stress-like effect reflected in reduced enzymatic activities of catalase, superoxide dismutase and glutathione S-transferase, and decreased acetylcholinesterase activity. In addition, increased levels of proinflammatory interleukins IL-6 and IL-1-⍺ occurred in the hippocampus, reveal the existence of brain inflammation. The concomitant administration of saffron extract to animals exposed to ZnO-NPs prevented the enhanced anxiety-related to the behaviour in the elevated plus-maze test, the open field test and preserved spatial learning abilities in the Morris water maze. Moreover, animals exposed to ZnO-NPs and saffron showed abnormal activity of several antioxidant enzymes as well as acetylcholinesterase activity, an effect that may underly the preserved anxiety-like behaviour and spatial learning abilities observed in these animals. Saffron extract has a potential beneficial therapeutic effect: antioxidant, anti-inflammatory and neuroprotective agent.

Effects of wildfire ashes on aquatic invertebrates: First molecular approach on Chironomus riparius larvae.

The 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.

Molecular biomarkers as tool for early warning by chlorpyrifos exposure on Alpine chironomids.

Pesticides 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.

Suborganismal responses of the aquatic midge Chironomus riparius to polyethylene microplastics.

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.

Altered gene expression in Chironomus riparius (insecta) in response to tire rubber and polystyrene microplastics.

The extent until which plastics are present in our surrounding environment completely exceeds our expectations. Plastic materials in the form of microplastics have been found in terrestrial, freshwater and marine environments and are transported through the atmosphere even to remote locations. However, we are still far from understanding the effects that they may have caused and are causing to biota. In the present study, we investigated the alterations in the expression of twelve genes in the aquatic insect Chironomus riparius after 36 h exposures to polystyrene and tire rubber microplastics at nominal concentrations of 1 and 10 mg L-1. The results indicated that several genes encoding for heat shock proteins (hsp90, Glycoprotein 93 (Gp93), hsc70, hsp60, hsp40, and the small HSP hsp17) were overexpressed respect to the control. In addition, the genes coding for manganese superoxide dismutase (SOD Mn, related to alleviation of oxidative stress) and for the FK506-binding protein of 39 kDa. (FKBP39, related to development and pupation) showed altered expression. Most of the alterations on gene expression level occurred at a concentration of 10 mg L-1 of tire rubber microplastics, although specific modifications arose at other concentrations of both rubber and polystyrene. On the contrary, one hsp gene (hsp10) and genes related to biotransformation and detoxification (Cyp9f2, Cyp12a2, and ABCB6) did not alter their expression in any of the treatments. Overall, the results of the gene expression indicated that microplastics (especially tire rubber) or their additives caused cellular stress that led to some alterations in the normal gene expression but did not cause any mortality after 36 h. These results highlight the need for more studies that describe the alterations caused by microplastics at the molecular level. Additionally, it opens questions about the effects caused to aquatic fauna in environmental realistic situations, especially in hot spots of microplastic contamination (e.g., tire rubber released in storm water runoff discharge points).

Persistent pesticides: effects of endosulfan at the molecular level on the aquatic invertebrate Chironomus riparius.

Although banned in multiple areas, due to its persistence in the environment, endosulfan constitutes a significant environmental concern. In this work, fourth instar Chironomus riparius larvae were exposed at environmentally relevant endosulfan concentrations of 0.1, 1, and 10 µg/L for 24 h to analyze the possible effects of this acaricide on gene expression and enzymatic activity. Transcriptional changes were studied through the implementation of a real-time polymerase chain reaction array with 42 genes related to several metabolic pathways (endocrine system, detoxification response, stress response, DNA reparation, and immune system). Moreover, glutathione-S-transferase (GST), phenoloxidase (PO), and acetylcholinesterase (AChE) activities were assessed. The five pathways were differentially altered by endosulfan exposure with significant changes in the E93, Dis, MAPR, Met, InR, GSTd3, GSTt3, MRP1, hsp70, hsp40, hsp24, ATM, PARP, Proph, and Def genes. Besides, all of the measured enzymatic activities were modified, with increased activity of GST, followed by PO and AChE. In summary, the results reflected the effects provoked in C. riparius at molecular level despite the absence of lethality. These data raise concerns about the strong alteration on different metabolic routes despite the low concentrations used. Therefore, new risk assessment strategies should consider include the effects at the sub-organismal level as endpoints in addition to the classical ecologically relevant parameters (such as survival). This endeavor will facilitate a comprehensive evaluation of toxicants in the environment.

Ibuprofen as an emerging pollutant on non-target aquatic invertebrates: Effects on Chironomus riparius.

The concern about pharmaceuticals has been increased over the last decade due to their burgeoning consumption. Ibuprofen has an extensive presence in surface water with risks for the aquatic biota. This study focuses on the effects of ibuprofen at environmental concentrations on the survival, transcriptional level, and enzymatic activity for 24, 96 h on Chironomus riparius. Ibuprofen developed a substantial effect on survival by all the conditions. mRNA levels of EcR, Dronc, and Met (endocrine system), hsp70, hsp24, and hsp27 (stress response), and Proph and Def (immune system) were modified, joined to increased GST and PO activity. The results confirmed alterations on the development of C. riparius, as well as two essential mechanisms, involved in protection against external toxicological challenge. Ibuprofen poses an incipient risk to C. riparius and could at an organismal level by compromising their survival, development, and ability to respond to adverse conditions on the future populations.

Unveiling complex responses at the molecular level: Transcriptional alterations by mixtures of bisphenol A, octocrylene, and 2'-ethylhexyl 4- (dimethylamino)benzoate on Chironomus riparius.

Living organisms are exposed to mixtures of pollutants in the wild. Inland aquatic ecosystems contain many compounds from different sources that pollute the water column and the sediment. However, majority of toxicological research is focused on the effects of single exposures to toxicants. Furthermore, studies have been principally oriented toward ecologically relevant effects of intoxication, and lack an analysis of the cellular and molecular mechanisms involved in the response to toxicants. Effects of single, binary, and ternary mixtures of three compounds, bisphenol A, octocrylene, and 2'-ethylhexyl 4- (dimethylamino)benzoate, were assessed using a Real-Time PCR array. Forty genes, and additional six reference genes, were included in the array. The genes were selected based on their association with hormone responses, detoxification mechanisms, the stress response, DNA repair, and the immune system. The study was performed on Chironomus riparius, a benthic dipteran with an essential role in the food web. Transcriptional responses were assessed both 24 and 96 h post-exposure, to determinate short- and medium-term cellular responses. Individual fourth instar larvae were exposed to 0.1 and 1 mg/L of each of the toxic compounds and compound mixtures. A weak response was detected at 24 h, which was stronger in larvae exposed to mixtures than to individual toxicants. The response at 96 h was complex and principally involved genes related to the endocrine system, detoxification mechanisms, and the stress response. Furthermore, exposure to mixtures of compounds altered the expression patterns of an increased number of genes than did individual compound exposures, which suggested complex interactions between compounds affected the regulation of transcriptional activity. The results obtained highlight the importance of analyzing the mechanisms involved in the response to mixtures of compounds over extended periods and offer new insights into the basis of the physiological responses to pollution.

Endosulfan exposure alters transcription of genes involved in the detoxification and stress responses in Physella acuta.

Endosulfan 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.

Combined effects of benzophenone-3 and temperature on gene expression and enzymatic activity in the aquatic larvae Chironomus riparius.

Climate change and pollution are two of the main environmental problems living organisms currently face. Temperature can modify a toxicant's effects and the organism's response to it. Global warming is expected to increase the temperature of freshwater ecosystems. In this work, 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-stress response in this animal, and opens new possibilities in the toxicity evaluation of this organism in line with the real scenario that organisms face today.

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.

Ultraviolet 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 (OD-PABA) 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 OD-PABA, 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.