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.

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.

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.

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.

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.

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.

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.

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.

Pyrosequencing-based transcriptomic resources in the pond snail Lymnaea stagnalis, with a focus on genes involved in molecular response to diquat-induced stress.

Due to their ability to explore whole genome response to drugs and stressors, omics-based approaches are widely used in toxicology and ecotoxicology, and identified as powerful tools for future ecological risk assessment and environmental monitoring programs. Understanding the long-term effects of contaminants may indeed benefit from the coupling of genomics and eco-evolutionary hypotheses. Next-generation sequencing provides a new way to investigate pollutants impact, by targeting early responses, screening chemicals, and directly quantifying gene expression, even in organisms without reference genome. Lymnaea stagnalis is a freshwater mollusk in which access to genomic resources is critical for many scientific issues, especially in ecotoxicology. We used 454-pyrosequencing to obtain new transcriptomic resources in L. stagnalis and to preliminarily explore gene expression response to a redox-cycling pesticide, diquat. We obtained 151,967 and 128,945 high-quality reads from control and diquat-exposed individuals, respectively. Sequence assembly provided 141,999 contigs, of which 124,387 were singletons. BlastX search revealed significant match for 34.6 % of the contigs (21.2 % protein hits). KEGG annotation showed a predominance of hits with genes involved in energy metabolism and circulatory system, and revealed more than 400 putative genes involved in oxidative stress, cellular/molecular stress and signaling pathways, apoptosis, and metabolism of xenobiotics. Results also suggest that diquat may have a great diversity of molecular effects. Moreover, new genetic markers (putative SNPs) were discovered. We also created a Ensembl-like web-tool for data-mining ( http://genotoul-contigbrowser.toulouse.inra.fr:9095/Lymnaea_stagnalis/index.html ). This resource is expected to be relevant for any genomic approach aimed at understanding the molecular basis of physiological and evolutionary responses to environmental stress in L. stagnalis.

Parental exposure to pesticides and progeny reaction norm to a biotic stress gradient in the freshwater snail Lymnaea stagnalis.

Human-induced environmental stress may lead to rapid evolutionary processes, and can affect the ability of natural populations to respond to other environmental change or stress. We used quantitative genetics tools, pesticide exposure and a gradient of biotic stress to investigate these questions in the freshwater snail Lymnaea stagnalis. The study focused on the genetic component of variance for life-history traits within populations, and the ability of different lines to respond differently to stress. The effect of parental exposure to a xenobiotic stress on the reaction norm of the progeny to another stress was also estimated (parental non-genetic effect). First, under laboratory conditions, inter-family variance suggested significant heritability for most traits. Second, under outdoor exposure to various pesticides, variation among families was significant for individual growth. Clutch size and hatching rate of the clutches laid in the laboratory after exposure showed similar results, and moreover, family interacted significantly with pesticides. Third, under a gradient of biotic stress (food and competition), inter-family variation was again significant for growth, and a significant interaction with biotic stress was observed for juvenile growth and ultimate size. Family heterogeneity and family × environment interactions indicate the possibility of differential evolutionary responses among lines, through different reaction norms. Stressful conditions did not affect the estimated heritability, and for pesticides, no transgenerational effect was detected on progeny growth in response to the biotic stress. Focused on short-term evolutionary responses, the present study illustrates a possible way of incorporating evolutionary approaches into ecotoxicological risk assessment procedures, for example, by accounting for inter-family variation.

Population-level effects of spinosad and Bacillus thuringiensis israelensis in Daphnia pulex and Daphnia magna: comparison of laboratory and field microcosm exposure conditions.

Because exposure to toxicants not only results in mortality but also in multiple sublethal effects, the use of life-table data appears particularly suitable to assess global effects on exposed populations. The present study uses a life table response approach to assess population-level effects of two insecticides used against mosquito larvae, spinosad (8 µg/l) and Bacillus thuringiensis var. israelensis (Bti, 0.5 µl/l), on two non target species, Daphnia pulex and Daphnia magna (Crustacea: Cladocera), under laboratory versus field microcosms conditions. Population growth rates were inferred from life table data and Leslie matrices under a model with resource limitation (ceiling). These were further used to estimate population risks of extinction under each tested condition, using stochastic simulations. In laboratory conditions, analyses performed for each species confirmed the significant negative effect of spinosad on survival, mean time at death, and fecundity as compared to controls and Bti-treated groups; for both species, population growth rate λ was lower under exposure to spinosad. In field microcosms, 2 days after larvicide application, differences in population growth rates were observed between spinosad exposure conditions, and control and Bti exposure conditions. Simulations performed on spinosad-exposed organisms led to population extinction (minimum abundance = 0, extinction risk = 1), and this was extremely rapid (time to quasi-extinction = 4.1 one-week long steps, i.e. one month). Finally, D. magna was shown to be more sensitive than D. pulex to spinosad in the laboratory, and the effects were also detectable through field population demographic simulations.

Effects of the mixture of diquat and a nonylphenol polyethoxylate adjuvant on fecundity and progeny early performances of the pond snail Lymnaea stagnalis in laboratory bioassays and microcosms.

Effects of the bipyridylium herbicide diquat and tank-mix adjuvant Agral90 were investigated on various life history traits of the freshwater pulmonate snail Lymnaea stagnalis. Trait expression was measured in simple laboratory bioassays on small size groups of snails, and under more complex, indoor microcosm conditions, on larger groups of snails. Microcosms were provided with sediment, plants, and fish, thus allowing a more complex level of intra and inter-specific interactions to develop. Treatments were performed with substances alone or in mixture, at concentrations ranging from 4.4 to 222.2microgl(-1) for diquat, and from 10 to 500microgl(-1) for Agral 90, under a fixed ratio design. Adult growth was negatively affected by diquat and its mixture with Agral 90 both at the highest concentrations (222.2 and 500microgl(-1), respectively). Fecundity expressed differently in bioassays and microcosms, but no effect of the chemicals could be observed on this trait. Progeny development was impaired by 222.2microgl(-1) diquat and its mixture with 500microgl(-1) Agral 90, as reflected by longer development time and reduced hatching rate of clutches laid by the exposed animals, as compared to the controls. Hatching data suggested that diquat bioavailability was lower in microcosms than under bioassay conditions. Consistently, chemical analysis showed that diquat disappeared more rapidly from the water in microcosms than in bioassays. Moreover, the differential expression of several life history traits under bioassays and microcosms conditions was probably also influenced by the level of intraspecific interaction, which differed among the systems. When significant, the effect of diquat was attenuated by the presence of Agral 90, indicating antagonistic interaction between the two substances. Such a deviation from additivity was partly validated statistically.

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.

Influence of isolation on the recovery of pond mesocosms from the application of an insecticide. I. Study design and planktonic community responses.

The influence of relative isolation on the ecological recovery of freshwater outdoor mesocosm communities after an acute toxic stress was assessed in a 14-month-long study. A single concentration of deltamethrin was applied to 8 out of 16 outdoor 9-m3 mesocosms to create a rapid decrease of the abundance of arthropods. To discriminate between external and internal recovery mechanisms, four treated and four untreated (control) mesocosms were covered with 1-mm mesh screen lids. The dynamics of planktonic communities were monitored in the four types of ponds. The abundance of many phytoplankton taxa increased after deltamethrin addition, but the magnitude of most increases was relatively small, probably due to low nutrient availability and the survival of rotifers. The greatest impact on zooplankton was seen in Daphniidae and, to a lesser extent, calanoid copepods. Recovery (defined as when statistical analysis failed to detect a difference in the abundance between the deltamethrin-treated ponds and corresponding control ponds for two consecutive sampling dates) of Daphniidae was observed in the water column 105 and 77 d after deltamethrin addition in open and covered mesocosms, respectively, and <42 d for both open and covered ponds at the surface of the sediments. Rotifers did not proliferate, probably because of the survival of predators (e.g., cyclopoid copepods). These results confirm that the recovery of planktonic communities after exposure to a strong temporary chemical stress mostly depends upon internal mechanisms (except for larvae of the insect Chaoborus sp.) and that recovery dynamics are controlled by biotic factors, such as the presence of dormant forms and selective survival of predators.

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.