Lower Levels Of Biological Organization Research Articles

Stable isotopes reveal sex- and context-dependent amino acid routing in green anole lizards (Anolis carolinensis).

Allocation of acquired resources to phenotypic traits is affected by resource availability and current selective context. While differential investment in traits is well documented, the mechanisms driving investment at lower levels of biological organization, which are not directly related to fitness, remain poorly understood. We supplemented adult male and female Anolis carolinensis lizards with an isotopically labelled essential amino acid (13C-leucine) to track routing in four tissues (muscle, liver, gonads, and spleen) under different combinations of resource availability (high and low-calorie diets) and exercise training (sprint training and endurance capacity). We predicted sprint training should drive routing to muscle, and endurance training to liver and spleen, and that investment in gonads should be of lower priority in each of the cases of energetic stress. We found complex interactions between training regime, diet, and tissue type in females, and between tissue type and training and tissue type and diet in males, suggesting that males and females adjust their 13C-leucine routing strategies differently in response to similar environmental challenges. Importantly, our data show evidence of increased 13C-leucine routing in training contexts not to muscle as we expected, but to the spleen, which turns over blood cells, and to the liver, which supports metabolism under differing energetic scenarios. Our results reveal the context-specific nature of long-term tradeoffs associated with increased chronic activity. They also illustrate the importance of considering the costs of locomotion in studies of life history strategies.

Genomic and Epigenomic Influences on Resilience Across Scales: lessons from the responses of fishes to environmental stressors.

Understanding the factors that influence the resilience of biological systems to environmental change is an increasingly critical concern in the face of increasing human impacts on ecosystems and the organisms that inhabit them. However, most considerations of biological resilience have focused at the community and ecosystem levels, whereas here we discuss how including consideration of processes occurring at lower levels of biological organization may provide insights into factors that influence resilience at higher levels of biological organization. Specifically, we explore how processes at the genomic and epigenomic levels may cascade up to influence resilience at higher levels and ask how the concepts of 'resistance', or the capacity of a system to minimize change in response to a disturbance, and 'recovery', or the ability of a system to return to its original state following a disturbance and avoid tipping points and resulting regime shifts map to these lower levels of biological organization. Overall, we argue that substantial changes at these lower levels may be required to support resilience at higher levels, using selected examples of genomic and epigenomic responses of fish to climate-change relevant stressors such as high temperature and hypoxia at the levels of the genome, epigenome and organism.

Eco-evolutionary emergence of macroecological scaling in plankton communities.

Macroecological scaling patterns, such as between prey and predator biomass, are fundamental to our understanding of the rules of biological organization and ecosystem functioning. Although these scaling patterns are ubiquitous, how they arise is poorly understood. To explain these patterns, we used an eco-evolutionary predator-prey model parameterized using data for phytoplankton and zooplankton. We show that allometric scaling relationships at lower levels of biological organization, such as body-size scaling of nutrient uptake and predation, give rise to scaling relationships at the food web and ecosystem levels. Our predicted macroecological scaling exponents agree well with observed values across ecosystems. Our findings explicitly connect scaling relationships at different levels of biological organization to ecological and evolutionary mechanisms, yielding testable hypotheses for how observed macroecological patterns emerge.

How does temperature rise affect a freshwater catfish Rhamdia quelen? A proteomic approach

Outside of scientific circles, climate change is a hotly debated topic due to all its consequences. Changes in the temperature can affect aquatic organisms and it is important to understand and to detect earlier signals. This study aimed to analyze how a Neotropical fish species responds to temperature increases, using proteomic analysis as a tool. For this, fish of the species Rhamdia quelen, male and female, were exposed to two temperatures: 25 °C and 30 °C. After 96 h, the animals were anesthetized, euthanized and the liver was collected for proteomic analysis. Using freely available online software and databases (e.g. MetaboAnalyst, Gene Ontology and UniProt), we define the altered proteins in both sexes: 42 in females and 62 in males. Data are available via ProteomeXchange with identifier PXD046475. Differences between the two temperatures were observed mainly in the amino acid metabolic pathways. The cellular process and the immune response was altered, indicating that effects at lower levels of biological organization could serve as a predictor of higher-level effects when temperature rise affects wildlife populations. Thus, we conclude that the increase in temperature is capable of altering important cellular and physiological processes in R. quelen fish, with this response being different for males and females.

Adverse Outcome Pathways for Chronic Copper Toxicity to Fish and Amphibians.

In the present review, we synthesize information on the mechanisms of chronic copper (Cu) toxicity using an adverse outcome pathway framework and identify three primary pathways for chronic Cu toxicity: disruption of sodium homeostasis, effects on bioenergetics, and oxidative stress. Unlike acute Cu toxicity, disruption of sodium homeostasis is not a driving mechanism of chronic toxicity, but compensatory responses in this pathway contribute to effects on organism bioenergetics. Effects on bioenergetics clearly contribute to chronic Cu toxicity with impacts at multiple lower levels of biological organization. However, quantitatively translating these impacts into effects on apical endpoints such as growth, amphibian metamorphosis, and reproduction remains elusive and requires further study. Copper-induced oxidative stress occurs in most tissues of aquatic vertebrates and is clearly a significant driver of chronic Cu toxicity. Although antioxidant responses and capacities differ among tissues, there is no clear indication that specific tissues are more sensitive than others to oxidative stress. Oxidative stress leads to increased apoptosis and cellular damage in multiple tissues, including some that contribute to bioenergetic effects. This also includes oxidative damage to tissues involved in neuroendocrine axes and this damage likely alters the normal function of these tissues. Importantly, Cu-induced changes in hormone concentrations and gene expression in endocrine-mediated pathways such as reproductive steroidogenesis and amphibian metamorphosis are likely the result of oxidative stress-induced tissue damage and not endocrine disruption. Overall, we conclude that oxidative stress is likely the primary driver of chronic Cu toxicity in aquatic vertebrates, with bioenergetic effects and compensatory response to disruption of sodium homeostasis contributing to some degree to observed effects on apical endpoints. Environ Toxicol Chem 2022;41:2911-2927. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Tissue tectonics and the multi-scale regulation of developmental timing.

Development encompasses processes that occur at multiple length scales, including gene-regulatory interactions, cell movements and reorganization, cell signalling and growth. It is essential that the timing of events in all of these different processes is coordinated to generate well-patterned tissues and organs. However, how the timing of intrinsic cell state changes is coordinated with events occurring at the multi-tissue and whole-organism level is unknown. Here, we argue that an important mechanism that accounts for the integration of timing across levels of organization is provided by tissue tectonics, i.e. how morphogenetic events driving tissue shape changes result in the relative displacement of signalling and responding tissues and coordinate developmental timing across scales. In doing so, tissue tectonics provides a mechanism by which the cell specification events intrinsic to cells can be modulated by the temporal exposure to extracellular signals. This exposure is in turn regulated by higher-order properties of the embryo, such as their physical properties, rates of growth and the combination of dynamic cell behaviours, impacting tissue morphogenesis. Tissue tectonics creates a downward flow of information from higher to lower levels of biological organization, providing an instance of downward causation in development.

Prioritization of contaminants and biological process targets in the North Sea using toxicity data from ToxCast

The increasing number of chemicals detected in the marine environment underlines the need for appropriate prioritization strategies prior to further testing and potential inclusion into monitoring programs. Here, a prioritization strategy is proposed for chemicals detected in the North Sea over the last decade, through the development of a Concern Index (CI) using exposure and toxicity data obtained from peer-review publications and the ToxCast database, respectively. A total of 158 chemicals were ranked and the most sensitive tested assay endpoints were identified. Additionally, similar analysis was performed for the classes of chemicals and Biological Process Targets (BPTs). By first ranking chemicals currently acknowledged for their high toxicity to the aquatic environment, i.e. naphthalene, salicylic acid and simazine, the obtained results not only reinforce the risk posed by these but also promote a confident extrapolation from mammalian in vitro toxicity data to fish. Furthermore, genes targeted by the most sensitive assays, related to basic cell maintenance processes and immune defense, are highly evolutionarily conserved across species. The identification of these assays further reinforces the importance of a shift from traditional toxicity endpoints to lower levels of biological organization, allowing the detection of adverse effects at lower concentrations.

Chronic toxicity of technical atrazine to the fathead minnow (Pimephales promelas) during a full life-cycle exposure and an evaluation of the consistency of responses

Fathead minnows (Pimephales promelas) were continuously exposed to the herbicide atrazine (0.15, 0.25, 0.46, 0.99, and 2.0 mg a.i./L, plus dilution water and solvent controls) for a complete life cycle (274 days). Concentrations of atrazine up to 2.0 mg a.i./L did not significantly reduce hatching success, larval survival at 30 or 60 days post-hatch, or reproduction (eggs/spawn, total eggs, spawns/female, or eggs/female) in the F0 generation. However, at 60 days of exposure, total length and total survival to study completion were significantly reduced in ≥0.46 mg a.i./L and ≥ 0.99 mg a.i./L treatments, respectively. In the F1 generation, hatchability of embryos at ≥0.25 mg a.i./L (range 74–82%) was significantly less than that of pooled control organisms (86%). Following 30 days' post-hatch exposure, F1 survival was not significantly different from pooled control for any treatment. Finally, tissues representing major life stages had bioconcentration factors ranging from 3.7× (F1 embryos, <24 h) to 8.5× (F0 adults), indicating little to no evidence of bioconcentration. We developed a series of questions to assess the consistency of observed responses in order to place the data in context with the wider available and relevant literature (e.g., Observed between studies? Observed between species? Observed at lower levels of biological organization?). The analysis for consistency supports the conclusion that atrazine does not pose a significant chronic risk to freshwater fish in terms of growth, reproduction, or survivorship at concentrations of up to at least 100 μg/L.

Physicochemical characteristics, animal species diversity and oxidative stress responses in dominant fish from an impacted site on the Lagos Lagoon, Nigeria

The evaluation of biomarkers in resident fisheries of aquatic ecosystems is important for risk assessment of such ecosystems. In this study, surface water and sediment physicochemical characteristics, macrobenthic invertebrate and fish species diversity and oxidative stress of dominant fish were evaluated at an anthropogenic-impacted site (Iddo) as well as a reference site (after the Third Mainland Bridge) of the Lagos lagoon, Nigeria, over a period of three (3) months. Surface water analysis result showed that the Total dissolved solids (TDS) (5.54±2.85 mg/L), Salinity (5.09±2.56 o/oo), Conductivity (6.51±4.54 µS/cm), Nitrates (4.70±1.49 mg/L), Phosphates (3.03±0.60 mg/L), Biochemical oxygen demand (BOD) (18.62±3.77 mg/L) and Chemical oxygen demand (COD) (463.44±65.83 mg/L) were significantly higher at the test site compared to the reference site. The COD of the sediment were significantly higher (p&lt;0.05) at the test site compared to the reference while the other sediment physicochemical parameters except pH were higher at the test site compared to the reference site. Macrobenthic invertebrate species diversity were slightly higher (1.70) at the reference site compared to the test site (1.66) while species richness were higher at the test site (1.43) compared to the reference site (1.26). Fish species diversity (1.95) and richness (1.79) were higher at the test site compared to the reference site. Dominant fish species at the test and reference sites were Ethmalosa fimbriata (Bonga shad) and Sarotherodon melanotheron (Blackchin Tilapia) respectively. There were increased levels of antioxidant enzymes and biomolecules- glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and malondialdehyde (MDA) in the gills and liver of E. fimbriata indicative of oxidative stress. Further studies on biomarkers at lower levels of biological organisation are recommended for holistic evaluations and evidence-based intervention if necessary. This will support the achievement of the United Nations Sustainable Development Goal 14 (life below water).

Transgenerational exposure to ocean acidification induces biochemical distress in a keystone amphipod species (Gammarus locusta)

Atmospheric carbon dioxide (CO2) levels are increasing at the fastest rate ever recorded, causing higher CO2 dissolution in the ocean, leading to a process known as ocean acidification (OA). Unless anthropogenic CO2 emissions are reduced, they are expected to reach ~900 ppm by the century’s end, resulting in a 0.13–0.42 drop in the seawater pH levels. Since the transgenerational effects of high CO2 in marine organisms are still poorly understood at lower levels of biological organization (namely at the biochemical level), here we reared a key ecological relevant marine amphipod, Gammarus locusta, under control and high CO2 conditions for two generations. We measured several stress-related biochemical endpoints: i) oxidative damage [lipid peroxidation (LPO) and DNA damage]; ii) protein repair and removal mechanisms [heat shock proteins (HSPs) and ubiquitin (Ub)]; as well as iii) antioxidant responses [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione s-transferase (GST)] and total antioxidant capacity (TAC). The present results support the premise that exposure to high CO2 is expected to decrease survival rates in this species and cause within- and transgenerational oxidative damage. More specifically, the predicted upsurge of reactive oxygen and nitrogen species seemed to overwhelm the stimulated amphipod antioxidant machinery, which proved insufficient in circumventing protein damage within the parents. Additionally, negative effects of OA are potentially being inherited by the offspring, since the oxidative stress imposed in the parent’s proteome appears to be restricting DNA repair mechanisms efficiency within the offspring’s. Thus, we argue that a transgenerational exposure of G. locusta could further increase vulnerability to OA and may endanger the fitness and sustainability of natural populations.

Investigating a transcriptomic approach on marine mussel hemocytes exposed to carbon nanofibers: An in vitro/in vivo comparison

Manufactured nanomaterials are an ideal test case of the precautionary principle due to their novelty and potential environmental release. In the context of regulation, it is difficult to implement for manufactured nanomaterials as current testing paradigms identify risk late into the production process, slowing down innovation and increasing costs. One proposed concept, namely safe(r)-by-design, is to incorporate risk and hazard assessment into the design process of novel manufactured nanomaterials by identifying risks early. When investigating the manufacturing process for nanomaterials, differences between products will be very similar along key physicochemical properties and biological endpoints at the individual level may not be sensitive enough to detect differences whereas lower levels of biological organization may be able to detect these variations. In this sense, the present study used a transcriptomic approach on Mytilus edulis hemocytes following an in vitro and in vivo exposure to three carbon nanofibers created using different production methods. Integrative modeling was used to identify if gene expression could be in linked to physicochemical features. The results suggested that gene expression was more strongly associated with the carbon structure of the nanofibers than chemical purity. With respect to the in vitro/in vivo relationship, results suggested an inverse relationship in how the physicochemical impact gene expression.

Environmental health assessment of warming coastal ecosystems in the tropics – Application of integrative physiological indices

According to climate science, ocean warming is one of the current and future greatest threats to coastal ecosystems. Projection scenarios for the end of this century show that tropical intertidal ecosystems are particularly at risk. In this study we optimized and tested a holistic method for bio-monitoring present and projected thermal pressure in such ecosystems, in order to assess organism vulnerability to ocean warming. Several species representative of different animal groups (fish, crustaceans and gastropods) were collected from the field and subjected to an experimental trial for 28 days, testing two temperatures: control (present seawater summer temperature) and elevated temperature (+3 °C, projected seawater temperature anomaly for 2100). Muscle samples were collected weekly to quantify several biomarkers of: i) macromolecular damage (protein unfolding and denaturation, and lipid peroxidation), ii) reactive oxygen species (ROS) scavengers (antioxidant enzymes), and iii) body condition (energy reserves and body mass). These biomarkers were combined in integrated biomarker response (IBR) indices, either in three separate stress response categories (as previously defined) or in a unique combined analysis of overall physiological performance. Both approaches suggest that temperature affected IBRs, with increasing temperatures significantly impairing the overall health of individuals. Biomarkers of lower levels of biological organization indicated deleterious effects of temperature, whereas biomarkers of higher levels suggested maintenance of performance after chronic exposure. Overall indices combining the estimates of biomarkers across levels of biological organization are essential to predict the vulnerability of species, or populations, to climate warming. Such indices may assist managers and stakeholders in the establishment of monitoring programs and environmental policies toward the conservation of fragile coastal systems.

Introduction to the Symposium: Integrative Life-History of Whole-Organism Performance.

A strong case can be made for whole-organism performance traits (i.e., dynamic, ecologically relevant traits whose expression is shaped by underlying morphological factors) as being the ultimate integrative traits. This is not only because they capture the output of multiple lower levels of biological organization, but also because they are directly relevant to individual fitness in multiple ecological contexts, and are in many cases important proximate determinants of survival and/or reproductive success. But although many ecological and evolutionary phenomena can be examined through the lens of performance (and vice-versa), performance research has been surprisingly slow to incorporate concepts from the large and important field of life-history evolution. Such a synthesis is necessary, because shifts in resource allocation strategies can have implications for these highly ecologically relevant, functional traits, whose expression may trade-off against fecundity, immune function, or longevity, among other key life-history traits. The papers in this symposium showcase many of the ways in which life-history strategies can have direct consequences for the expression, maintenance, and evolution of whole-organism performance (and at least one case where they may not). By approaching the issue of life-history trade-offs from a number of diverse perspectives, this symposium reveals the scope for future explicit integration of life-history techniques with those of whole-organism performance studies for a more complete understanding of multivariate phenotypic evolution.

Adverse outcome pathways: Application to enhance mechanistic understanding of neurotoxicity.

Recent developments have prompted the transition of empirically based testing of late stage toxicity in animals for a range of different endpoints including neurotoxicity to more efficient and predictive mechanistically based approaches with greater emphasis on measurable key events early in the progression of disease. The adverse outcome pathway (AOP) has been proposed as a simplified organizational construct to contribute to this transition by linking molecular initiating events and earlier (more predictive) key events at lower levels of biological organization to disease outcomes. As such, AOPs are anticipated to facilitate the compilation of information to increase mechanistic understanding of pathophysiological pathways that are responsible for human disease.In this review, the sequence of key events resulting in adverse outcome (AO) defined as parkinsonian motor impairment and learning and memory deficit in children, triggered by exposure to environmental chemicals has been briefly described using the AOP framework. These AOPs follow convention adopted in an Organization for Economic Cooperation and Development (OECD) AOP development program, publically available, to permit tailored application of AOPs for a range of different purposes.Due to the complexity of disease pathways, including neurodegenerative disorders, a specific symptom of the disease (e.g. parkinsonian motor deficit) is considered as the AO in a developed AOP. Though the description is necessarily limited by the extent of current knowledge, additional characterization of involved pathways through description of related AOPs interlinked into networks for the same disease has potential to contribute to more holistic and mechanistic understanding of the pathophysiological pathways involved, possibly leading to the mechanism-based reclassification of diseases, thus facilitating more personalized treatment.

Emergent Properties in the Honey Bee Superorganism

For scientists, the superorganism concept is not so much a theory as it is a tool for studying how organisms evolve. There is a vector in evolution toward lower levels of biological organization be...

Linking effects of anthropogenic debris to ecological impacts.

Accelerated contamination of habitats with debris has caused increased effort to determine ecological impacts. Strikingly, most work on organisms focuses on sublethal responses to plastic debris. This is controversial because (i) researchers have ignored medical insights about the mechanisms that link effects of debris across lower levels of biological organization to disease and mortality, and (ii) debris is considered non-hazardous by policy-makers, possibly because individuals can be injured or removed from populations and assemblages without ecological impacts. We reviewed the mechanisms that link effects of debris across lower levels of biological organization to assemblages and populations. Using plastic, we show microplastics reduce the 'health', feeding, growth and survival of ecosystem engineers. Larger debris alters assemblages because fishing-gear and tyres kill animals and damage habitat-forming plants, and because floating bottles facilitate recruitment and survival of novel taxa. Where ecological linkages are not known, we show how to establish hypothetical links by synthesizing studies to assess the likelihood of impacts. We also consider how population models examine ecological linkages and guide management of ecological impacts. We show that by focusing on linkages to ecological impacts rather than the presence of debris and its sublethal impacts, we could reduce threats posed by debris.

Gene transcription patterns and energy reserves in Daphnia magna show no nanoparticle specific toxicity when exposed to ZnO and CuO nanoparticles.

There is still a lot of contradiction on whether metal ions are solely responsible for the observed toxicity of ZnO and CuO nanoparticles to aquatic species. While most experiments have studied nanoparticle effects at organismal levels (e.g. mortality, reproduction), effects at lower levels of biological organization may clarify the role of metal ions, nanoparticles and nanoparticle aggregates. In this study, the effect of ZnO and CuO nanoparticles was tested at two lower levels: energy reserves and gene transcription and compared with zinc and copper salts. Daphnia magna was exposed during 96h to 10% immobilization concentrations of all chemicals, after which daphnids were sampled for determination of glycogen, lipid and protein concentration and for a differential gene transcription analysis using microarray. The dissolved, nanoparticle and aggregated fraction in the medium was characterized. The results showed that ZnO nanoparticles had largely dissolved directly after addition to the test medium. The CuO nanoparticles mostly formed aggregates, while only a small fraction dissolved. The exposure to zinc (both nano and metal salt) had no effect on the available energy reserves. However, in the copper exposure, the glycogen, lipid and protein concentration in the exposed daphnids was lower than in the unexposed ones. When comparing the nanoparticle (ZnO or CuO) exposed daphnids to the metal salt (zinc or copper salt) exposed daphnids, the microarray results showed no significantly differentially transcribed gene fragments. The results indicate that under the current exposure conditions the toxicity of ZnO and CuO nanoparticles to D. magna is solely caused by toxic metal ions.

A weight‐of‐evidence approach to determine estuarine fish health using indicators from multiple levels of biological organization

ABSTRACT A prototype Estuarine Fish Health Index (EFHI) using biological indicators from multiple levels of biological organization is described. Three test estuaries of similar size and morphology but with different development pressures were sampled for this purpose. The EFHI integrates the well established Estuarine Fish Community Index (EFCI) developed for South African estuaries, which uses fish community level metrics to assess estuarine condition, with stress responses measured at lower levels of biological organization. Four sub‐organism level biomarkers were used: liver histopathology assessment, acetylcholinesterase and lipid peroxidase assays and an individual fish condition factor analysis. The data from these analyses and the results of the EFCI were integrated to determine if combining these inputs produces a more sensitive and predictive tool for assessing estuarine health than when used in isolation. Challenges and management applications of this new tool are explored and further studies to improve its prognostic capacity are suggested.Copyright © 2011 John Wiley &amp; Sons, Ltd.

How do consumers deal with stoichiometric constraints? Lessons from functional genomics using Daphnia pulex

Disaccord between the supply and demand of energy (carbon, C) and certain material elements (e.g. phosphorus, P) across trophic levels is common in most ecosystems and impacts the strength of trophic interactions and ecosystem functions such as productivity and nutrient recycling. Yet, we know little about mechanisms operating at the lower levels of biological organization that drive such higher-level ecological processes. Such information should help refine theories integrating biological processes at multiple levels of organization. Understanding the expression and functions of genes that underlie (to a large degree) physiological adjustments made by organisms to stoichiometric imbalances at trophic interfaces is a first step in this enterprise. Here, we investigate adjustments in gene expression to varying supply and demand of phosphorus relative to other dietary components in the keystone limnetic herbivore, Daphnia pulex. Daphniids were fed an algal diet of either LoC-HiP (molar C:P ∼100) or HiC-LoP (molar C:P ∼900) for 5 days, resulting in significant growth reductions under HiC-LoP conditions. Microarrays measured the transcriptional regulation of 8217 annotated protein-coding genes under contrasting dietary conditions and revealed 1818 differentially expressed (DE) genes; 19% are genes unique to the Daphnia lineage. We mapped DE genes onto a global chart of metabolic pathways to obtain a systems-level perspective on the responses to stoichiometric imbalances. Daphnia differentially regulated pathways were involved in sequestering limiting elements, and in dealing with the products of metabolic adjustments that may be triggered by nutrient stress in primary producers. Functional genomics at trophic interfaces illuminate the complexity of processes underlying stoichiometric constraints on energy and nutrient fluxes in ecosystems.

Reproduction and biochemical responses in Enchytraeus albidus (Oligochaeta) to zinc or cadmium exposures

To better understand chemical modes of action, emphasis has been given to stress responses at lower levels of biological organization. Cholinesterases and antioxidant defenses are among the most used biomarkers due to their crucial role in the neurocholinergic transmission and in cell homeostasis preventing DNA damage, enzymatic inactivation and lipid peroxidation. The main goal of this study was to investigate the effects of zinc and cadmium on survival and reproduction of E. albidus and to assess metals oxidative stress potential and neurotoxic effects at concentrations that affected reproduction. Both metals affected the enchytraeids’ survival and reproduction and induced significant changes in the antioxidant defenses as well as increased lipid peroxidation, indicating oxidative damage. This study demonstrates that determining effects at different levels of biological organization can give better information on the physiological responses of enchytraeids in metal contamination events and further unravel the mechanistic processes dealing with metal stress.