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.

Impact of single and combined exposure to priority pollutants on gene expression and post-embryonic development in Drosophila melanogaster.

Many priority pollutants are concentrated in the environment due to human activity. Most are highly toxic to various organisms, including endocrine disruptors EDCs, aromatic polycyclic hydrocarbons PAHs, pesticides. While the effects of single and binary exposure have been widely explored, several pollutants can be simultaneously present at the same time in the environment, in in more or less polluted matrices. Effective pollution control requires the presence and sources of contamination to be identified. Previously we used Drosophila melanogaster to investigate metal pollution. Here, we re-used Drosophila to identify the biomarkers of pollution, and to determine if they can be used for specific types of pollution. Single and combined exposure of Bis(2-ethylhexyl) phthalate (DEHP), bisphenol A, nonylphenol, benzo(a)pyrene, and glyphosate was investigated. The impact of these pollutants on post-embryonic development and the expression pattern of 38 molecular targets were examined using qPCR. During single exposure, different profiles were observed at the molecular level. In complex mixtures, the expression profile resembled that of bisphenol A. In contrast, relatively specific gene expression profiles were obtained for the effects of each pollutant separately. While direct pollutant-gene profiling remains difficult in mixtures, molecular biology analyses enhance pollution monitoring, and should be incorporated in toxicological studies.

Transcriptome analysis of aphids exposed to glandular trichomes in tomato reveals stress and starvation related responses.

Understanding the responses of insect herbivores to plant chemical defences is pivotal for the management of crops and pests. However, the mechanisms of interaction are not entirely understood. In this study, we compared the whole transcriptome gene expression of the aphid Macrosiphum euphorbiae grown on two different varieties of tomato that differ in their inducible chemical defences. We used two isogenic lines of tomato with a shared genetic background that only differ in the presence of type IV glandular trichomes and their associated acylsucrose excretions. This works also reports a de novo transcriptome of the aphid M. euphorbiae. Subsequently, we identified a unique and distinct gene expression profile for the first time corresponding to aphid´s exposure to type IV glandular trichomes and acylsugars. The analysis of the aphid transcriptome shows that tomato glandular trichomes and their associated secretions are highly efficient in triggering stress-related responses in the aphid, and demonstrating that their role in plant defence goes beyond the physical impediment of herbivore activity. Some of the differentially expressed genes were associated with carbohydrate, lipid and xenobiotic metabolisms, immune system, oxidative stress response and hormone biosynthesis pathways. Also, the observed responses are compatible with a starvation syndrome. The transcriptome analysis puts forward a wide range of genes involved in the synthesis and regulation of detoxification enzymes that reveal important underlying mechanisms in the interaction of the aphid with its host plant and provides a valuable genomic resource for future study of biological processes at the molecular level using this aphid.

How to thrive in unstable environments: Gene expression profile of a riparian earthworm under abiotic stress.

Nowadays, extreme weather events caused by climate change are becoming more frequent. This leads to the occurrence of extreme habitats to which species must adapt. This challenge becomes crucial for species living in unstable environments, such as the riparian earthworm Eiseniella tetraedra. Its cosmopolitan distribution exposes it to various environmental changes, such as freezing in subarctic regions or droughts in Mediterranean areas. Transcriptional changes under cold and desiccation conditions could therefore shed light on the adaptive mechanisms of this species. An experiment was performed for each condition. In the cold experiment, the temperature was lowered to -14 °C ± 2 °C (compared to 8 °C for control samples), and in the desiccation treatment, humidity was lowered from 60% to 15%. Comparisons of gene expression levels between earthworms under freezing conditions and control earthworms revealed a total of 84 differentially expressed genes and comparisons between the desiccation experiment and the control yielded 163 differentially expressed genes. However, no common responses were found between the two treatments. The results suggest that E. tetraedra can acclimate to low temperatures due to the upregulation of genes involved in glucose accumulation. However, downregulation of the respiratory chain suggests that this earthworm does not tolerate freezing conditions. Under desiccation conditions, genes involved in cell protection from apoptosis and DNA repair were upregulated. In contrast, lipid metabolism was downregulated, presumably to conserve resources by reducing the rate at which they are consumed.

Intergenerational Patterns of DNA Methylation in Procambarus clarkii Following Exposure to Genotoxicants: A Conjugation in Past Simple or Past Continuous?

Epigenome is susceptible to modulation by environmental pressures-namely, through alterations in global DNA methylation, impacting the organism condition and, ultimately, reverberating on the phenotype of the subsequent generations. Hence, an intergenerational study was conducted, aiming to clarify the influence of genotoxicants on global DNA methylation of the crayfish Procambarus clarkii. Two subsequent generations were exposed to the herbicide penoxsulam (Px; 23 µg·L-1) and to the genotoxicant model ethyl methanesulfonate (EMS; 5 mg·L-1). Px did not induce changes in DNA methylation of adult crayfish (F0). However, the hypomethylation occurring in unexposed F1 juveniles demonstrated that the history of exposure per se can modulate epigenome. In F1 descendants of the Px-exposed group, methylome (hypermethylated) was more affected in males than in females. EMS-induced hypomethylation in adult females (F0), also showed gender specificity. In addition, hypomethylation was also observed in the unexposed F1 crayfish, indicating an intergenerational epigenetic effect. The modulatory role of past exposure to penoxsulam or to EMS also showed a dependency on the crayfish developmental stage. Overall, this research revealed that indirect experiences (events occurring in a predecessor generation) can have an impact even greater than direct experiences (present events) on the epigenetic dynamics.

Single and mixed exposure to cadmium and mercury in Drosophila melanogaster: Molecular responses and impact on post-embryonic development.

Heavy metals, like many other chemical elements, are naturally present in the environment; however, the concentrations of these metals in various environmental matrices have increased through their intensive use in many human activities (such as industry, mining and agriculture). Among the heavy metals, cadmium (Cd) and mercury (Hg) induce a wide variety of defects in animals. While the effects of these heavy metals have been widely documented, a single exposure paradigm is typically used. Few studies have focused on evaluating combined exposure to these metals. However, in the environment, animals are confronted with a plethora of substances simultaneously; thus, the presence and origin of such substances must be determined to reduce the sources of contamination. Using the model of the fruit fly Drosophila melanogaster, for which many tools are readily available, we investigated how different concentrations of Cd and Hg in single and combined exposures impact post-embryonic development. In parallel, we evaluated the extended expression pattern of 38 molecular targets used as potential biomarkers of exposure through qPCR. Our results showed that both metals caused developmental delays and mortality in dose-dependent responses. Both metals were able to deregulate genes involved in hormonal control, general stress, and oxidative stress. Importantly, we confirmed synergistic interactions between Cd and Hg. Our results indicate the importance of assessing several biomarkers and their kinetics in mixtures. Drosophila represents a useful model for monitoring the toxicity of substances in polluted environments.

Characterization of the detrimental effects of type IV glandular trichomes on the aphid Macrosiphum euphorbiae in tomato.

BACKGROUND: Glandular trichomes are essential in plants' defence against pests however, the mechanisms of action are not completely understood. While there is considerable evidence of feeding and movement impairment by trichomes, the effect on other traits is less clear. We combined laboratory and greenhouse experiments with molecular analysis to understand how glandular trichomes affect the behavior, population growth, and the expression of biomarkers involved in detoxification, primary metabolism, and developmental pathways of the aphid Macrosiphum euphorbiae. We used two isogenic tomato lines that differ in the presence of type IV glandular trichomes and production of acylsucroses; i.e.,Solanum lycopersicum cv. 'Moneymaker' and an introgressed line from Solanum pimpinellifolium (with trichomes type IV). RESULTS: Type IV glandular trichomes affected host selection and aphid proliferation with aphids avoiding, and showing impaired multiplication on the genotype with trichomes. The exposure to type IV glandular trichomes resulted in the overexpression of detoxication markers (i.e., Hsp70, Hsp17, Hsp10); the repression of the energetic metabolism (GAPDH), and the activation of the ecdysone pathway; all these, underlying the key adaptations and metabolic trade-offs in aphids exposed to glandular trichomes. CONCLUSION: Our results demonstrate the detrimental effect of glandular trichomes (type IV) on the aphid and put forward their mode of action. Given the prevalence of glandular trichomes in wild and cultivated Solanaceae; and of the investigated molecular biomarkers in insects in general, our results provide relevant mechanisms to understand the effect of trichomes not only on herbivorous insects but also on other trophic levels.

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.

Developmental/reproductive effects and gene expression variations in Chironomus riparius after exposure to reclaimed water and its fortification with carbamazepine and triclosan.

The potential benefits of reclaimed water (RW) uses for environmental enhancement and restoration could become adverse impacts if RW does not meet the quality criteria that ensure wildlife preservation. RW can contain complex mixtures of micropollutants that may accumulate in sediment after environmental uses and affect benthic fauna. Therefore, we designed this study to assess the effects of RW on a sediment insect species used mainly in ecotoxicology (Chironomus riparius). Whole organism effects and gene expression were measured in a water sediment system after spiking RW as overlying water, which was renewed 3 times during the test. Development rate, emergence rate and fecundity were monitored after the 21-day exposure. Endocrine-related genes (EcR, ERR, E75, Vtg), cellular stress genes (hsp70, hsc70, hsp24, hsp10) and biotransformation genes (gp93, GSTd3, GPx, cyp4g) were assessed in larvae after the 10-day exposure. The experimental design also included single or binary fortifications of both test medium and RW, obtained by adding two emerging pollutants: carbamazepine (100 µg/L CBZ) and triclosan (20 µg/L TCS). The chemical characterisation of RW showed that 20 of the 23 screened emerging pollutants fell within the detection limit, 10 exceeded 0.01 µg/L (including CBZ) and three exceeded 0.1 µg/L (hydrochlorothiazide, atenolol, ibuprofen). The analytical measures of sediment (day 21) and overlying water (days 7, 14 and 21) were taken to know the water-sediment distribution of CBZ and TCS added to fortifications. CBZ distributed mainly in overlying water (110-164 µg/L and 73-100 µg/kg), while TCS showed a higher affinity to sediment (2.8-5.1 µg/L and 36-55 µg/kg). RW had significant effects in molecular terms (Vtg, hsp70, hsc70), but had no significant effects on the whole organism. Nevertheless, the single RW fortifications impaired both the development rate and fecundity, while the binary RW fortification impaired only fecundity. The most marked increase in EcR expression was observed for the binary RW fortification. Hsps, GSTd3 and cyp4g showed a similar tendency to that observed for EcR and Vtg in the binary and single RW fortifications. The binary mixture (CBZ and TCS together) in RW was toxic, but not in the medium tests. Therefore, the major concern of RW uses is apparently related to the interactivity between this complex matrix and any other pollutants possibly present in the environment where RW is applied. Our results underscore the need for raising awareness about RW effects, which can be achieved by ecotoxicological testing.

Exposure to heavy metal-contaminated sediments disrupts gene expression, lipid profile, and life history traits in the midge Chironomus riparius.

Despite the concern about anthropogenic heavy metal accumulation, there remain few multi-level ecotoxicological studies to evaluate their effects in fluvial ecosystems. The toxicity of field-collected sediments exhibiting a gradient of heavy metal contamination (Cd, Pb, and Zn) was assessed in Chironomus riparius. For this purpose, larvae were exposed throughout their entire life cycle to these sediments, and toxic effects were measured at different levels of biological organization, from the molecular (lipidomic analysis and transcriptional profile) to the whole organism response (respiration rate, shape markers, and emergence rate). Alterations in the activity of relevant genes, as well as an increase of storage lipids and decrease in membrane fluidity, were detected in larvae exposed to the most contaminated sediments. Moreover, reduced larval and adult mass, decrease of larval respiration rate, and delayed emergence were observed, along with increased mentum and mandible size in larvae and decreased wing loading in adults. This study points out the deleterious effects of heavy metal exposure at various levels of biological organization and provides some clues regarding the mode of toxic action. This integrative approach provides new insights into the multi-level effects on aquatic insects exposed to heavy metal mixtures in field sediments, providing useful tools for ecological risk assessment in freshwater ecosystems.

Complex patterns in tolerance and resistance to pests and diseases underpin the domestication of tomato.

A frequent hypothesis explaining the high susceptibility of many crops to pests and diseases is that, in the process of domestication, crops have lost defensive genes and traits against pests and diseases. Ecological theory predicts trade-offs whereby resistance and tolerance go at the cost of each other. We used wild relatives, early domesticated varieties, traditional local landraces and cultivars of tomato (Solanum lycopersicum) to test whether resistance and tolerance trade-offs were phylogenetically structured or varied according to degree of domestication. We exposed tomato genotypes to the aphid Macrosiphum euphorbiae, the cotton leafworm Spodoptera littoralis, the root knot nematode Meloidogyne incognita and two common insect-transmitted plant viruses, and reconstructed their phylogenetic relationships using Genotyping-by-Sequencing. We found differences in the performance and effect of pest and diseases but such differences were not related with domestication degree nor genetic relatedness, which probably underlie a complex genetic basis for resistance and indicate that resistance traits appeared at different stages and in unrelated genetic lineages. Still, wild and early domesticated accessions showed greater resistance to aphids and tolerance to caterpillars, nematodes and diseases than modern cultivars. Our findings help to understand how domestication affects plant-pest interactions and underline the importance of tolerance in crop breeding.

Chironomus riparius exposure to field-collected contaminated sediments: From subcellular effect to whole-organism response.

The toxicity of three field-collected sediments differentially contaminated with pesticides, heavy metals, phtalates and polycyclic aromatic hydrocarbons (PAHs), was assessed in Chironomus riparius. For this purpose, C. riparius larvae were exposed throughout their entire life cycle to sediments collected in three sites along the Saulx river in France, and the toxic effects were measured at different levels of biological organization: from the molecular (lipidomic analysis and transcriptional variations) to the whole organism response (respiration rate, shape markers and emergence rate). In the sediment characterized by an intermediate level of contamination with PAHs and phtalates, we detected an increase of the cell stress response and delayed emergence of males. In the group exposed to the most contaminated sediment with PAHs, phtalates and pesticides, genes related to endocrine pathways, cell stress response and biotransformation processes were overexpressed, while female wing shape was affected. Field-collected sediment exposure did not induce significant effects on mentum shape markers or on the lipid profile. The present study provides new insights into the multilevel effects of differentially contaminated sediments in insects. This integrative approach will certainly contribute to improved assessment of the risk that complex mixtures of pollutants pose to the aquatic ecosystem.

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.

Transcriptional deregulation of genetic biomarkers in Chironomus riparius larvae exposed to ecologically relevant concentrations of di(2-ethylhexyl) phthalate (DEHP).

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental pollutant used worldwide as a plasticizer and solvent in many formulations. Based on available toxicological data, it has been classified as toxic for reproduction and as an endocrine disruptor. Despite this, ecotoxicological studies in aquatic wildlife organisms are still scarce. In the present work, the toxic molecular alterations caused by DEHP in aquatic larvae of the midge Chironomus riparius have been studied, by analyzing the transcriptional activity of genes related to some vital cellular pathways, such as the ribosomal machinery (rpL4, rpL13), the cell stress response (hsc70, hsp70, hsp40, hsp27), the ecdysone hormone pathway (EcR), the energy metabolism (GAPDH), and detoxication processes (CYP4G). Environmentally relevant concentrations (10-3 to 105 µg/L) and exposure conditions (24 to 96 h) have been tested, as well as the toxic effects after DEHP withdrawal. Although the compound caused no mortality, significant changes were detected in almost all the studied biomarkers: e.g. strong repression of hsp70; general inhibition of EcR; GAPDH activity loss in long exposures; among others. Our data show a general transcriptional downregulation that could be associated with an adaptive response to cell damage. Besides, the activity of the compound as an ecdysone antagonist and its delayed effects over almost all the biomarkers analyzed are described as novel toxic targets in insects.

The ribosome biogenesis pathway as an early target of benzyl butyl phthalate (BBP) toxicity in Chironomus riparius larvae.

Butyl benzyl phthalate (BBP) is a ubiquitous contaminant whose presence in the environment is expected for decades, since it has been extensively used worldwide as a plasticizer in the polyvinyl chloride (PVC) industry and the manufacturing of many other products. In the present study, the interaction of BBP with the ribosome biogenesis pathway and the general transcriptional profile of Chironomus riparius aquatic larvae were investigated by means of changes in the rDNA activity (through the study of the internal transcribed spacer 2, ITS2) and variations in the expression profile of ribosomal protein genes (rpL4, rpL11, and rpL13) after acute 24-h and 48-h exposures to a wide range of BBP doses. Furthermore, cytogenetic assays were conducted to evaluate the transcriptional activity of polytene chromosomes from salivary gland cells, with special attention to the nucleolus and the Balbiani rings (BRs) of chromosome IV. BBP caused a dose and time-dependent toxicity in most of the selected biomarkers, with a general depletion in the gene expression levels and the activity of BR2 after 48-h treatments. At the same time, decondensation and activation of some centromeres took place, while the activity of nucleolus remained unaltered. Withdrawal of the xenobiotic allowed the larvae to reach control levels in the case of rpL4 and rpL13 genes, which were previously slightly downregulated in 24-h tests. These data provide the first evidence on the interaction of BBP with the ribosome synthesis pathways, which results in a significant impairment of the functional activity of ribosomal protein genes. Thus, the depletion of ribosomes would be a long-term effect of BBP-induced cellular damage. These findings may have important implications for understanding the adverse biological effects of BBP in C. riparius, since they provide new sensitive biomarkers of BBP exposure and highlight the suitability of this organism for ecotoxicological risk assessment, especially in aquatic ecosystems.

Ecdysone-Related Biomarkers of Toxicity in the Model Organism Chironomus riparius: Stage and Sex-Dependent Variations in Gene Expression Profiles.

Despite being considered a model organism in toxicity studies, particularly in assessing the environmental impact of endocrine disrupting compounds (EDCs) and other chemicals, the molecular basis of development is largely unknown in Chironomus riparius. We have characterized the expression patterns of important genes involved in the ecdysone pathway from embryos to pupa, but specially during the different phases of C. riparius fourth larval instar, according to the development of genital and thoracic imaginal discs. Real-Time PCR was used to analyze: EcR and usp, two genes encoding the two dimerizing partners of the functional ecdysone receptor; E74, an early response gene induced by ecdysteroids; vg (vitellogenin), an effector gene; hsp70 and hsc70, two heat-shock genes involved in the correct folding of the ecdysone receptor; and rpL13, as a part of the ribosomal machinery. Our results show for the first time stage and sex-dependent variations in ecdysone-responsive genes, specially during the late larval stage of C. riparius. The induction in the expression of EcR and usp during the VII-VIII phase of the fourth instar is concomitant with a coordinated response in the activity of the other genes analyzed, suggesting the moment where larvae prepare for pupation. This work is particularly relevant given that most of the analyzed genes have been proposed previously in this species as sensitive biomarkers for the toxicological evaluation of aquatic ecosystems. Identifying the natural regulation of these molecular endpoints throughout the Chironomus development will contribute to a more in-depth and accurate evaluation of the disrupting effects of EDCs in ecotoxicological studies.

The plasticizer benzyl butyl phthalate (BBP) alters the ecdysone hormone pathway, the cellular response to stress, the energy metabolism, and several detoxication mechanisms in Chironomus riparius larvae.

Butyl benzyl phthalate (BBP) has been extensively used worldwide as a plasticizer in the polyvinyl chloride (PVC) industry and the manufacturing of many other products, and its presence in the aquatic environment is expected for decades. In the present study, the toxicity of BBP was investigated in Chironomus riparius aquatic larvae. The effects of acute 24-h and 48-h exposures to a wide range of BBP doses were evaluated at the molecular level by analysing changes in genes related to the stress response, the endocrine system, the energy metabolism, and detoxication pathways, as well as in the enzyme activity of glutathione S-transferase. BBP caused a dose and time-dependent toxicity in most of the selected biomarkers. 24-h exposures to high doses affected larval survival and lead to a significant response of several heat-shock genes (hsp70, hsp40, and hsp27), and to a clear endocrine disrupting effect by upregulating the ecdysone receptor gene (EcR). Longer treatments with low doses triggered a general repression of transcription and GST activity. Furthermore, delayed toxicity studies were specially relevant, since they allowed us to detect unpredictable toxic effects, not immediately manifested after contact with the phthalate. This study provides novel and interesting results on the toxic effects of BBP in C. riparius and highlights the suitability of this organism for ecotoxicological risk assessment, especially in aquatic ecosystems.

Transcriptional responses, metabolic activity and mouthpart deformities in natural populations of Chironomus riparius larvae exposed to environmental pollutants.

Biomarkers are an important tool in laboratory assays that link exposure or effect of specific toxicants to key molecular and cellular events, but they have not been widely used in invertebrate populations exposed to complex mixtures of environmental contaminants in their natural habitats. The present study focused on a battery of biomarkers and their comparative analysis in natural populations of the benthic larvae of Chironomus riparius (Diptera), sampled in three differentially polluted rivers (the Con, Sar, and Louro in Galicia, Spain). In our study, some parameters were identified, such as hsp70 gene activity, GST enzymatic activity, total glycogen content and mouthpart deformities, which showed significant differences among populations from the three rivers that differed in the levels and types of sedimentary contaminants analyzed (metals, organic-chlorine pesticides, alkylphenols, pharmaceutical, and personal care products). In contrast to these sensitive biomarkers, other parameters showed no significant differences (hsc70 gene, EcR gene, P450 gene, RNA:DNA ratio, total protein content), and were stable even when comparing field and nonexposed laboratory populations. The hsp70 gene seems to be particularly sensitive to conditions of pollutant exposure, while its constitutive counterpart hsc70 showed invariable expression, suggesting that the hsc70/hsp70 ratio may be a potential indicator of polluted environments. Although further studies are required to understand the correlation between molecular responses and the ecological effects of pollutants on natural populations, the results provide new data about the biological responses to multiple-stressor environments. This field study adds new molecular endpoints, including gene expression, as suitable tools that, complementing other ecotoxicological parameters, may help to improve the methodologies of freshwater monitoring under the increasing burden of xenobiotics.