Sub-individual Level Research Articles

Responses of freshwater organisms to multiple stressors in a climate change scenario: a review on small-scale experiments.

This review summarizes how salinity and temperature, two key global factors driven by climate change in freshwater systems, interact with other stressors on organisms in controlled small-scale factorial experiments at the population, individual, or subindividual level (excluding mesocosm and field studies). Despite the growing interest, research following all these criteria remains limited with 156 publications of which 50% analyzed stressors + salinity, 46% stressors + temperature, and only 4% involved the triple combination. Research on the combined effect of temperature and salinity predominantly focused on metals, pesticides, and, to a lesser extent, emergent contaminants, such as microplastics and nanomaterials, encompassing various biological models and responses. In general, increased temperature amplifies the single effect of stressors, whereas salinity leads to a higher diversity of responses, with similar proportions of synergisms and antagonisms. Fish (Salmoniformes, Perciformes, and Cypriniformes) were the most studied organisms. Among Crustacea, only cladocerans of the genera Daphnia and Ceriodpahnia were considered. The present review highlights the need to include other species that play key roles in freshwater food webs and to increase triple combination studies to understand complex interactions and develop adaptation and mitigation strategies to preserve the environment and its services in this changing world.

Multi-level toxicity assessment of polylactic acid (PLA) microplastics on the cladoceran Daphnia magna

The accumulation of plastics waste in the environment has raised a worrisome concern, moving the society to seek out for sustainable solutions, such as the transition from the use of fossil-based, conventional plastics to bioplastics (BPs). However, once in the environment bioplastics have the same probability to accumulate and experience weathering processes than conventional plastics, leading to the formation of microplastics (MPs). However, to date the information on the potential toxicity of MPs originated from the weathering of bioplastics is limited. Thus, this study aimed at investigating the adverse effects induced by the exposure to MPs made of a bioplastic polymer, the polylactic acid (PLA), towards the freshwater cladoceran Daphnia magna. Organisms were exposed for 21 days to three concentrations (0.125 µg/mL, 1.25 µg/mL and 12.5 µg/mL) of PLA microplastics (hereafter PLA-MPs). A multi-level approach was performed to investigate the potential effects through the biological hierarchy, starting from the sub-individual up to the individual level. At the sub-individual level, changes in the oxidative status (i.e., the amount of reactive oxygen species and the activity of antioxidant and detoxifying enzymes) and oxidative damage (i.e., lipid peroxidation) were explored. Moreover, the total caloric content as well as the content of protein, carbohydrate and lipid content assess were used to investigate the effects on energy reserves. At individual level the changes in swimming activity (i.e., distance moved and swimming speed) were assessed. Our results showed that the exposure to PLA-MPs induced a slight modulation in the oxidative status and energy reserves, leading to an increase in swimming behavior of treated individuals compared to control conspecifics. These results suggest that the exposure to MPs made of a bioplastic polymer can induce adverse effects similar to those caused by conventional polymers.

Are tolerance processes limiting the responses of Hediste diversicolor to cadmium exposure? A multimarker approach

Cadmium (Cd) is considered a priority hazardous substance under the European Community Directive 2013/39 due to its ecotoxicity. The ragworm Hediste diversicolor (O.F. Müller, 1776), a common species in estuaries and coastal lagoons, plays an important ecological role in these ecosystems and is a suitable bioindicator of environmental chemical contamination. In this study, H. diversicolor was chosen as an ecotoxicological model with the aim of evaluating the responses to Cd contamination, considering a multi-biomarker approach (mortality, biometry, behaviour, Cd bioaccumulation, oxidative stress and damage, and energy metabolism). Also, the hypothesis of different tolerances resulting in different responses was evaluated, by collecting worms from three systems distinctly impacted by metal contamination (Mondego estuary, Óbidos Lagoon and Sado estuary - Portugal). Animals were exposed under laboratory conditions to cadmium (10, 50 and 100µg/L), for 10 days. Significant differences were observed in responses amongst worms originating from the different sites. Organisms from the less impacted systems revealed greater effects on mortality, biomass decrease and burrowing behaviour, as well as higher bioaccumulation potential, after exposure to Cd. Biochemical and behaviour impairments were observed as a consequence of Cd exposure, although not in a concentration-dependant manner. The results obtained in this study reinforce the importance of integrating endpoint responses, at the individual and sub-individual levels, to assess potential changes induced by pollutants in the physiological status and fitness of H. diversicolor and help to predict what their ecological consequences might be.

Effects of aqueous extracts of wildfire ashes on tadpoles of Pelophylax perezi: Influence of plant coverage

Wildfires have been pointed out as an important source of diffuse contamination to aquatic ecosystems, namely through the input of toxic compounds such as polycyclic aromatic hydrocarbons and metals. However, amphibians' responses to this disturbance have been largely ignored. Hence, this study intended to assess how ashes from Pinus sp. and Eucalyptus sp. plantation forests affect tadpoles of Pelophylax perezi. Tadpoles were exposed 14 days to serial concentrations (26.9 %-100 %) of aqueous extracts of ashes (AEA, with 10 g L-1 of ashes) containing Eucalypt (ELS) and Pine (PLS) ashes. The following endpoints were measured: mortality, malformations, developmental stage, body length and weight. Effects at sub-individual level were also monitored for oxidative stress, neurotoxicity, and energetic metabolism. Chemical characterization of the AEA of ELS showed higher concentrations of As, Cd, Co, Cr, Pb and V, while PLS showed higher concentrations of Cu, Mn, Ni and Zn. Concerning the lethal effects of AEAs on tadpoles, both extracts were able to induce mortality at high concentrations (76.9 and/or 100 % of AEA), although a high variability in the response was found. A significant mortality in tadpoles exposed to ELS was observed at the concentration of 76.9 %. For organisms exposed to PLS, though a mortality above 20 % was registered at the two highest tested concentrations, it was not significantly different from the control. No significant sub-lethal effects were observed in the ELS treatments. Contrasting, exposure to PLS induced a decrease in body length, weight, glutathione-S-transferase activity and an increase in oxygen consumption. Overall, the distinct effects of ELS and PLS suggest an influence of vegetation cover in ash toxicity. In conclusion, exposure to both ash extracts negatively affected sublethal responses of tadpoles of P. perezi. Future research is needed to assess how these effects at individual level may translate into effects at population level.

Differential biochemical and behavioral responses induced by cocaine and benzoylecgonine exposure to the red swamp crayfish Procambarus clarkii

Cocaine (COC) and its main metabolite, the benzoylecgonine (BE), are the main illicit drugs measured in aquatic system worldwide, with concentrations up to hundreds of ng/L. Although their current environmental concentrations are low these molecules can induce adverse effects at sub-individual level in non-target organisms. In contrast, the information at individual and behavioral level are still scant. The present study aimed at investigating biochemical and behavioral effects induced by 14-days exposure to environmentally relevant concentrations (50 ng/L and 500 ng/L) of COC and BE towards Procambarus clarkii. At sub-individual level, the activity of antioxidant and detoxifying (superoxide dismutase - SOD, catalase - CAT, glutathione peroxidase - GPx and glutathione S-transferases - GST) enzymes, as well as the levels of lipid peroxidation (LPO), were measured as oxidative stress-related endpoints. We also measured the acetylcholinesterase (AChE) activity to check for neurotoxic effect of COC and BE. At individual level, the modulation of some behavioral tasks (i.e., response to external stimuli, changes in feeding activity and exploration of a new environment) were assessed. Although both COC and BE exposure did not induce an oxidative stress situation, a significant inhibition of AChE activity was noted, resulting in behavioral changes in crayfish exposed to COC only. Crayfish exposed to the higher COC concentration showed an increase in the boldness and a decrease in the feeding activity, suggesting that COC may act according to its psychotropic mode of action.

Integration of Genotoxic Biomarkers in Environmental Biomonitoring Analysis Using a Multi-Biomarker Approach in Three-Spined Stickleback (Gasterosteus aculeatus Linnaeus, 1758).

Water is impacted by a variety of increasing pressures, such as contaminants, including genotoxic pollutants. The proposed multi-biomarker approach at a sub-individual level gives a complementary indicator to the chemical and ecological parameters of the Water Framework Directive (WFD, 2000/60/EC). By integrating biomarkers of genotoxicity and erythrocyte necrosis in the sentinel fish species the three-spined stickleback (Gasterosteus aculeatus) through active biomonitoring of six stations of the Artois-Picardie watershed, north France, our work aimed to improve the already existing biomarker approach. Even if fish in all stations had high levels of DNA strand breaks, the multivariate analysis (PCA), followed by hierarchical agglomerative clustering (HAC), improved discrimination among stations by detecting an increase of nuclear DNA content variation (Etaing, St Rémy du Nord, Artres and Biache-St-Vaast) and erythrocyte necrosis (Etaing, St Rémy du Nord). The present work highlighted that the integration of these biomarkers of genotoxicity in a multi-biomarker approach is appropriate to expand physiological parameters which allow the targeting of new potential effects of contaminants.

THE ROLE OF CONCEPTUAL METAPHOR IN CONCEPT PROFILING

The article systematizes the main methodological aspects of the study of conceptual metaphor. Attention is focused on solving the following tasks: to reveal the essence of ‘conceptual metaphor’, to determine the types of conceptual metaphor, to systematize the methodological aspects of the study of conceptual metaphor. It is argued that metaphor is a cognitive tool that allows to present an abstract concept difficult for comprehensionthrough another more empirically studied concept. The cognitive-linguistic view of metaphor is considered, according to which metaphor works atthree levels: supra-individual, individual and sub-individual. The supra-individual level corresponds to how a given language and culture reflect decontextualized metaphorical patterns,itcorrelates with the level of domains and frames. The individual level corresponds to the metaphorical cognitive system used by individual speakers, correlates with the use of mental processes in mental space, when universal aspects of the meaning of metaphorical expression in a particular communicative situation contains the most specific information acquiredwith experience. The sub-individual level corresponds to the universal aspects of various kinds of embodiment, corresponds to the level of image-schemes, generalizing models of which help to explain the meaning of abstract concepts. Conceptual metaphors connect abstract concepts with physical ones (based on perception), as a result of which the conceptual system becomes embodied, thoroughly based on perception. It is concluded that concepts consist of metaphorical structures that we have in our long-term memory and that provide a conceptual substrate of meaning in general and meaning ofa particular language in particular. Image-schemes, domains and frames, taken together, constitute the content of concepts, which gets an emotional color in the mental space of each individual speaker

Within-Species Trait Variation Can Lead to Size Limitations in Seed Dispersal of Small-Fruited Plants

The inability of small-gaped animals to consume very large fruits may limit seed dispersal of the respective plants. This has often been shown for large-fruited plant species that remain poorly dispersed when large-gaped animal species are lost due to anthropogenic pressure. Little is known about whether gape-size limitations similarly influence seed dispersal of small-fruited plant species that can show a large variation in fruit size within species. In this study, fruit sizes of 15 plant species were compared with the gape sizes of their 41 animal dispersers in the temperate, old-growth Białowieża Forest, Poland. The effect of gape-size limitations on fruit consumption was assessed at the plant species level, and for a subset of nine plant species, also at the individual level, and subindividual level (i.e., fruits of the same plant individual). In addition, for the species subset, fruit-seed trait relationships were investigated to determine whether a restricted access of small-gaped animals to large fruits results in the dispersal of fewer or smaller seeds per fruit. Fruit sizes widely varied among plant species (74.2%), considerably at the subindividual level (17.1%), and to the smallest extent among plant individuals (8.7%). Key disperser species should be able to consume fruits of all plant species and all individuals (except those of the largest-fruited plant species), even if they are able to consume only 28-55% of available fruits. Fruit and seed traits were positively correlated in eight out of nine plant species, indicating that gape size limitations will result in 49% fewer (in one) or 16–21% smaller seeds (in three plant species) dispersed per fruit by small-gaped than by large-gaped main dispersers, respectively. Our results show that a large subindividual variation in fruit size is characteristic for small-fruited plant species, and increases their connectedness with frugivores at the level of plants species and individuals. Simultaneously, however, the large variation in fruit size leads to gape-size limitations that may induce selective pressures on fruit size if large-gaped dispersers become extinct. This study emphasizes the mechanisms by which gape-size limitation at the species, individual and subindividual level shape plant-frugivore interactions and the co-evolution of small-fruited plants.

N-acetyl-β-d-glucosaminidase activity in Palaemon serratus - Methodological optimisation and intrinsic variability

Chitinolytic enzymes fulfil a key role in the moulting process of crustaceans, in degrading the endocuticle during apolysis. Measuring the enzyme activity is an interesting manner to monitor the moult process at sub-individual level, complementary to the classical observation of the integument morphogenesis, ecdysis success, or moult cycle duration. The present study aimed to optimise the methodology of using N-acetyl-β-D-glucosaminidase (NAGase) activity to monitor moulting in the marine prawn Palaemon serratus, and to compare NAGase activity levels along the moult cycle of both male and female specimens. First, to optimise protocols for five different organs, different reaction medium compositions were tested, considering the type buffer, concentration of the substrate, and the load in enzymatic extract. Second, levels of NAGase activity were closely monitored during eight moulting stages in male prawns. Variations in NAGase activity were observed during the moult cycle, with an increase in activity in the late premoult phase of approximately 2.4-fold the level of the intermoult phase. This response profile was observed for each tested organ. The levels of NAGase activity of male and female specimens were compared during three stages of the premoult phase. The patterns observed for both sexes were similar for all the tested organs.

Effects of triclosan on early development of Solea senegalensis: from biochemical to individual level

Harmful effects of triclosan (TCS) have been reported on several organisms; however, effects on early life stages of marine vertebrates are limited. Therefore, the objective of this work was to assess the effects of TCS during early development of the flatfish Solea senegalensis after initial characterization of cholinesterases (ChEs) and determination of selected biochemical markers baseline levels. Characterization of ChEs and determination of biochemical markers baseline levels of cholinergic activity, energy metabolism and oxidative stress were analysed in sole at 3 days after hatching (dah) and at the onset and end of metamorphosis. To assess TCS effects, fish were exposed during 96h to 30-500 μg L-1 TCS until 3 dah. Fish at 13 dah were exposed during 48h to 200-1,500 μg L-1 TCS and maintained until complete metamorphosis. Effects on survival, malformations, length, metamorphosis progression and biochemical markers were evaluated. The main ChE active form present in sole early life stages is acetylcholinesterase and baseline levels of oxidative stress and energy metabolism biomarkers changed according to fish developmental stage. Triclosan induced malformations (EC50 = 180 μg L-1 at 3 dah), decreased growth (95 μg L-1 at 3 dah; 548 μg L-1 at 24 dah) and affected metamorphosis progression (391 μg L-1 at 17 dah). Impairment of antioxidant system was observed, with TCS affecting catalase at the end of metamorphosis test, however, no oxidative damage on lipids was detected. Glutathione S-transferase was the most sensitive endpoint during early larval test (LOEC = 30 μg L-1). Exposure to TCS affected S.senegalensis at individual and sub-individual levels, both at early larval stage and during the critical period of metamorphosis.

Is there a link between acetylcholinesterase, behaviour and density populations of the ragworm Hediste diversicolor?

The main objective of the present study was to explore the potential link between acetylcholinesterase (AChE) activity and burrowing behaviour of the ragworm Hediste diversicolor, which may have consequences at higher levels of biological organisation. Two complementary studies were conducted. AChE activity, at the sub-individual level, and behavioural responses, at the individual level, were evaluated in worms from the Loire estuary (France), whereas density and biomass of H. diversicolor were determined at the population level. A Spearman positive correlation between both biomarkers (AChE and burrowing) suggested that inhibition of AChE activity was linked to behaviour impairments. At the population level, lower AChE and behaviour activities were detected in worms corresponding to lower population density and biomass. These results provide direct empirical field evidence demonstrating the sensitivity of behaviour of H. diversicolor as a biomonitor of estuarine health status assessment.

Patrones de herbivoría en Vassobia breviflora (Solanaceae): variación en el daño foliar y selección natural mediada por herbívoros

Herbivore mediated-selection shapes the evolution of defensive plant traits. Knowledge about the role of herbivores as mediators of selection is scarce and even more if herbivore functional groups are considered. The objectives of this work were (1) to describe the variation in foliar traits between populations and both between and intra-plants within a population, (2) to explore the relationship between the variation in the herbivory level and foliar traits, (3) to determine the relationship between leaf traits and damage patterns and (4) estimate the selection regimes by different herbivore functional groups. We conducted this study in four populations of Vassobia breviflora in northwestern Argentina (Yungas). The foliar traits considered were size, leaf area (af), shape (leaf length / width ratio; laf) and proportion of leaf area removed (pafr) (N = 1582 leaves, 57 plants). The herbivores consumed 15.6 % of the leaf area and 76.8 % of the variation in the pafr occurred at the sub-individual level. The damage pattern was dominated by cutter herbivores (70 %), followed by a dotted herbivory pattern (22 %), mixed 5 % and 1 % miner. Nonlinear selection was detected for laf (γii = -0.180; EE = 0.76; P < 0.05), and correlational selection between the cutter damage and af (γij = -1.297; SE = 0.62; P < 0.05) and between the dotted damage and af (γij = -1.130; SE = 0.76; P < 0.05). Natural selection favored plants with small leaves and high foliar removal and large leaves with less damage and selection against larger leaves with greater damage was detected. In addition, deduced from the relationship between the damage type and the relative fitness, the selection would favor the dotted damage over the cutter one. The plants would resolve the conflict with the herbivores according to the damage type and natural selection would regulate the foliar display as a strategy to deal with the herbivore diversity.

Linking sub-individual and supra-individual effects in Daphnia magna exposed to sub-lethal concentration of chlorpyrifos.

The main objective of the present study was to investigate possible links between sub-individual and supra-individual levels (i.e. population level) biomarkers in D.magna exposed to sublethal concentrations of the insecticide chlorpyrifos (CPF). To achieve the aim, 8-day old individuals were exposed for 96 h to two environmentally relevant concentrations of CPF (50 and 250 ng/L). Sub-individual level effects were investigated by measuring the activity of antioxidant (SOD, CAT, and GPx) and detoxifying (GST) enzymes, as well as by measuring the acetylcholinesterase (AChE) inhibition. In addition, the effects at supra-individual level were assessed by using a video-tracking system and analyzing changes in swimming capabilities (i.e. percentage of activity time, distance moved, and velocity). Our data have shown that daphnids exposed to both CPF concentrations were in a condition of stress which was highlighted by changes in both sub- and supra-individual biomarkers. Moreover, our results highlighted that the lowest tested CPF concentration did not modulate the antioxidant and detoxifying enzymes, whereas, an inhibition of AChE and a decrease of some parameters related to swimming behaviour (distance moved and velocity) were noted. On the contrary, significant changes in all the sub-individual biomarkers were measured at the highest tested concentration. In addition, organisms recovered the movement capability (distance moved) and also activate a mechanism of avoidance (increased swimming velocity). On the other hand, a reduction in the percent of active time was measured and this was attributed to the energy spent by organisms to activate antioxidant and detoxifying enzymes and the mechanism of avoidance. Based on these results, our study suggests the existence of a link between sub- and supra-individual levels, as the activation or non-activation in the antioxidant and detoxifying enzymes activities can led to different modifications of the swimming behaviour in D.magna.

Phytotoxicity of silver nanoparticles to Lemna minor: Surface coating and exposure period-related effects

Silver nanoparticles (Ag NPs) exponential production raises concern about their environmental impact. The effects of Ag NPs to aquatic plants remain scarcely studied, especially in extended exposures. This paper aims to evaluate Ag NPs effects in Lemna minor at individual and sub-individual levels, focusing on three variables: Ag form (NPs versus ions - Ag+), NPs surface coating (citrate vs polyvinylpyrrolidone - PVP) and exposure period (7 vs 14days). Endpoints were assessed at individual level (specific growth rate, chlorosis incidence and number of fronds per colony) and sub-individual level (enzymatic activities of catalase (CAT), guaiacol peroxidase (GPx) and glutathione-S-transferase (GST)). Generally, plants exposed to all Ag forms underwent decays on growth rate and fronds per colony, and increases on chlorosis, GPX and GST, but no effects on CAT. The most sensitive endpoints were specific growth rate and GPx activity, showing significant effects down to 0.05mg/L for Ag NPs and 3μg/L for Ag+, after 14days. Ag+ showed higher toxicity with a 14d-EC50 of 0.0037mg Ag/L. Concerning surface coating, PVP-Ag NPs were more deleterious on growth rate and fronds per colony, whereas citrate-Ag NPs affected more the chlorosis incidence and GPx and GST activities. The exposure period significantly affected chlorosis: 14days triggered a chlorosis increase in Ag+-exposed plants and a decrease in Ag NPs-exposed plants when compared to 7days. Ag NPs induced an oxidative stress status in cells, thus ensuing upregulated enzymatic activity as a self-defense mechanism. Since Ag NPs dissolution might occur on a steady and continuous mode along time, and the average longevity of fronds, we propose longer exposures periods than the recommended by the OECD guideline. This approach would provide more relevant and holistic evidences on the overall response of freshwater plants to Ag NPs in an ecological relevant scenario.

Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints

The guidelines for the Environmental Risk Assessment (ERA) of pharmaceuticals and personal care products (PPCP) recommend the use of standard ecotoxicity assays and the assessment of endpoints at the individual level to evaluate potential effects of PPCP on biota. However, effects at the sub-individual level can also affect the ecological fitness of marine organisms chronically exposed to PPCP. The aim of the current study was to evaluate the environmental risk of two PPCP in marine sediments: triclosan (TCS) and ibuprofen (IBU), using sub-individual and developmental endpoints. The environmental levels of TCS and IBU were quantified in marine sediments from the vicinities of the Santos submarine sewage outfall (Santos Bay, São Paulo, Brazil) at 15.14 and 49.0ngg-1, respectively. A battery (n=3) of chronic bioassays (embryo-larval development) with a sea urchin (Lytechinus variegatus) and a bivalve (Perna perna) were performed using two exposure conditions: sediment-water interface and elutriates. Moreover, physiological stress through the Neutral Red Retention Time Assay (NRRT) was assessed in the estuarine bivalve Mytella charruana exposed to TCS and IBU spiked sediments. These compounds affected the development of L.variegatus and P.perna (75ngg-1 for TCS and 15ngg-1 for IBU), and caused a significant decrease in M.charruana lysosomal membrane stability at environmentally relevant concentrations (0.08ngg-1 for TCS and 0.15ngg-1 for IBU). Chemical and ecotoxicological data were integrated and the risk quotient estimated for TCS and IBU were higher than 1.0, indicating a high environmental risk of these compounds in sediments. These are the first data of sediment risk assessment of pharmaceuticals and personal care products of Latin America. In addition, the results suggest that the ERA based only on individual-level and standard toxicity tests may overlook other biological effects that can affect the health of marine organisms exposed to PPCP.

Effects of Organic Pesticides on Enchytraeids (Oligochaeta) in Agroecosystems: Laboratory and Higher-Tier Tests

Enchytraeidae (Oligochaeta, Annelida) are often considered to be typical forestliving organisms, but they are regularly found in agroecosystems of the temperate regions of the world. Although less known than their larger relatives, the earthworms, these saprophagous organisms play similar roles in agricultural soils (but at a smaller scale), e.g., influencing soil structure and organic matter dynamics via microbial communities, and having a central place in soil food webs. Their diversity is rarely studied or often underestimated due to difficulties in distinguishing the species. New genetic techniques reveal that even in anthropogenically highly influenced soils, more than ten species per site can be found. Because of their close contact with the soil pore water, a high ingestion rate and a thin cuticle, they often react very sensitively to a broad range of pesticides. Firstly we provide a short overview of the diversity and abundance of enchytraeid communities in agroecosystems. Afterwards, we explore the available data on enchytraeid sensitivity towards pesticides at different levels of biological organization, focusing on pesticides used in (mainly) European agroecosystems. Starting with non-standardized studies on the effects of pesticides on the sub-individual level, we compile the results of standard laboratory tests performed following OECD and ISO guidelines as well as those of higher-tier studies (i.e., semi-field and field tests). The number of comparable test data is still limited, because tests with enchytraeids are not a regulatory requirement in the European Union. While focusing on the effects of pesticides, attention is also given to their interactions with environmental stressors (e.g., climate change). In conclusion, we recommend to increase the use of enchytraeids in pesticide risk assessment because of their diversity and functional importance as well as their increasingly simplified use in (mostly standardized) tests at all levels of biological organization.

Detecting copper toxicity in sediments: from the subindividual level to the population level

Summary Sediments accumulate chemicals that can be toxic to biota and often contribute to aquatic ecosystem decline. Measuring mortality in laboratory‐bred organisms is a common way to assess sediment toxicity. However, mortality‐based responses of resilient laboratory organisms may not reflect indigenous macroinvertebrate responses, which can be relatively more sensitive to sediment toxicants. A possible solution is to also measure responses at the subindividual level. Several organism responses to sediment copper toxicity were assessed in a field‐based microcosm. Responses of laboratory‐bred chironomids and snails deployed in microcosms were compared at subindividual (metabolomic and gene expression), individual (survival and dry weight) and population (reproduction) levels, and contrasted to the abundance of colonizing macroinvertebrates in the microcosms. Colonizing macroinvertebrate abundance showed a range of sensitivities based on EC50 (effect dose 50% change). Chironomidae made up 94·5% of the microcosm macroinvertebrates, with Paratanytarsus the most sensitive genus (EC50: 89 mg kg−1 copper) and Procladius the least sensitive (EC50: 681 mg kg−1). Survival of laboratory‐bred organisms was the least sensitive response, comparable to decreased abundance of the least sensitive macroinvertebrate. Juvenile production in the snail, Potamopyrgus antipodarum, was the most sensitive population‐level response (EC50: 121 mg kg−1), in contrast the snail Physella acuta was relatively more tolerant (EC50: 298 mg kg−1). Changes in subindividual responses (gene expression and metabolite abundance) in laboratory‐bred chironomid, Chironomus tepperi, were evident at 60 mg kg−1. These changes likely reflect the direct effects of copper exposure and represent metal‐specific responses. Synthesis and applications. We showed that copper toxicity in sediments could be readily detected through changes in gene expression and metabolites in laboratory‐bred chironomids exposed in field‐based microcosms. These responses were more sensitive than mortality, and detected copper levels that caused microcosm chironomid populations to decline. These novel approaches will provide managers with new tools to better assess sediment toxicity.

Neonicotinoid Insecticides and Their Impacts on Bees: A Systematic Review of Research Approaches and Identification of Knowledge Gaps.

It has been suggested that the widespread use of neonicotinoid insecticides threatens bees, but research on this topic has been surrounded by controversy. In order to synthesize which research approaches have been used to examine the effect of neonicotinoids on bees and to identify knowledge gaps, we systematically reviewed research on this subject that was available on the Web of Science and PubMed in June 2015. Most of the 216 primary research studies were conducted in Europe or North America (82%), involved the neonicotinoid imidacloprid (78%), and concerned the western honey bee Apis mellifera (75%). Thus, little seems to be known about neonicotinoids and bees in areas outside Europe and North America. Furthermore, because there is considerable variation in ecological traits among bee taxa, studies on honey bees are not likely to fully predict impacts of neonicotinoids on other species. Studies on crops were dominated by seed-treated maize, oilseed rape (canola) and sunflower, whereas less is known about potential side effects on bees from the use of other application methods on insect pollinated fruit and vegetable crops, or on lawns and ornamental plants. Laboratory approaches were most common, and we suggest that their capability to infer real-world consequences are improved when combined with information from field studies about realistic exposures to neonicotinoids. Studies using field approaches often examined only bee exposure to neonicotinoids and more field studies are needed that measure impacts of exposure. Most studies measured effects on individual bees. We suggest that effects on the individual bee should be linked to both mechanisms at the sub-individual level and also to the consequences for the colony and wider bee populations. As bees are increasingly facing multiple interacting pressures future research needs to clarify the role of neonicotinoids in relative to other drivers of bee declines.

Spatial and temporal distribution patterns of nectar-inhabiting yeasts: how different floral microenvironments arise in winter-blooming Helleborus foetidus

Yeasts frequently colonise floral nectar, where they can reach high densities. Recent investigations have further shown that yeast metabolism alters nectar properties by decreasing its total sugar content, modifying sugar composition, or raising nectar local temperature. However, the distribution patterns of nectar yeasts remain poorly investigated at multiple spatial and temporal scales. Here, we study natural variation of the nectar yeasts in a single host plant, Helleborus foetidus , in a mountainous region. We quantified spatio-temporal variation in the frequency and abundance of yeast species across six populations located along an altitudinal gradient. Variance partitioning techniques were used to estimate the relative magnitude of variation in yeast abundance between individual plants, flowers within plants, and nectaries within flowers. Although yeast frequency and abundance varied widely across sites and dates, the largest part of total variance occurred at the sub-individual level (i.e., flowers on the same plant). Pollinator composition and activity seemed the main factors explaining the observed patterns of yeast frequency and abundance across floral nectar samples. • Nectar yeast incidence varied widely across sites and dates for a single host plant. • However, the largest part of total variance occurred at the sub-individual level. • Pollinator features were the main factors explaining yeast abundance patterns. • Nectar yeast patchy distribution may affect pollinator choice and plant fitness. • Sub-individual variation may disfavour plant selective forces against nectar yeasts.

Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: Exposure assessment and reprotoxic effects of water column contamination

This study highlights the usefulness of gastropods for water quality monitoring. Gastropods were caged upstream and downstream of an effluent discharge. Exposure was assessed by measurement of organic contaminants in water. Contamination of the Potamopyrgus antipodarum mudsnail was also measured using innovative techniques at the end of the 42 days of exposure. Biological effects were measured at the individual level (growth, reproduction) and subindividual level (energy reserves, vitellin-like proteins, steroid levels, expression of genes involved in estrogen signaling pathways), thus providing a better understanding of reprotoxic effects. The effluent was mainly contaminated by pharmaceutical compounds, as was the mudsnail. The highest concentrations were measured for oxazepam and were higher than 2mg/kg downstream of the effluent discharge. Alkylphenols, bisphenol A, and vertebrate-like sex-steroid hormones were also bioaccumulated by the mudsnail downstream of the effluent. The combined use of water and snail contamination provided a complete exposure assessment. Exposure was further linked to biological effects. The mudsnail was shown to be a better adapted species for in situ exposures than Valvata piscinalis. Reproduction was sharply decreased after 6 weeks of exposure in the mudsnail. Feeding issues were excluded, confirming the toxic origin. These effects were related to estrogen signaling pathways using genomic analysis. Genes coding for proteins involved in nongenomic signaling pathways were inhibited, and those of genomic pathway repressors were induced. These results suggest that the chemical contamination due to the effluent discharge altered steroid control of reproduction and blocked the transition between oocyte and unshelled embryo, resulting in a drastic decrease of embryo production, while survival was not affected.