Toxicogenomics of the Freshwater Oligochaete, Tubifex tubifex (Annelida, Clitellata), in Acute Water-Only Exposure to Arsenic.

A toxicogenomic approach was used for toxicity evaluation of arsenic in the aquatic environment, and differential gene expression was investigated from 24 h and 96 h water-only acute toxicity tests with the aquatic oligochaete, Tubifex tubifex (Annelida, Clitellata). Several toxicological endpoints (survival and autotomy) of the oligochaete and tissue residues were measured, and dose-response modelling of gene expression data was studied. A reference transcriptome of the aquatic oligochaete, T. tubifex, was reconstructed for the first time, and genes related to cell stress response (Hsc70, Hsp10, Hsp60, and Hsp83), energy metabolism (COX1), oxidative stress (Cat, GSR, and MnSOD), and the genes involved in the homeostasis of organisms (CaM, RpS13, and UBE2) were identified and characterised. The potential use of the genes identified for risk assessment in freshwater ecosystems as early biomarkers of arsenic toxicity is discussed.

Unveiling Molecular Effects of the Secondary Metabolite 2-Dodecanone in the Model Hymenopteran Nasonia vitripennis.

Over the past decade, multiple studies have suggested that the secondary metabolites produced by plants against herbivorous insects could be used as biopesticides. However, as the molecular mechanism of action of these compounds remains unknown, it is difficult to predict how they would affect non-target insects; thus, their innocuity needs to be clarified. Here, we investigate, from the molecular level to the organism, the responses of a useful parasitic insect Nasonia vitripennis (Walker, 1836) being exposed at the pupae stage for 48 h (up to 6 days) to sublethal doses (5 µg/L and 500 µg/L) of 2-Dodecanone. 2-Dodecanone altered the gene expression of genes related to ecdysone-related pathways, biotransformation, and cell homeostasis. A significant induction of ecdysone response-genes (EcR, usp, E78, Hr4, Hr38) was detected, despite no significant differences in ecdysteroid levels. Regarding the cell homeostasis processes, the gene l(2)efl was differentially altered in both experimental conditions, and a dose-dependent induction of hex81 was observed. 2-Dodecanone also triggered an induction of Cyp6aQ5 activity. Finally, 2-Dodecanone exposure had a significant effect on neither development time, energy reserves, nor egg-laying capacity; no potential genotoxicity was detected. For the first time, this study shows evidence that 2-Dodecanone can modulate gene expression and interfere with the ecdysone signalling pathway in N. vitripennis. This could lead to potential endocrine alterations and highlight the suitability of this organism to improve our general understanding of the molecular effects of plant defences in insects. Our findings provide new insights into the toxicity of 2-Dodecanone that could potentially be explored in other species and under field conditions for plant protection and pest management as a means to reduce reliance on synthetic pesticides.

Characterization and expression of heat shock and immune genes in natural populations of Prodiamesa olivacea (Diptera) exposed to thermal stress.

This paper characterizes the heat stress response (HSR) and explores the impact of temperatures on the immune response of larvae from two chironomid species, Prodiamesa olivacea and Chironomus riparius. Genes involved in crucial metabolic pathways were de novo identified in P. olivacea: Hsp27, Hsp60, Hsp70, Hsc70, Cdc37, and HSF for the heat stress response (HSR) and TOLL, PGRP, C-type lectin, and JAK/hopscotch for the immune system response (ISR). Quantitative real-time PCR was used to evaluate the expression levels of the selected genes in short-term treatments (up to 120') at high temperatures (35 °C and 39 °C). Exposing P. olivacea to elevated temperatures resulted in HSR induction with increased expression of specific heat shock genes, suggesting the potential of HSPs as early indicators of acute thermal stress. Surprisingly, we found that heat shock represses multiple immune genes, revealing the antagonist relation between the heat shock response and the innate immune response in P. olivacea. Our results also showed species-dependent gene responses, with more significant effects in P. olivacea, for most of the biomarkers studied, demonstrating a higher sensitivity in this species to environmental stress conditions than that of C. riparius. This work shows a multi-species approach that enables a deeper understanding of the effects of heat stress at the molecular level in aquatic dipterans.

Prodiamesa olivacea: de novo biomarker genes in a potential sentinel organism for ecotoxicity studies in natural scenarios.

Along with traditional ecotoxicological approaches in model organisms, toxicological studies in non-model organisms are being taken into consideration in order to complement them and contribute to more robust approaches. This allows us to figure out the complexity of the exposures involved in natural ecosystems. In this context, in the present research we have used the model species Chironomus riparius (Chironomidae, Diptera) and the non-model species Prodiamesa olivacea (Chironomidae, Diptera) to assess the aquatic toxic effects of acute 4-h and 24-h exposures to 1 µgL-1 of three common environmental pollutants: butyl benzyl phthalate (BBP), bisphenol A (BPA), and benzophenone 3 (BP3). Individuals of both species were collected from a contaminated river (Sar) in Galicia (Spain). Regarding Chironomus, there are four OECD standardized tests for the evaluation of water and sediment toxicity, in which different species in this genus can be used to assess classical toxicity parameters such as survival, immobilization, reproduction, and development. In contrast, Prodiamesa is rarely used in toxicity studies, even though it is an interesting toxicological species because it shares habitats with Chironomus but requires less extreme conditions (e.g., contamination) and higher oxygen levels. These different requirements are particularly interesting in assessing the different responses of both species to pollutant exposure. Quantitative real-time PCR was used to evaluate the transcriptional changes caused by xenobiotics in different genes of interest. Since information about P. olivacea in genomic databases is scarce, its transcriptome was obtained using de novo RNAseq. Genes involved in biotransformation pathways and the oxidative stress response (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a14-like and Cyp6a2-like) were de novo identified in this species. Our results show differential toxic responses depending on the species and the xenobiotic, being P. olivacea the dipteran that showed the most severe effects in most of the studied biomarker genes. This work represents a multi-species approach that allows us to deepen in the toxicity of BBP, BPA, and BP3 at the molecular level. Besides, it provides an assessment of the tolerance/sensitivity of natural populations of model and non-model insect species chronically exposed to complex mixtures of pollutants in natural scenarios. These findings may have important implications for understanding the adverse biological effects of xenobiotics on P. olivacea, providing new sensitive biomarkers of exposure to BBP, BPA, and BP3. It also highlights the suitability of Prodiamesa for ecotoxicological risk assessment, especially in aquatic ecosystems.

Effects on tadpole snail gene expression after exposure to vinclozolin.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests.

Combining the assessment of apical endpoints and gene expression in the freshwater snail Physa acuta after exposure to reclaimed water.

Post-treatment wastewater reuses are diverse. Recreational and environmental restoration uses of reclaimed water (RW) can be potentially harmful to aquatic organisms. In this work the freshwater snail Physa acuta was exposed to RW (100%) and its dilution (RW 50%). A simple laboratory mixture of three emerging pollutants was used to address the complex problem of mixture toxicity of RW. Hence fortified reclaimed water (FRW), obtained by adding fluoxetine (400 µg FLX/L), perfluorooctane sulphonic acid (90 µg PFOS/L) and methylparaben (9 µg MP/L), was tested at two dilution percentages: 100% and 50%. The effects of the laboratory mixture of FLX, PFOS and MP on the test medium were also studied. Long-lasting effects, together with early molecular responses, were assessed. Fecundity (cumulative egg production) over 21 days and the hatching of produced eggs (F1) after another 21-day embryonic exposure were monitored. The gene expression of three genes was analysed after 24 h of exposure: two endocrine-related nuclear receptors (ERR and RXR) and one stress protein gene (Hsp70). This reproduction test, with additional assessments of the F1 recovered eggs' hatching success, showed that both RW and FRW significantly reduced fecundity. F1 hatching was affected only by FRW. The gene expression results showed that the RXR response was strikingly similar to the fecundity response, which suggests that this nuclear receptor is involved in the reproductive pathways of gastropods. ERR remained virtually unaltered. Hsp70 was overexpressed by the laboratory mixture in the test medium, but no effect was observed in the fortification of RW. This opposite effect and lack of response for F1 hatching produced by the laboratory mixture in the test medium highlighted the difficulty of predicting mixture effects. The experimental approach allowed us to test the effects caused by RW on P. acuta at different biological organisation levels. Thus, the combination of molecular biomarkers and ecological relevant endpoints is a good strategy to test complex mixtures like RW as it provides a framework to link mechanisms of action and whole organism effects when it is almost impossible to detect the pollutant(s) that cause toxic effects.

The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae).

Bisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled "BPA-free". Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 µg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.

Genotoxic effects of vinclozolin on the aquatic insect Chironomus riparius (Diptera, Chironomidae).

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 µg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates.

Ultraviolet filters and heat shock proteins: effects in Chironomus riparius by benzophenone-3 and 4-methylbenzylidene camphor.

Benzophenone-3 (BP3) and 4-methylbenzylidene camphor (4MBC) are common ultraviolet filters (UV filters), compounds considered as emergent contaminants, used in different products like plastics and personal care products. The levels of these compounds are rising in the wild, but the effects they have on invertebrates are poorly understood. Chironomus riparius is a benthic insect widely used in toxicology, and several studies have been previously performed in our laboratory to determine the effects these compounds have on this organism at the molecular level. We have shown that UV filters can alter the mRNA levels of heat shock protein 70 (Hsp70), one of the most studied heat shock proteins. Although these proteins are crucial for the survival of organisms, little data is available on the effects these emergent contaminants have on them, especially in invertebrates. Here, we analyzed the transcriptional activity of 12 genes covering the different groups of heat shock protein [Hsp10, Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, Hsp27, Hsp34, Hsp40, Hsp60, Hsc70 (3), and Hsc70 (4)] in response to 0.1 and 1 mg/L concentrations of BP3 and 4MBC at 8 and 24 h. The results showed that some small Hsp (sHsp) genes were altered by these compounds, while the genes of proteins present in mitochondria, Hsp10 and Hsp60, did not change. sHsps are also involved in developmental processes, so the observed variations could be due to the endocrine disruption activity described for these compounds rather than to a stress response.

UV filters induce transcriptional changes of different hormonal receptors in Chironomus riparius embryos and larvae.

Organic ultraviolet (UV) filters are emerging contaminants that are ubiquitous in fresh and marine aquatic systems due to their extensive use in cosmetics, plastics, paints, textiles, and many other industrial products. The estrogenic effects of organic UV filters have been long demonstrated in vertebrates, and other hormonal activities may be altered, according to more recent reports. The impact of UV filters on the endocrine system of invertebrates is largely unknown. We have previously reported that some UV filters may affect ecdysone-related genes in the aquatic insect Chironomus riparius, an ecotoxicologically important model organism. To further analyze other possible effects on endocrine pathways, we first characterized four pivotal genes related with hormonal pathways in insects; thereafter, these genes were assessed for alterations in transcriptional activity after exposure to 4-methylbenzylidene camphor (4MBC) or benzophenone-3 (BP-3), two extensively used sunscreens. We found that both chemicals disturbed the expression of all four genes analyzed: hormonal receptor 38 (HR38), methoprene-tolerant (Met), membrane-associate progesterone receptor (MAPR) and insulin-like receptor (INSR), measured by changes in mRNA levels by real-time PCR. An upregulatory effect at the genomic level was detected in different developmental stages. Interestingly, embryos appeared to be more sensitive to the action of the UV filters than larvae. Our results suggest that the risk of disruption through different endocrine routes is not negligible, considering the significant effects of UV filters on key hormonal receptor and regulatory genes. Further effort is needed to develop environmental risk assessment studies on these pollutants, particularly for aquatic invertebrate model organisms.

Vinclozolin alters the expression of hormonal and stress genes in the midge Chironomus riparius.

Vinclozolin is a fungicide used in agriculture that can reach aquatic ecosystems and affect the organisms living there. Its effects have been intensively studied in vertebrates, where it acts as an antiandrogen, but there is a lack of information about its mechanistic effects on invertebrates. In this work, we analyzed the response of genes related to the endocrine system, the stress response, and the detoxification mechanisms of Chironomus riparius fourth instar larvae after 24h and 48h exposures to 20 (69.9nM), 200 (699nM), and 2000µg/L (6.99µM) of Vinclozolin. Survival analysis showed that this compound has low toxicity, as it was not lethal for this organism at the concentrations used. However, this fungicide was shown to modify the transcriptional activity of the ecdysone response pathway genes EcR, E74, and Kr-h1 by increasing their mRNA levels. While no changes were observed in disembodied, a gene related with the ecdysone synthesis metabolic pathway, Cyp18A1, which is involved in the inactivation of the active form of ecdysone, was upregulated. Additionally, the expression of two genes related to other hormones, FOXO and MAPR, did not show any changes when Vinclozolin was present. The analysis of stress response genes showed significant changes in the mRNA levels of Hsp70, Hsp24, and Gp93, indicating that Vinclozolin activates the cellular stress mechanisms. Finally, the expressions of the genes Cyp4G and GstD3, which encode enzymes involved in phase I and phase II detoxification, respectively, were analyzed. It was found that their mRNA levels were altered by Vinclozolin, suggesting their involvement in the degradation of this compound. For the first time, these results show evidence that Vinclozolin can modulate gene expression, leading to possible significant endocrine alterations of the insect endocrine system. These results also offer new clues about the mode of action of this compound in invertebrates.