Adaptive plastic responses to metal contamination in a multistress context: a field experiment in fish.

Wild populations often differ in their tolerance to environmental stressors, but intraspecific variability is rarely taken into account in ecotoxicology. In addition, plastic responses to multiple stressors have rarely been investigated in realistic field conditions. In this study, we compared the responses to metal contamination of gudgeon populations (Gobio occitaniae) differing in their past chronic exposure to metal contamination, using a reciprocal transplant experiment and an immune challenge mimicking a parasite attack to test for potential effects of multiple stressors across biological levels. We measured fish survival and traits involved in metal bioaccumulation, oxidative stress, immunity, cell apoptosis, and energy management to decipher underpinning physiological mechanisms across biological levels (i.e., gene expression, cell, organism). Fish from the two replicate High Contamination sites had higher survival when transferred into contaminated sites, suggesting a local adaptation to the contaminated site, possibly explained by higher levels of detoxification and antioxidant capacity but with potential higher apoptosis costs compared to their naïve counterparts. We found no evidence of co- or maladaptation to the immune stressor, suggesting no specific costs to face pathogens. In the emerging field of evolutionary ecotoxicology, this study underlines the need to consider intraspecific variability to better understand the effects of pollution in heterogeneous populations.

Changes in fish skin microbiota along gradients of eutrophication in human-altered rivers.

The skin microbiota plays a major role in health of organisms but it is still unclear how such bacterial assemblages respond to changes in environmental conditions and anthropogenic perturbations. In this study, we investigated the effects of the eutrophication of freshwater ecosystems on the skin microbiota of fish. We sampled wild gudgeon Gobio occitaniae from 17 river sites along an eutrophication gradient and compared their skin microbiota diversity and composition, using a 16s rRNA gene metabarcoding approach. Results showed a tendency for higher taxonomic and phylogenetic diversity in highly eutrophic sites linked to the presence of suspended organic matters. We also highlighted significant links between eutrophication and skin microbiota taxonomic composition and beta-diversity. In contrast, skin microbiota characteristics did not correlate with host factors such as age or sex, although microbiota beta-diversity did vary significantly according to host parasite load. To conclude, our study highlights the importance of environmental factors, especially eutrophication, on the diversity and composition of skin mucus bacterial communities. Because changes in the skin microbiota may induce potential deleterious consequences on host health and population persistence, our results confirm the importance of accounting for host-microbiota interactions when examining the consequences of anthropogenic activities on aquatic fauna.

Intraspecific variability of responses to combined metal contamination and immune challenge among wild fish populations.

Wild organisms are increasingly exposed to multiple anthropogenic and natural stressors that can interact in complex ways and lead to unexpected effects. In aquatic ecosystems, contamination by trace metals has deleterious effects on fish health and commonly co-occurs with pathogens, which affect similar physiological and behavioral traits. However, the combined effects of metal contamination and parasitism are still poorly known. In addition, the sensitivity to multiple stressors could be highly variable among different fish populations depending on their evolutionary history, but this intraspecific variability is rarely taken into account in existing ecotoxicological studies. Here, we investigated i) the interactive effects of metal contamination (i.e., realistic mixture of Cd, Cu and Zn) and immune challenge mimicking a parasite attack on fish health across biological levels. In addition, we compared ii) the physiological and behavioral responses among five populations of gudgeon fish (Gobio occitaniae) having evolved along a gradient of metal contamination. Results show that single stressors exposure resulted in an increase of immune defenses and oxidative stress at the expense of body mass (contamination) or fish swimming activity (immune challenge). Multiple stressors had fewer interactive effects than expected, especially on physiological traits, but mainly resulted in antagonistic effects on fish swimming activity. Indeed, the immune challenge modified or inhibited the effects of contamination on fish behavior in most populations, suggesting that multiple stressors could reduce behavioral plasticity. Interestingly, the effects of stressors were highly variable among populations, with lower deleterious effects of metal contamination in populations from highly contaminated environments, although the underlying evolutionary mechanisms remain to be investigated. This study highlights the importance of considering multiple stressors effects and intraspecific variability of sensitivity to refine our ability to predict the effects of environmental contaminants on aquatic wildlife.

Dose- and time-dependent effects of an immune challenge on fish across biological levels.

Due to global changes, fish are increasingly exposed to immune challenges associated with disease outbreaks in aquatic ecosystems. Adjustments in physiology and behavior are generally critical to maintaining homeostasis after an immune challenge, but there is limited knowledge on the specific thresholds and dynamics of responses across levels of biological organization in fish. In this study, we tested how different concentrations of an antigens mixture (phytohemagglutinin and lipopolysaccharide) affected innate immunity with potential consequences on oxidative stress, energy reserves, body condition, and behavior across time, using the common gudgeon (Gobio sp.) as model species. The immune challenge induced a transitory increase in lytic enzyme activity (i.e., lysozyme) and local immune response (i.e., skin swelling) 2 days after the antigen injection. The available energy stored in muscle was also reduced 4 days after injection, without inducing oxidative stress at the cellular level. Overall, the immune challenge induced limited costs at the molecular and cellular levels but had strong effects at the whole organism level, especially on behavior. Indeed, fish swimming activity and sociability were affected in a dose- and time-dependent manner. These results suggest that immune challenges have dose-dependent effects across levels of biological organization and that behavior is a key response trait to cope with pathogen-induced immune costs in the wild, although fitness consequences remain to be tested.

Direct and indirect effects of multiple environmental stressors on fish health in human-altered rivers.

Freshwater fish face multiple challenges in human-altered rivers such as trace metal contamination, temperature increase and parasitism. These multiple stressors could have unexpected interactive effects on fish health due to shared physiological pathways, but few studies investigated this question in wild fish populations. In this study, we compared 16 populations of gudgeon (Gobio occitaniae) distributed along perturbation gradients in human-altered rivers in the South of France. We tested the effects of single and combined stressors (i.e., metal contamination, temperature, parasitism) on key traits linked to fish health across different biological levels using a Structural Equation Modelling approach. Parasitism and temperature alone had limited deleterious effects on fish health. In contrast, fish living in metal-contaminated sites had higher metal bioaccumulation and higher levels of cellular damage in the liver through the induction of an inflammatory response. In addition, temperature and contamination had interactive negative effects on growth. These results suggest that trace metal contamination has deleterious effects on fish health at environmentally realistic concentrations and that temperature can modulate the effects of trace metals on fish growth. With this study, we hope to encourage integrative approaches in realistic field conditions to better predict the effects of natural and anthropogenic stressors on aquatic organisms.

Health indicators and contaminant levels of a critically endangered species in the Gironde estuary, the European sturgeon.

The European sturgeon, Acipenser sturio, is a highly endangered species that almost disappeared in the last decades. Thanks to yearly restocking of the population, this species is still found in the Gironde estuary (France), where juveniles grow during several years before leaving to the ocean. The aims of this study were to evaluate the pressure exerted on these fish by known organic and inorganic contaminants during their stay at the Gironde estuary, and to get information on the fish's health in this context. Monthly captures over the year 2014 provided 87 fish from the cohorts 2012 and 2013 mainly, and from cohorts 2008, 2009, and 2011, all fish born in hatchery. We report the very first analyses of contaminant levels and of biological markers measured in the blood of these fish. Low inorganic contamination was found, composed of seven metals mainly Zn (< 5 µg mL-1), Fe (< 1.5 µg mL-1), Cu (< 0.8 µg mL-1), Se (< 0.8 µg mL-1), As (< 0.25 µg mL-1), Co (< 0.14 µg mL-1), and Mn (< 0.03 µg mL-1). Concerning persistent organic contaminants, the sum of seven PCBs varied from 1 to 10 ng g-1 plasma, that of eight OCPs from 0.1 to 1 ng g-1, and that of eight PBDEs from 10 to 100 pg g-1. Higher levels of contaminants were measured during spring as compared to summer. The sex steroid hormone plasma levels (estradiol, testosterone, and 11-ketotestosterone) were quite low, which was predictable for juveniles. The transcription of reproduction-involved genes (EstR, AR, LHR, sox9) in blood cells was demonstrated for the first time. Some of them were correlated with organic contaminant levels PCBs and OCPs. Other gene transcriptions (sodCu and bax) were correlated with PCBs and OCPs. However, the DNA damage level measured here as comet tail DNA and micronuclei ratio in red blood cells were in the very low range of the values commonly obtained in fish from pristine areas. The data presented here can serve as a reference base for future monitoring of this population of sturgeons.

Combined effects of temperature increase and immune challenge in two wild gudgeon populations.

In the context of global changes, aquatic ecosystems are increasingly exposed to multiple stressors that can have unexpected interactive effects on aquatic organisms. Among these stressors, the occurrence of heat waves and pathogens is changing rapidly in freshwater rivers, but their combined effects on fish health are still understudied. In this study, we experimentally tested the crossed effects of increased temperature (mimicking a heat wave) and a standardized immune challenge (mimicking a parasite attack) on wild gudgeon (Gobio occitaniae) physiology and behaviour across biological levels from molecules to the whole individual. We also investigated the potential variation of sensitivity among populations by comparing two wild populations from contrasted thermal regimes. Combined stressors (i.e. temperature increase and immune challenge) had contrasted effects on fish physiology and behaviour compared to single stressors, but only at the individual level. In particular, the immune challenge inhibited the effect of the temperature on fish behaviour (activity, exploration and foraging) but amplified the negative effect of temperature on fish survival. No interactions were found at other biological levels. This study thus shows that it is essential to consider biotic stressors such as pathogens to better anticipate the effects of global changes on aquatic organisms. In addition, there was a high variability of response between the two gudgeon populations, suggesting that future studies should take into account population variability to better predict the responses of aquatic wildlife to current and future stressors.

Adaptive response under multiple stress exposure in fish: From the molecular to individual level.

Aquatic systems are subjected to various sources of stress due to global changes, such as increasing temperature and pollution. A major challenge for the next decade will be to evaluate the combined effects of these multiple stressors on organisms and ecosystems. For organisms submitted to chemical, biological or physical stressors, the capacity to set up an efficient adaptive response is a fundamental prerequisite for their long-term survival and performance. In this study, goldfish (Carassius auratus) were subjected to individual and combined pesticide mixtures and increased temperatures to evaluate their adaptive response in multistress conditions from the molecular to the individual level. Fish were exposed for 16 days to a mixture of pesticides at environmental relevant concentrations (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin and tebuconazole) and at two temperatures (22 °C and 32 °C). Three major physiological traits of the stress response were measured: the hormonal response (i.e. plasma cortisol), the metabolic balance from molecular to individuals' levels (metabolomics, cellular energy allocation, energy reserves and global condition indexes), and the cellular defense system induction (SOD, CAT and GST). Results show that (1) environmentally relevant concentrations of pesticides lead to significant responses in fish at all biological levels; (2) the metabolic response depends on the nature of stress (thermal vs. chemical); and (3) fish may be unable to set up an efficient adaptive response when chemical and thermal stresses were combined, with adverse outcomes at the individuals' level.

Short term exposure to multi-walled carbon nanotubes induce oxidative stress and DNA damage in Xenopus laevis tadpoles.

The potential impact of Multiwalled Carbon NanoTubes (MWCNTs) was investigated on Xenopus laevis tadpoles exposed to 0.1, 1 and 10mg/L. Oxidative stress was measured in entire larvae exposed and DNA damage (Comet assay) was carried out in erythrocytes of circulating blood from 2h to 24h according to standardized recommendations. Results showed significant H2O2 production when larvae were exposed to 1mg/L and 10mg/L of MWCNTs after 4h and 2h of exposure, respectively. Antioxidant enzyme activities showed significant induction of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) from only 2h of exposure to 10mg/L of MWCNTs. In presence of 1mg/L of MWCNTs, only GR and CAT activities were significantly induced at 4h. Enzyme activities do not follow a simple dose-effect relation, but the time of induction is shortened in relation with the tested concentration. The Comet assay results showed significant DNA damages with a dose dependent response. The profiles of DNA damages show fluctuations, in course of time, which are characteristics of oxidative stress response in relation with the continuous balance between damage and compensation process.