Neglected impacts of plant protection products on invertebrate aquatic biodiversity: a focus on eco-evolutionary processes.

The application of plant protection products (PPPs) may have delayed and long-term non-intentional impacts on aquatic invertebrates inhabiting agricultural landscapes. Such effects may induce population responses based on developmental and transgenerational plasticity, selection of genetic resistance, as well as increased extirpation risks associated with random genetic drift. While the current knowledge on such effects of PPPs is still scarce in non-target aquatic invertebrate species, evidences are accumulating that support the need for consideration of evolutionary components of the population response to PPPs in standard procedures of risk assessment. This mini-review, as part of a contribution to the collective scientific assessment on PPP impacts on biodiversity and ecosystem services performed in the period 2020-2022, presents a brief survey of the current results published on the subject, mainly in freshwater crustaceans, and proposes some research avenues and strategies that we feel relevant to fill this gap.

The use of copper as plant protection product contributes to environmental contamination and resulting impacts on terrestrial and aquatic biodiversity and ecosystem functions.

Copper-based plant protection products (PPPs) are widely used in both conventional and organic farming, and to a lesser extent for non-agricultural maintenance of gardens, greenspaces, and infrastructures. The use of copper PPPs adds to environmental contamination by this trace element. This paper aims to review the contribution of these PPPs to the contamination of soils and waters by copper in the context of France (which can be extrapolated to most of the European countries), and the resulting impacts on terrestrial and aquatic biodiversity, as well as on ecosystem functions. It was produced in the framework of a collective scientific assessment on the impacts of PPPs on biodiversity and ecosystem services in France. Current science shows that copper, which persists in soils, can partially transfer to adjacent aquatic environments (surface water and sediment) and ultimately to the marine environment. This widespread contamination impacts biodiversity and ecosystem functions, chiefly through its effects on phototrophic and heterotrophic microbial communities, and terrestrial and aquatic invertebrates. Its effects on other biological groups and biotic interactions remain relatively under-documented.

Impacts of neonicotinoids on biodiversity: a critical review.

Neonicotinoids are the most widely used class of insecticides in the world, but they have raised numerous concerns regarding their effects on biodiversity. Thus, the objective of this work was to do a critical review of the contamination of the environment (soil, water, air, biota) by neonicotinoids (acetamiprid, clothianidin, imidacloprid, thiacloprid, thiamethoxam) and of their impacts on terrestrial and aquatic biodiversity. Neonicotinoids are very frequently detected in soils and in freshwater, and they are also found in the air. They have only been recently monitored in coastal and marine environments, but some studies already reported the presence of imidacloprid and thiamethoxam in transitional or semi-enclosed ecosystems (lagoons, bays, and estuaries). The contamination of the environment leads to the exposure and to the contamination of non-target organisms and to negative effects on biodiversity. Direct impacts of neonicotinoids are mainly reported on terrestrial invertebrates (e.g., pollinators, natural enemies, earthworms) and vertebrates (e.g., birds) and on aquatic invertebrates (e.g., arthropods). Impacts on aquatic vertebrate populations and communities, as well as on microorganisms, are less documented. In addition to their toxicity to directly exposed organisms, neonicotinoid induce indirect effects via trophic cascades as demonstrated in several species (terrestrial and aquatic invertebrates). However, more data are needed to reach firmer conclusions and to get a clearer picture of such indirect effects. Finally, we identified specific knowledge gaps that need to be filled to better understand the effects of neonicotinoids on terrestrial, freshwater, and marine organisms, as well as on ecosystem services associated with these biotas.

Linking ecotoxicological effects on biodiversity and ecosystem functions to impairment of ecosystem services is a challenge: an illustration with the case of plant protection products.

There is growing interest in using the ecosystem services framework for environmental risk assessments of chemicals, including plant protection products (PPPs). Although this topic is increasingly discussed in the recent scientific literature, there is still a substantial gap between most ecotoxicological studies and a solid evaluation of potential ecotoxicological consequences on ecosystem services. This was recently highlighted by a collective scientific assessment (CSA) performed by 46 scientific experts who analyzed the international science on the impacts of PPPs on biodiversity, ecosystem functions, and ecosystem services. Here, we first point out the main obstacles to better linking knowledge on the ecotoxicological effects of PPPs on biodiversity and ecological processes with ecosystem functions and services. Then, we go on to propose and discuss possible pathways for related improvements. We describe the main processes governing the relationships between biodiversity, ecological processes, and ecosystem functions in response to effects of PPP, and we define categories of ecosystem functions that could be directly linked with the ecological processes used as functional endpoints in investigations on the ecotoxicology of PPPs. We then explore perceptions on the possible links between these categories of ecosystem functions and ecosystem services among a sub-panel of the scientific experts from various fields of environmental science. We find that these direct and indirect linkages still need clarification. This paper, which reflects the difficulties faced by the multidisciplinary group of researchers involved in the CSA, suggests that the current gap between most ecotoxicological studies and a solid potential evaluation of ecotoxicological consequences on ecosystem services could be partially addressed if concepts and definitions related to ecological processes, ecosystem functions, and ecosystem services were more widely accepted and shared within the ecotoxicology community. Narrowing this gap would help harmonize and extend the science that informs decision-making and policy-making, and ultimately help to better address the trade-off between social benefits and environmental losses caused by the use of PPPs.

Insights into the mechanisms of within-species variation in sensitivity to chemicals: A case study using daphnids exposed to CMIT/MIT biocide.

Living organisms adapt to their environment, and this adaptive response to environmental changes is influenced by both genomic and epigenomic components. As adaptation underpins tolerance to stressors, it is crucial to consider biological adaptation in evaluating the adverse outcomes of environmental chemicals, such as biocides. Daphnid studies have revealed differences in sensitivity to environmental chemicals between conspecific populations or clones, as well as between species. This study aimed to identify whether sensitivity to chemicals is subject to intraspecific variation, and whether this sensitivity depends on the genetic and epigenetic backgrounds of the daphnid population. We used an integrative approach to assess the comparative toxicity of a mixture of 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT), a commonly used isothiazolinone biocide, by measuring mortality, reproduction, physiological traits, global DNA methylation, and proteomic expression at the species and strain levels. The results showed that the variation in sensitivity to CMIT/MIT between conspecific strains (Daphnia pulex; DPR vs. DPA strains) could exceed that observed between congeneric species (D. magna vs. D. pulex DPR strain). Under the control conditions, DPR (the strain most sensitive to CMIT/MIT) was characterized by a larger body size, a higher heart rate, and a higher level of global DNA methylation compared to its counterpart (DPA), and proteome profiles differed between the two strains. Particularly, the study identified strain-specific epigenetic and proteomic responses to LC20 of CMIT/MIT, demonstrating putative critical proteins and biological pathways associated with the observed differences in phenotype and sensitivity to CMIT/MIT. Downregulation of certain proteins (e.g., SAM synthase, GSTs, hemoglobin, and cuticle proteins) and DNA hypomethylation can be proposed as key events (KEs) of adverse outcome pathway (AOP) for isothiazolinone toxicity. Our findings indicate that both genetic variations and epigenetic modifications can lead to intraspecific variation in sensitivity to chemicals, and this variation should be considered in the ecological risk assessment framework for chemical substances. We suggest conducting further analysis on methylated gene regions and observing transgenerational effects to verify the role of crosstalk between genetic and epigenetic factors in phenotypic and protein expressions. DATA AVAILABILITY: Proteomic data is available in supplementary materials.

Main conclusions and perspectives from the collective scientific assessment of the effects of plant protection products on biodiversity and ecosystem services along the land-sea continuum in France and French overseas territories.

Preservation of biodiversity and ecosystem services is critical for sustainable development and human well-being. However, an unprecedented erosion of biodiversity is observed and the use of plant protection products (PPP) has been identified as one of its main causes. In this context, at the request of the French Ministries responsible for the Environment, for Agriculture and for Research, a panel of 46 scientific experts ran a nearly 2-year-long (2020-2022) collective scientific assessment (CSA) of international scientific knowledge relating to the impacts of PPP on biodiversity and ecosystem services. The scope of this CSA covered the terrestrial, atmospheric, freshwater, and marine environments (with the exception of groundwater) in their continuity from the site of PPP application to the ocean, in France and French overseas territories, based on international knowledge produced on or transposable to this type of context (climate, PPP used, biodiversity present, etc.). Here, we provide a brief summary of the CSA's main conclusions, which were drawn from about 4500 international publications. Our analysis finds that PPP contaminate all environmental matrices, including biota, and cause direct and indirect ecotoxicological effects that unequivocally contribute to the decline of certain biological groups and alter certain ecosystem functions and services. Levers for action to limit PPP-driven pollution and effects on environmental compartments include local measures from plot to landscape scales and regulatory improvements. However, there are still significant gaps in knowledge regarding environmental contamination by PPPs and its effect on biodiversity and ecosystem functions and services. Perspectives and research needs are proposed to address these gaps.

Variation of Tolerance to Isothiazolinones Among Daphnia pulex Clones.

Isothiazolinones are a family of broad-spectrum biocides widely used in industry and consumer products. Chloro- and methyl-isothiazolinones (CMIT and MIT) are documented as strong irritants, yet they are still used in a wide variety of applications, including cosmetics, cleansers, hygienic products, and various industrial applications. The subsequent substantial release of these molecules from urban sources into freshwater environments, and their potential impacts on aquatic species, have nevertheless received little attention so far, with few studies reporting on the toxicity of either CMIT or MIT to nontarget organisms. The present study addresses this current knowledge gap by evaluating the acute toxicity to Daphnia pulex (Cladocera) of CMIT/MIT (3:1) and MIT, the two formulations most commonly used by manufacturers. In addition, genetic diversity is known to be a major component of variability in phenotypic responses, although it is largely overlooked in typical toxicity tests. Thus the potential range of responses inherent to genetic diversity is rarely considered. Therefore, to account for intraspecific variations in sensitivity, our design involved eight clonal lines of D. pulex stemming from distinct natural populations or commercial strains. Clones exhibited strong variation in their responses, with median lethal concentration (LC50) values ranging from 0.10 to 1.84 mg/L for the mixture CMIT/MIT, and from 0.68 to 2.84 mg/L for MIT alone. These intraspecific ranges of LC50 values challenge the use of single clones of daphnids in standard ecotoxicological tests and the predictions based on their results. The present study brings new evidence that assessing ecological risk of chemicals while ignoring genotype diversity is neither ecologically relevant, nor a representative evaluation of the diversity of potential adverse outcomes. Environ Toxicol Chem 2023;42:805-814. © 2023 SETAC.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis.

BACKGROUND: The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. RESULTS: In this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. CONCLUSIONS: Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Riverscape genetics in brook lamprey: genetic diversity is less influenced by river fragmentation than by gene flow with the anadromous ecotype.

Understanding the effect of human-induced landscape fragmentation on gene flow and evolutionary potential of wild populations has become a major concern. Here, we investigated the effect of riverscape fragmentation on patterns of genetic diversity in the freshwater resident European brook lamprey (Lampetra planeri) that has a low ability to pass obstacles to migration. We tested the hypotheses of (i) asymmetric gene flow following water current and (ii) an effect of gene flow with the closely related anadromous river lamprey (L. fluviatilis) ecotype on L. planeri genetic diversity. We genotyped 2472 individuals, including 225 L. fluviatilis, sampled from 81 sites upstream and downstream barriers to migration, in 29 western European rivers. Linear modelling revealed a strong positive relationship between genetic diversity and the distance from the river source, consistent with expected patterns of decreased gene flow into upstream populations. However, the presence of anthropogenic barriers had a moderate effect on spatial genetic structure. Accordingly, we found evidence for downstream-directed gene flow, supporting the hypothesis that barriers do not limit dispersal mediated by water flow. Downstream L. planeri populations in sympatry with L. fluviatilis displayed consistently higher genetic diversity. We conclude that genetic drift and slight downstream gene flow drive the genetic make-up of upstream L. planeri populations whereas gene flow between ecotypes maintains higher levels of genetic diversity in L. planeri populations sympatric with L. fluviatilis. We discuss the implications of these results for the design of conservation strategies of lamprey, and other freshwater organisms with several ecotypes, in fragmented dendritic river networks.

Divergence of seminal fluid gene expression and function among natural snail populations.

Seminal fluid proteins (SFPs) can trigger drastic changes in mating partners, mediating post-mating sexual selection and associated sexual conflict. Also, cross-species comparisons have demonstrated that SFPs evolve rapidly and hint that post-mating sexual selection drives their rapid evolution. In principle, this pattern should be detectable within species as rapid among-population divergence in SFP expression and function. However, given the multiple other factors that could vary among populations, isolating divergence in SFP-mediated effects is not straightforward. Here, we attempted to address this gap by combining the power of a common garden design with functional assays involving artificial injection of SFPs in the simultaneously hermaphroditic freshwater snail, Lymnaea stagnalis. We detected among-population divergence in SFP gene expression, suggesting that seminal fluid composition differs among four populations collected in Western Europe. Furthermore, by artificially injecting seminal fluid extracted from these field-derived snails into standardized mating partners, we also detected among-population divergence in the strength of post-mating effects induced by seminal fluid. Both egg production and subsequent sperm transfer of partners differed depending on the population origin of seminal fluid, with the response in egg production seemingly closely corresponding to among-population divergence in SFP gene expression. Our results thus lend strong intraspecific support to the notion that SFP expression and function evolve rapidly, and confirm L. stagnalis as an amenable system for studying processes driving SFP evolution.

Toward sustainable environmental quality: Priority research questions for Europe.

The United Nations' Sustainable Development Goals have been established to end poverty, protect the planet, and ensure prosperity for all. Delivery of the Sustainable Development Goals will require a healthy and productive environment. An understanding of the impacts of chemicals which can negatively impact environmental health is therefore essential to the delivery of the Sustainable Development Goals. However, current research on and regulation of chemicals in the environment tend to take a simplistic view and do not account for the complexity of the real world, which inhibits the way we manage chemicals. There is therefore an urgent need for a step change in the way we study and communicate the impacts and control of chemicals in the natural environment. To do this requires the major research questions to be identified so that resources are focused on questions that really matter. We present the findings of a horizon-scanning exercise to identify research priorities of the European environmental science community around chemicals in the environment. Using the key questions approach, we identified 22 questions of priority. These questions covered overarching questions about which chemicals we should be most concerned about and where, impacts of global megatrends, protection goals, and sustainability of chemicals; the development and parameterization of assessment and management frameworks; and mechanisms to maximize the impact of the research. The research questions identified provide a first-step in the path forward for the research, regulatory, and business communities to better assess and manage chemicals in the natural environment. Environ Toxicol Chem 2018;37:2281-2295. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Mollusc shells as metagenomic archives: The true treasure is the chest itself.

Mollusc shells, beyond the treasure of information inherently conveyed through their morphology and chemical composition also have the capacity to preserve DNA sequences over the long term in their inner structure. This has been clearly demonstrated for the first time in the study published in this issue of Molecular Ecology Resources by Der Sarkissian et al. (). With a methodology specifically dedicated to ancient DNA and solid matrices, the authors were able to successfully extract and amplify DNA from marine shells spanning the last 7,000 years. Furthermore, using metagenomic analyses, they could identify important factors affecting DNA recovery. Using reference genomes and sequences in a targeted approach to assign high-throughput sequencing reads, the authors revealed both the presence of endogenous mollusc DNA and a potent pathogen of Manilla clam. Collectively, the results presented in this study open extremely promising research avenues, from palaeogenomics and evolutionary biology to ecological genomics at population and community levels, as well as the opportunity to fine-tune diagnostic tools for conservation and aquaculture purposes. Last but not least, this study also offers exciting perspectives in epigenomics and the evolution of regulatory processes in the context of adaptation to global change. It can be easily expected that the approach developed by Der Sarkissian et al. () will be pursued and extensively investigated in the near future by the scientific community interested in these issues.

Validation of a two-generational reproduction test in Daphnia magna: An interlaboratory exercise.

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.

Genetic variation of Lymnaea stagnalis tolerance to copper: A test of selection hypotheses and its relevance for ecological risk assessment.

The use of standardized monospecific testing to assess the ecological risk of chemicals implicitly relies on the strong assumption that intraspecific variation in sensitivity is negligible or irrelevant in this context. In this study, we investigated genetic variation in copper sensitivity of the freshwater snail Lymnaea stagnalis, using lineages stemming from eight natural populations or strains found to be genetically differentiated at neutral markers. Copper-induced mortality varied widely among populations, as did the estimated daily death rate and time to 50% mortality (LT50). Population genetic divergence in copper sensitivity was compared to neutral differentiation using the QST-FST approach. No evidence for homogenizing selection could be detected. This result demonstrates that species-level extrapolations from single population studies are highly unreliable. The study provides a simple example of how evolutionary principles could be incorporated into ecotoxicity testing in order to refine ecological risk assessment.

Environmental versus anthropogenic effects on population adaptive divergence in the freshwater snail Lymnaea stagnalis.

Repeated pesticide contaminations of lentic freshwater systems located within agricultural landscapes may affect population evolution in non-target organisms, especially in species with a fully aquatic life cycle and low dispersal ability. The issue of evolutionary impact of pollutants is therefore conceptually important for ecotoxicologists. The impact of historical exposure to pesticides on genetic divergence was investigated in the freshwater gastropod Lymnaea stagnalis, using a set of 14 populations from contrasted environments in terms of pesticide and other anthropogenic pressures. The hypothesis of population adaptive divergence was tested on 11 life-history traits, using Q(ST)-F(ST) comparisons. Despite strong neutral differentiation (mean F(ST) = 0.291), five adult traits or parameters were found to be under divergent selection. Conversely, two early expressed traits showed a pattern consistent with uniform selection or trait canalization, and four adult traits appeared to evolve neutrally. Divergent selection patterns were mostly consistent with a habitat effect, opposing pond to ditch and channel populations. Comparatively, pesticide and other human pressures had little correspondence with evolutionary patterns, despite hatching rate impairment associated with global anthropogenic pressure. Globally, analyses revealed high genetic variation both at neutral markers and fitness-related traits in a species used as model in ecotoxicology, providing empirical support for the need to account for genetic and evolutionary components of population response in ecological risk assessment.

Population genetics of Lymnaea stagnalis experimentally exposed to cocktails of pesticides.

Freshwater invertebrates may be regularly exposed to pesticides in agricultural landscapes, as water bodies such as ditches and ponds are the final recipient of many chemicals, through various transfer pathways. Local evolutionary impacts may hence be expected on populations, especially for species with a completely aquatic life cycle. We tested the hypothesis that exposure to combinations of pesticides used in crop protection programmes could increase the effect of random genetic drift in a non-target species, via demographic impacts. To do so, experimental populations of the freshwater snail Lymnaea stagnalis were created from a common genetic pool and exposed for three successive generations to treatments corresponding to two different crop protection plans (conventional and low pesticide input). Population genetic parameters were estimated in each generation on the basis of ten polymorphic microsatellite loci. Effects consistent with increased random genetic drift were observed for one sampling campaign performed in the third generation, i.e., decreased genetic variability and increased population differentiation in the group of populations exposed to the treatment programme whose demographic impact was the most effective on L. stagnalis. Otherwise, no clear pattern emerged and even opposed effects could be observed. All populations were found significantly inbred, mostly due to biparental inbreeding. Conversely, selfing was generally not significant, and did not express preferentially under high pesticide pressure. We conclude from this study that population genetics should be used very cautiously in the context of ecological risk assessment, especially when applied to natural populations.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis.

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 µg l(-1)) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased significantly (or non-significantly for cat) after 5 h of exposure, and went back to control levels afterwards, suggesting the onset of an early response to oxidative stress associated to the unbalance of reactive oxygen species (ROS) in hepatocytes. Although increases obtained for Gred and SOD activities were globally consistent with their respective transcript expressions, up-regulation of transcription was not always correlated with increase of enzymatic activity, indicating that diquat might affect steps downstream of transcription. However, constitutive levels of enzymatic activities were at least maintained. In conclusion, diquat was shown to affect expression of the whole set of studied transcripts, reflecting their suitability as markers of early response to oxidative stress in L. stagnalis.