Marie-Agnès Coutellec

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.

Heterosis and inbreeding depression in bottlenecked populations: a test in the hermaphroditic freshwater snail Lymnaea stagnalis

Small population size is expected to induce heterosis, due to the random fixation and accumulation of mildly deleterious mutations, whereas within‐population inbreeding depression should decrease due to increased homozygosity. Population bottlenecks, although less effective, may have similar consequences. We tested this hypothesis in the self‐fertile freshwater snail Lymnaea stagnalis, by subjecting experimental populations to a single bottleneck of varied magnitude. Although patterns were not strong, heterosis was significant in the most severely bottlenecked populations, under stressful conditions. This was mainly due to hatching rate, suggesting that early acting and highly deleterious alleles were involved. Although L. stagnalis is a preferential outcrosser, inbreeding depression was very low and showed no clear relationship with bottleneck size. In the less reduced populations, inbreeding depression for hatching success increased under high inbreeding. This may be consistent with the occurence of synergistic epistasis between fitness loci, which may contribute to favour outcrossing in L. stagnalis.

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.

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.

Special issue on long-term ecotoxicological effects: an introduction

Two years after the Special Issue published in JournalEcotoxicology on Evolutionary Process in Ecotoxicology(vol 20 (3); see Coutellec and Barata 2011), the presentissue proposes a timely update on long-term biologicalimpacts of pollutants, including delayed, trans-genera-tional, and evolutionary effects. The number of articlescontributed in the present issue confirms that this questionis gaining ground in the field of ecotoxicology. Despite theawareness of the scientific community that evolutionarychange can occur rapidly (e.g., Carroll et al. 2007), and thataddressing the long term ecotoxicological impact is rele-vant to improve the significance of ecological risk assess-ment of chemicals (e.g., Bickham 2011), this research fieldhas been poorly explored until recent time. Most often,long term trans-generational or evolutionary responses topollution have been limited to experimental studies basedon model species appropriate to multi-generation exposure(primarily waterflea or other short-lived invertebrates) andto molecules with some knowledge or expectation on theirmode of action (e.g., metals, candidate genes involved intolerance and adaptation). While these remain undeniablysound models, current advances in molecular technologyallow tackling long term trans-generational or evolutionaryeffects under more realistic conditions, involving naturalecosystems, presence of multistressors in the field, andusing species of particular ecological relevance (includinglonger-lived species), or by tracing back evolutionaryprocesses in wild populations (see van Straalen and Feder2012).Long-term effects of pollutants: inferring causativerelationshipsOne recurrent problem in assessing the evolutionary impactof a given chemical is that cause-effect relationships arestill extremely difficult to determine in the field, as manyfactors can be responsible for an observed pattern of neu-tral or selectable genetic variation. Nevertheless, we simplycannot ignore such impact just because it is difficult toassess. The current endeavor of scientists towards fillingthis gap is further motivated by the fact that the environ-ment needs to be considered in its changing nature (i.e.,with regard to climate or biological invasions). The ever-increasing demand imposed by humans on natural resour-ces also requires that some trade-off can be reachedbetween human impact and conservation (see Bijlsma andLoeschcke 2012). Reliable tools are needed to enabledecision makers to apply conditions under which this trade-off could be optimized in order to ensure a sustainable useand management of ecosystems and resources. This isclearly a new challenge, and in this respect, long-term andevolutionary ecotoxicology represents a building block ofsuch a strategy, providing concepts and tools with bothindicative and predictive value, that could be implementedin environmental risk assessment (see also Breitholtz et al.2006; Bickham 2011).In the present issue, long-term effects of pollutants areeither documented under natural situations or tested usingexperimental designs, and some contributions combined

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.

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.