Chironomus Riparius Larvae Research Articles

The sediment of a river having ‘’good ecological status” turned out to be toxic to midge Chironomus riparius larvae: Implication for environmental monitoring?

Globally, monitoring of the surface waters is largely limited to the physico-chemical analysis of water in rivers and lakes. Sediment state in the aquatic systems including sediment chemical content or the structure and diversity of benthic communities or ecotoxicological studies with natural sediments remains largely overlooked by the monitoring programs. Hence we assessed the potential toxicity of three riverine sediments on the life-cycle traits (emergence and reproduction) of midge Chironomus riparius via an ecotoxicological testing method over two generations (according to OECD test 233 guidelines). In addition, the riverine sediments were spiked with polyamide (nylon) microplastic particles (1 g kg−1) to analyze an additive effect of microplastic on the sediment toxicity. As model river systems, three rivers (Karchaghbyur, Gavaraget, Argichi) in the Lake Sevan basin (Armenia) were selected. Results of ecotoxicity testing were compared with the indices of water quality (derived from the physico-chemical analysis) and the indices of the ecological status of the rivers (derived from the analysis of benthic communities). The results of testing demonstrated an unexpectedly low emergence of midges after the first generation exposed to the sediment of the river having ‘’good ecological status’’ - the Argichi. Sediments of the Karchaghbyur and Gavaraget rivers impeded the emergence and reproduction of midges after the second generation. An addition of polyamide particles to the sediments did not significantly affect the life-cycle traits of C. riparius indicating the primary effect of the sediments’ condition. The discrepancy of biotesting result with that of the other two methods (which indicated ‘’average water quality’’ and “good ecological status”) underlies the importance of designing more comprehensive monitoring programs for better assessment and protection of aquatic systems and resources.

Sludge degradation, nutrient removal and reduction of greenhouse gas emission by a Chironomus-Azolla wastewater treatment cascade.

Wastewater treatment plants (WWTPs) are a point source of nutrients, emit greenhouse gases (GHGs), and produce large volumes of excess sludge. The use of aquatic organisms may be an alternative to the technical post-treatment of WWTP effluent, as they play an important role in nutrient dynamics and carbon balance in natural ecosystems. The aim of this study was therefore to assess the performance of an experimental wastewater-treatment cascade of bioturbating macroinvertebrates and floating plants in terms of sludge degradation, nutrient removal and lowering GHG emission. To this end, a full-factorial experiment was designed, using a recirculating cascade with a WWTP sludge compartment with or without bioturbating Chironomus riparius larvae, and an effluent container with or without the floating plant Azolla filiculoides, resulting in four treatments. To calculate the nitrogen (N), phosphorus (P) and carbon (C) mass balance of this system, the N, P and C concentrations in the effluent, biomass production, and sludge degradation, as well as the N, P and C content of all compartments in the cascade were measured during the 26-day experiment. The presence of Chironomus led to an increased sludge degradation of 44% compared to 25% in the control, a 1.4 times decreased transport of P from the sludge and a 2.4 times increased transport of N out of the sludge, either into Chironomus biomass or into the water column. Furthermore, Chironomus activity decreased methane emissions by 92%. The presence of Azolla resulted in a 15% lower P concentration in the effluent than in the control treatment, and a CO2 uptake of 1.13 kg ha-1 day-1. These additive effects of Chironomus and Azolla resulted in an almost two times higher sludge degradation, and an almost two times lower P concentration in the effluent. This is the first study that shows that a bio-based cascade can strongly reduce GHG and P emissions simultaneously during the combined polishing of wastewater sludge and effluent, benefitting from the additive effects of the presence of both macrophytes and invertebrates. In addition to the microbial based treatment steps already employed on WWTPs, the integration of higher organisms in the treatment process expands the WWTP based ecosystem and allows for the inclusion of macroinvertebrate and macrophyte mediated processes. Applying macroinvertebrate-plant cascades may therefore be a promising tool to tackle the present and future challenges of WWTPs.

DNA damage and molecular level effects induced by polystyrene (PS) nanoplastics (NPs) after Chironomus riparius (Diptera) larvae

In this work, we analyzed the early molecular effects of polystyrene (PS) nanoplastics (NPs) on an aquatic primary consumer (larvae of Chironomus riparius, Diptera) to evaluate their potential DNA damage and the transcriptional response of different genes related to cellular and oxidative stress, endocrine response, developmental, oxygen transport, and immune response. After 24-h exposures of larvae to doses of PS NPs close to those currently found in the environment, the results revealed a large genotoxic effect. This end was evidenced after significant increases in DNA strand breaks of C. riparius larvae quantified by the comet assay, together with results obtained when analyzing the expression of four genes involved in DNA repair (xrrc1, ATM, DECAY and NLK) and which were reduced in the presence of these nanomaterials. Consequently, this reduction trend is likely to prevent the repair of DNA damage caused by PS NPs. In addition, the same tendency to reduce the expression of genes involved in cellular stress, oxidative stress, ecdysone pathway, development, and oxygen transport was observed. Taken together, these results suggest that PS NPs reduce the expression of hormonal target genes and a developmental gene. We show, for the first time, effects of PS NPs on the endocrine system of C. riparius and suggest a possible mechanism of blocking ecdysteroid hormones in insects. Moreover, the NPs were able to inhibit the expression of hemoglobin (Hb C), a protein involved in oxygen transport, and activate a gene of the humoral immune system. These data reveal for the first time the genomic effects of PS NPs in the aquatic invertebrate C. riparius, at the base of the food chain.

The Multifaceted Effects of Short-Term Acute Hypoxia Stress: Insights into the Tolerance Mechanism of Propsilocerus akamusi (Diptera: Chironomidae).

Plenty of freshwater species, especially macroinvertebrates that are essential to the provision of numerous ecosystem functions, encounter higher mortality due to acute hypoxia. However, within the family Chironomidae, a wide range of tolerance to hypoxia/anoxia is displayed. Propsilocerus akamusi depends on this great tolerance to become a dominant species in eutrophic lakes. To further understand how P. akamusi responds to acute hypoxic stress, we used multi-omics analysis in combination with histomorphological characteristics and physiological indicators. Thus, we set up two groups-a control group (DO 8.4 mg/L) and a hypoxic group (DO 0.39 mg/L)-to evaluate enzyme activity and the transcriptome, metabolome, and histomorphological characteristics. With blue-black chromatin, cell tightness, cell membrane invagination, and the production of apoptotic vesicles, tissue cells displayed typical apoptotic features in the hypoxic group. Although lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), catalase (CAT), and Na+/K+ -ATPase (NKA) activities were dramatically enhanced under hypoxic stress, glycogen content, and superoxide dismutase (SOD) activities were significantly reduced compared to the control group. The combined analysis of the transcriptome and metabolome, which further demonstrated, in addition to carbohydrates, including glycogen, the involvement of energy metabolism pathways, including fatty acid, protein, trehalose, and glyoxylate cycles, provided additional support for the aforementioned findings. Lactate is the end product of glycogen degradation, and HIF-1 plays an important role in promoting glycogenolysis in acute hypoxic conditions. However, we discovered that the ethanol tested under hypoxic stress likely originates from the symbiodinium of P. akamusi. These results imply that some parameters related to energy metabolism, antioxidant enzyme activities, and histomorphological features may be used as biomarkers of eutrophic lakes in Chironomus riparius larvae. The study also provides a scientific reference for assessing toxicity and favoring policies to reduce their impact on the environment.

Toxicity and risk management of oil-spiked sediments by diluted bitumen for two freshwater benthic invertebrates

Diluted bitumen (dilbit) is an unconventional oil produced by the oil sands industry in Canada. Despite the knowledge available on hydrocarbon toxicity, the effects of diluted bitumen on benthic organisms are still largely unknown. Moreover, in Quebec there are only provisional threshold values of 164 mg/kg C10–C50 for chronic effects and 832 mg/kg for acute effects. The protectiveness of these values for benthic invertebrates has not been tested for heavy unconventional oils such as dilbit. Two benthic organisms, the larvae of Chironomus riparius and Hyalella azteca, were exposed to these two concentrations and to an intermediate concentration (416 mg/kg) of two dilbits (DB1 and DB2) and a heavy conventional oil (CO). The aim of the study was to assess the sublethal and lethal effects of spiked sediment by dilbit. The oil was rapidly degraded in the sediment, especially in the presence of C. riparius. Amphipods were much more sensitive to oil than chironomids. LC50-14d values for H. azteca were 199 mg/kg C10–C50 for DB1, 299 mg/kg for DB2 and 8.42 mg/kg for CO compared to LC50-7d values for C. riparius of 492 mg/kg for DB1, 563 mg/kg for DB2 and 514 mg/kg for CO. The size of the organisms was reduced compared to controls for both species. The defense enzymes (GST, GPx, SOD and CAT) were not good biomarkers in these two organisms for this type of contamination. The current provisional sediment quality criteria seem too permissive for heavy oils and should be lowered.

Microplastics altered cellular responses, physiology, behaviour, and regeneration of planarians feeding on contaminated prey

Freshwater benthic environments are among the major sinks of microplastics (MPs, < 5 mm) sourced on inland anthropogenic activities. The ecotoxicological effects of MPs on benthic macroinvertebrates have been assessed preferably in collectors, shredders, and filter-feeders, but resulting in insufficient knowledge on the potential trophic transfer and its effects on macroinvertebrates with predator behaviour such as planarians. This work evaluated the behavioural (feeding, locomotion), physiological (regeneration) and biochemical responses (aerobic metabolism, energy reserves, oxidative damage) of the planarian Girardia tigrina after consuming contaminated live prey Chironomus riparius larvae previously exposed to microplastics of polyurethane (PU-MPs; 7–9 μm in size; 375 mg PU-MPs/kg). After the feeding period (3 h), planarians consumed 20 % more contaminated prey than uncontaminated prey, probably related to increased curling/uncurling movements of larvae (that might be more appellative to planarians). Histological analysis revealed planarians' limited intake of PU-MPs, mainly detected near the pharynx. The consumption of contaminated prey (and intake of PU-MPs) did not result in oxidative damage but slightly increased the aerobic metabolism and energy reserves which show that the consumption of more prey was sufficient to cope with the potential adverse effects of internalized MPs. Moreover, no effects were observed in the locomotion of planarians in good agreement with the hypothesis of sufficient energy acquired by the exposed planarians. Despite the previous, it seems that the energy acquired was not allocated for planarians' regeneration since a significant delay in the regeneration of the auricles was observed for planarians feeding on contaminated prey. Therefore, further studies should be performed considering the potential long-term effects (i.e., reproduction/fitness) and the effects of MPs that might result from continuous feeding on contaminated prey, representing a more realistic exposure scenario.

Tire and Road Wear Particle-Containing Sediments with High Organic Content Impact Behavior and Survival of Chironomid Larvae (Chironomus riparius)

Tire and road wear particles (TRWP), which contribute significantly to microplastic emission, are receiving more attention, but details about particle composition, translocation from source to sink, and particularly the possible effects on ecosystems are largely unknown. We examined the influence of native TRWP-containing sediments from two settling ponds on the mortality and behavior of the aquatic larvae of Chironomus riparius. Both sediments, whether pure or mixed with different proportions of quartz sand and suspended in water, led to increased mortalities with increasing concentrations and were shown to be oxygen consuming. Artificial aeration significantly reduced larval mortality in both sediments. Chironomid larvae show high tolerance to anoxic and polluted environments due to physiological and behavioral adaptations, such as the construction of vertical sediment tubes (chimneys), in which they create oxic compartments. A significant correlation was found between the proportion of contaminated sediment and the number of chimneys: the more contaminated sediment, the fewer chimneys were constructed. The number of chimneys per surviving larva decreased with an increased proportion of contaminated sediment in parallel to increased larval mortality. We hypothesize that contents of these sediments negatively impact the larvae’s ability to survive at low oxygen concentrations due to impairments of essential behavioral and physiological processes.

Metal Exposure and Sex Shape the Fatty Acid Profile of Midges and Reduce the Aquatic Subsidy to Terrestrial Food Webs.

Aquatic micropollutants can be transported to terrestrial systems and their consumers by emergent aquatic insects. However, micropollutants, such as metals, may also affect the flux of physiologically important polyunsaturated fatty acids (PUFAs). As certain PUFAs have been linked to physiological fitness and breeding success of terrestrial consumers, reduced fluxes from aquatic systems could affect terrestrial populations and food webs. We chronically exposed larvae of the aquatic insect Chironomus riparius to a range of environmentally relevant sediment contents of cadmium (Cd) or copper (Cu) in a 28-day microcosm study. Since elevated water temperatures can enhance metals' toxic effects, we used two temperature regimes, control and periodically elevated temperatures (heat waves) reflecting an aspect of climate change. Cd and Cu significantly reduced adult emergence by up to 95% and 45%, respectively, while elevated temperatures had negligible effects. Both metal contents were strongly reduced (∼90%) during metamorphosis. Furthermore, the chironomid FA profile was significantly altered during metamorphosis with the factors sex and metal exposure being relevant predictors. Consequently, fluxes of physiologically important PUFAs by emergent adults were reduced by up to ∼80%. Our results suggest that considering fluxes of physiologically important compounds, such as PUFAs, by emergent aquatic insects is important to understand the implications of aquatic micropollutants on aquatic-terrestrial meta-ecosystems.

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.

Toxic effects of a cyanobacterial strain on Chironomus riparius larvae in a multistress environment

Cyanobacteria and their toxic metabolites present a global threat to water habitats, but their impact on aquatic organisms in a multistress environment has been poorly investigated. Here we present the results of a survey on the effects of the toxic cyanobacterial strain Trichormus variabilis (heterotypic synonym Anabaena variabilis), and its toxic metabolite, cyanotoxin microcystin-LR, on Chironomus riparius larvae in a multistress environment. An environmentally relevant concentration of microcystin-LR (0.01 mg/L) caused an increase in larvae mortality in an acute toxicity test, which became greater in the presence of environmental stressors (NO3−, NH4+, PO43− and Cd2+), pointing to an additive effect of these agents. Chronic exposure of C. riparius larvae to the microcystin-LR producing strain of T. variabilis in a multistress environment led to a reduction in the larval mass and hemoglobin concentration, and it induced DNA damage in larval somatic cells. The results revealed the additive effect of microcystin-LR in combination with all three tested stressors (NO3−, NH4+, PO43−), and the deleterious effect of chronic exposure of C. riparius larvae to the microcystin-LR producing T. variabilis in a multistress environment. However, the present study further emphasizes the importance of investigating interactions between stressors and cyanotoxins, and their effect on aquatic organisms.

Polystyrene Microplastics Modulate the Toxicity of the Hydrophilic Insecticide Thiacloprid for Chironomid Larvae and Also Influence Their Burrowing Behavior

As there is still little knowledge of interactions between microplastics (MP) and hydrophilic compounds, we propose ways the toxicity of hydrophilic pesticides can be modulated by MP, when sorption can be excluded. Larvae of Chironomus riparius were exposed to thiacloprid (TH, 1 µg/L) and polystyrene microplastic particles (PS; &lt;50 µm; 150,000 and 1,000,000 particles/L) for 96 h, solely or in co-exposure. Burrowing behavior and mortality were observed. Larvae in treatments containing PS established themselves quicker in the sediment and kept the ability to rebury for a longer time compared to control and TH, respectively. While TH elevated the mortality, exposure to PS alone did not affect the survival of the larvae. In co-exposure of TH and PS, a concentration of 150,000 particles/L significantly reduced the toxicity of 1 µg/L TH after 96 h, an effect that was not observed at 1,000,000 particles/L. Therefore, we hypothesize that this modulation of the toxicity of TH eventually may have resulted from a combination of a ‘protective MP layer’ in the gut and a higher retention time of particles in larvae exposed to 150,000 particles/L than in those exposed to 1,000,000 particles/L due to the lower number of ingestible particles in the former.

Contribution of sediment contamination to multi-stress in lowland waters

Water bodies in densely populated lowland areas are often impacted by multiple stressors. At these multi-stressed sites, it remains challenging to quantify the contribution of contaminated sediments. This study, therefore, aimed to elucidate the contribution of sediment contamination in 16 multi-stressed drainage ditches throughout the Netherlands. To this end an adjusted TRIAD framework was applied, where 1) contaminants and other variables in the sediment and the overlying water were measured, 2) whole-sediment laboratory bioassays were performed using larvae of the non-biting midge Chironomus riparius, and 3) the in situ benthic macroinvertebrate community composition was determined. It was hypothesized that the benthic macroinvertebrate community composition would respond to all jointly present stressors in both water and sediment, whereas the whole-sediment bioassays would only respond to the stressors present in the sediment. The benthic macroinvertebrate community composition was indeed related to multiple stressors in both water and sediment. Taxa richness was positively correlated with the presence of PO4-P in the water, macrophyte cover and some pesticides. Evenness, the number of Trichoptera families and the SPEARpesticides were positively correlated to the C:P ratios in the sediment, whilst negative correlations were observed with various contaminants in both the water and sediment. The whole-sediment bioassays with C. riparius positively related to the nutrient content of the sediment, whereas no negative relations to the sediment-associated contaminants were observed, even though the lowered SPEARpesticides index indicated contaminant effects in the field. Therefore, it was concluded that sediment contamination was identified as one of the various stressors that potentially drove the benthic macroinvertebrate community composition in the multi-stressed drainage ditches, but that nutrients may have masked the adverse effects caused by low and diverse sediment contaminants on C. riparius in the bioassays.

Effects of Wildfire Ashes on Aquatic Invertebrates: First Molecular Approach on Chironomus Riparius Larvae

Effects of Wildfire Ashes on Aquatic Invertebrates: First Molecular Approach on Chironomus Riparius Larvae

A Multidisciplinary Approach for the Assessment of Origin, Fate and Ecotoxicity of Metal(loid)s from Legacy Coal Mine Tailings.

Over the course of history, the development of human societies implied the exploitation of mineral resources which generated huge amounts of mining wastes leading to substantial environmental contamination by various metal(loid)s. This is especially the case of coal mine tailings which, subjected to weathering reactions, produce acid mine drainage (AMD), a recurring ecological issue related to current and past mining activities. In this study, we aimed to determine the origin, the fate and the ecotoxicity of metal(loid)s leached from a historical coal tailing heap to the Beuveroux river (Franche-Comté, France) using a combination of mineralogical, chemical and biological approaches. In the constitutive materials of the tailings, we identified galena, tetrahedrite and bournonite as metal-rich minerals and their weathering has led to massive contamination of the water and suspended particles of the river bordering the heap. The ecotoxicity of the AMD has been assessed using Chironomus riparius larvae encaged in the field during a one-month biomonitoring campaign. The larvae showed lethal and sub-lethal (growth and emergence inhibition and delay) impairments at the AMD tributary and near downstream stations. Metal bioaccumulation and subcellular fractionation in the larvae tissues revealed a strong bioavailability of, notably, As, Pb and Tl explaining the observed biological responses. Thus, more than 70 years after the end of mining operations, the coal tailings remain a chronic source of contamination and environmental risks in AMD effluent receiving waters.

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.

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 molecularlevel 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.

Genotoxic effects and transcriptional deregulation of genetic biomarkers in Chironomus riparius larvae exposed to hydroxyl- and amine-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimers

The physical-chemical properties of polyamidoamine (PAMAM) dendrimers give them attractive qualities for multiple applications, including biomedical purposes. Their use has significantly increased in recent years, and there is currently a lack of information on their potential adverse effects on biological systems, which raises particular concerns about the risks to human health and the environment from their use and disposal. Studies focusing on ecotoxicological effects are particularly scarce. In the present work, we have used the model insect Chironomus riparius to assess the toxicity of hydroxyl-terminated and amine-terminated generation 3 PAMAM dendrimers: PAMAM G3-OH and PAMAM G3-NH2. Fourth-instar larvae were exposed to 10 or 100 ppm G3-OH or G3-NH2 dendrimers for 24 h (acute toxicity) or 24 + 24 h (delayed toxicity). Using quantitative real-time PCR, we analyzed transcriptional alterations in genes related to relevant physiological pathways: EcR, GAPDH, GPx, GST, hsp10, hsp24, Gp93, and rpL4. We also used the comet assay to assess the possible genotoxic characteristics of the dendrimers and their ability to induce DNA breakage. Regarding the effects on transcriptional activity, our results showed that G3-NH2 dendrimers induced greater (and in many cases opposite) alterations than G3-OH dendrimers, especially in acute 24-hour exposures. Delayed toxicity studies showed that both dendrimers produced transcriptional alterations even 24 h after the withdrawal of the compound and frequently contrary to what was observed acutely. In both cases, the lowest concentration studied (10 ppm) was the most toxic. On the other hand, the comet assay allowed us to detect genotoxic effects for the two dendrimers, thus demonstrating their ability to interact with DNA. This work provides new information about the possible disruptive effects of PAMAM G3 dendrimers on the health of exposed organisms, alerting the need to carry out more studies to deepen their toxic characteristics and mode of action, especially in natural environments.

Immune response triggered by the ingestion of polyethylene microplastics in the dipteran larvae Chironomus riparius

The activation of insects’ immune system due to the ingestion of microplastics (MPs) has only been evidenced by the upregulation of specific genes. The activation of phenoloxidase (PO) system is one of the primary responses involved in insects' innate immunity when facing parasites and pathogens, and ingestion of MPs can trigger a similar process. This study aimed at addressing the activities of basal PO and total PO (PO+ prophenoloxidase – proPO), in Chironomus riparius larvae (a model species in ecotoxicology) exposed to sediments spiked with polyethylene microplastics (PE-MPs; size-range 32–63 µm; concentrations: 1.25; 5; to 20 g kg–1) for 48 h. The ingestion of PE-MPs by larvae triggered a significant increase of basal PO activity at 5 and 20 g PE-MPs kg–1, by 26% and 29%, respectively, whereas total PO increased significantly in the latter (+48%), suggesting de novo synthesis of proPO by organisms. Considering the particle size, the immune response's activation is probably linked to damage in the epithelial cells of the gut lumen. This research work provides the first evidence on the activation of the insect's innate immune system after ingestion of MPs and underlines the PO activity as a good indicator of the immune response induced by MPs' ingestion.

Chironomus riparius Proteome Responses to Spinosad Exposure.

The potential of proteome responses as early-warning indicators of insecticide exposure was evaluated using the non-biting midge Chironomus riparius (Meigen) as the model organism. Chironomus riparius larvae were exposed to environmentally relevant concentrations of the neurotoxic pesticide spinosad to uncover molecular events that may provide insights on the long-term individual and population level consequences. The iTRAQ labeling method was performed to quantify protein abundance changes between exposed and non-exposed organisms. Data analysis revealed a general dose-dependent decrease in the abundance of globin proteins as a result of spinosad exposure. Additionally, the downregulation of actin and a larval cuticle protein was also observed after spinosad exposure, which may be related to previously determined C. riparius life-history traits impairment and biochemical responses. Present results suggest that protein profile changes can be used as early warning biomarkers of pesticide exposure and may provide a better mechanistic interpretation of the toxic response of organisms, aiding in the assessment of the ecological effects of environmental contamination. This work also contributes to the understanding of the sublethal effects of insecticides in invertebrates and their molecular targets.

Protein Fe-S Centers as a Molecular Target of Toxicity of a Complex Transition Metal Oxide Nanomaterial with Downstream Impacts on Metabolism and Growth.

Oxidative stress is frequently identified as a mechanism of toxicity of nanomaterials. However, rarely have the specific underlying molecular targets responsible for these impacts been identified. We previously demonstrated significant negative impacts of transition metal oxide (TMO) lithium-ion battery cathode nanomaterial, lithium cobalt oxide (LCO), on the growth, development, hemoglobin, and heme synthesis gene expression in the larvae of a model sediment invertebrate Chironomus riparius. Here, we propose that alteration of the Fe-S protein function by LCO is a molecular initiating event leading to these changes. A 10 mg/L LCO exposure causes significant oxidation of the aconitase 4Fe-4S center after 7 d as determined from the electron paramagnetic resonance spectroscopy measurements of intact larvae and a significant reduction in the aconitase activity of larval protein after 48 h (p < 0.05). Next-generation RNA sequencing identified significant changes in the expression of genes involved in 4Fe-4S center binding, Fe-S center synthesis, iron ion binding, and metabolism for 10 mg/L LCO at 48 h (FDR-adjusted, p < 0.1). We propose an adverse outcome pathway, where the oxidation of metabolic and regulatory Fe-S centers of proteins by LCO disrupts metabolic homeostasis, which negatively impacts the growth and development, a mechanism that may apply for these conserved proteins across species and for other TMO nanomaterials.