A psychiatric drug found in waste-water plant effluents alters the migratory behavior of critically endangered Anguilla anguilla juveniles.

Migratory fishes cross or settle in several environments potentially polluted. Psychiatric drugs, which represent one growing pollution and are found in discharges from waste-water treatment plants, may alter individual behaviors. Here, we assessed behavioral alterations in the upstream migratory behavior of Anguilla anguilla caused by diazepam, an anxiolytic. We monitored the swimming activity, swimming behavior, and boldness to assess whether diazepam impacts them or not. Our 7-day behavioral follow-up allowed us to test the kinetics of the potential effects of diazepam. We found diazepam reduced swimming activity and altered individual swimming behavior, with fewer individuals swimming against the current, so swimming upstream. Those effects varied over time and were stronger at the end of our monitoring, suggesting chemical pollutants encountered in estuaries may act as a chemical burden for individuals, despite metabolisation. We also found diazepam favored bolder behavior in glass eels. Our results provide new knowledge on chemical pollution and psychiatric drugs inducing behavioral alterations. Those alterations may have ecological and evolutionary consequences for glass eels, by diminishing predator avoidance and impacting spatial colonization, and thus, local density.

Metabolomics to study the sublethal effects of diazepam and irbesartan on glass eels (Anguilla anguilla).

Since glass eels are continuously exposed to contamination throughout their migratory journey in estuaries, to a certain extent the fall in the population of this endangered species might be attributed to this exposure, which is especially acute in estuaries under high urban pressure. In this work, metabolomics was used to address the main objective of this study, to evaluate the effects of two pharmaceuticals previously identified as potential concerning chemicals for fish (diazepam and irbesartan) on glass eels. An exposure experiment to diazepam, irbesartan and their mixture was carried out over 7 days followed by 7 days of depuration phase. After exposure, glass eels were individually sacrificed using a lethal bath of anesthesia, and then an unbiased sample extraction method was used to extract separately the polar metabolome and the lipidome. The polar metabolome was submitted to targeted and non-targeted analysis, whereas for the lipidome only the non-targeted analysis was carried out. A combined strategy using partial least squares discriminant analysis and univariate and multivariate statistical analysis (ANOVA, ASCA, t-test, and fold-change analysis) was used to identify the metabolites altered in the exposed groups with respect to the control group. The results of the polar metabolome analysis revealed that glass eels exposed to the diazepam-irbesartan mixture were the most impacted ones, with altered levels for 11 metabolites, some of them belonging to the energetic metabolism, which was confirmed to be sensitive to these contaminants. Additionally, the dysregulation of the levels of twelve lipids, most of them with energetic and structural functions, was also found after exposure to the mixture, which might be related to oxidative stress, inflammation, or alteration of the energetic metabolism.

Influence of energetic status on ontogenetic niche shifts: emergence from the redd is linked to metabolic rate in brown trout.

Ontogenetic niche shift should occur when the ratio of growth opportunities to mortality risk becomes higher in the subsequent habitat. While most studies have focused on size to understand the timing of these shifts, an endogenous factor like energetic status (interaction between energy available and energy requirements) appears as a natural candidate to integrate and analyze the growth trade-off between habitats. In this study, we measure energetic content and metabolic rate of individual brown trout (Salmo trutta) fry at emergence from gravel to investigate the influence of energetic status on the timing of this critical ontogenetic niche shift. In addition, as offspring energetic status is subject to parental effects, we examine how females could maximize their own fitness by influencing offspring emergence timing. Our results demonstrate that emergence from gravel is influenced by energetic status. Individuals that emerge first have a higher energetic content but deplete it faster because of a higher metabolic rate. We also find that female fecundity is positively related to emergence period duration. Moreover, our results suggest that females may decrease kin competition during the critical period of emergence by influencing the energetic status of offspring, thus, maximizing their own fitness. Our results help elucidate the mechanisms underlying early ontogenetic niche shifts in juvenile fish and suggest reasons why maternal investment can be so variable within populations.

Prioritization based on risk assessment to study the bioconcentration and biotransformation of pharmaceuticals in glass eels (Anguilla anguilla) from the Adour estuary (Basque Country, France).

The presence of contaminants of emerging concern in the aquatic environment directly impacts water-living organisms and can alter their living functions. These compounds are often metabolized and excreted, but they can also be accumulated and spread through the food chain. The metabolized contaminants can also lead to the formation of new compounds with unknown toxicity and bioaccumulation potential. In this work, we have studied the occurrence, bioconcentration, and biotransformation of CECs in glass eels (Anguilla anguilla) using UHPLC-HRMS. To select the target CECs, we first carried out an environmental risk assessment of the WWTP effluent that releases directly into the Adour estuary (Bayonne, Basque Country, France). The risk quotients of every detected contaminant were calculated and three ecotoxicologically relevant contaminants were chosen to perform the exposure experiment: propranolol, diazepam, and irbesartan. An experiment of 14 days consisting of 7 days of exposure and 7 days of depuration was carried out to measure the bioconcentration of the chosen compounds. The quantitative results of the concentrations in glass eel showed that diazepam and irbesartan reached BCF ≈10 on day 7, but both compounds were eliminated after 7 days of depuration. On the other hand, propranolol's concentration remains constant all along with the experiment, and its presence can be detected even in the non-exposed control group, which might suggest environmental contamination. Two additional suspect screening strategies were used to identify metabolization products of the target compounds and other xenobiotics already present in wild glass eels. Only one metabolite was identified, nordiazepam, a well-known diazepam metabolite, probably due to the low metabolic rate of glass eels at this stage. The xenobiotic screening confirmed the presence of more xenobiotics in wild glass eels, prominent among them, the pharmaceuticals exemestane, primidone, iloprost, and norethandrolone.

Video tracking in the extreme: a new possibility for tracking nocturnal underwater transparent animals with fluorescent elastomer tags.

Initially developed so that an individual could be recognized in mark-recapture studies of aquatic animals, fluorescent visible implant elastomer (VIE) tags are used here for a new application in ethometry: the study of the behavior of transparent animals in dim light or in darkness using automatic tracking technology. The application and validation of this multitracking method is tested in the context of research on the estuarine migratory behavior of the glass eel (Anguilla anguilla), a crucial point to better understand the dynamics of this endangered species. The method makes it possible to measure the activity (notably the distance and speed) of four individuals as a function of tidal and nycthemeral rhythms in the same flume (a circular aquarium simulating river or estuarine conditions) across a wide time scale (from seconds to weeks).

Modulation of Energy Metabolism and Epigenetic Landscape in Rainbow Trout Fry by a Parental Low Protein/High Carbohydrate Diet.

It is now recognized that parental diets could highly affect offspring metabolism and growth. Studies in fish are, however, lacking. In particular, the effect of a parental diet high in carbohydrate (HC) and low in protein (LP) on progeny has never been examined in higher trophic level teleost fish. Thus, two-year old male and female rainbow trout (Oncorhynchus mykiss) were fed either a control diet (0% carbohydrate and 63.89% protein) or a diet containing 35% carbohydrate and 42.96% protein (HC/LP) for a complete reproductive cycle for females and over a 5-month period for males. Cross-fertilizations were then carried out. To evaluate the effect of the parental diet on their offspring, different phenotypic and metabolic traits were recorded for offspring before their first feeding and again three weeks later. When considering the paternal and maternal HC/LP nutrition independently, fry phenotypes and transcriptomes were only slightly affected. The combination of the maternal and paternal HC/LP diets altered the energy metabolism and mitochondrial dynamics of their progeny, demonstrating the existence of a synergistic effect. The global DNA methylation of whole fry was also highly affected by the HC/LP parental diet, indicating that it could be one of the fundamental mechanisms responsible for the effects of nutritional programming.

New insights into methylmercury induced behavioral and energy-related gene transcriptional responses in European glass eel (Anguilla anguilla).

The effect of methylmercury (MeHg) was investigated in glass eel migration behavior and metabolism. To migrate up estuary, glass eels synchronize their swimming activity to the flood tide and remain on or in the substratum during ebb tide. Following seven days of exposure to MeHg (100 ng L-1), glass eels migration behavior was expressed by their swimming synchronization to the water current reversal every 6.2 h (mimicking the alternation of flood and ebb tides) and their swimming activity level. In relation to their behavior, we then analyzed the energy-related gene expression levels in individual head, viscera and muscle. Results showed that MeHg decreased the number of glass eels synchronized to the change in water current direction and their swimming activity level. This last effect was more pronounced in non-synchronized fish than in synchronized ones, supporting the idea that non-synchronized glass eels could be more vulnerable to stress. As regard the expression of energy-related genes, no significant difference was observed between control and MeHg-exposed fish. In contrast, when the swimming activity levels were plotted against transcriptional responses, positive correlations were evidenced in viscera and especially in the head of exposed glass eels but not in control. Finally, it is noteworthy that non-synchronized glass eels displayed lower expression level of metabolism genes than their synchronized counterpart, but only in the head. Altogether, these results support the interest of focusing on the head to investigate the facultative migration behavior in glass eels and the effect of environmental stressors on this rhythmic behavior.

Migratory behavior, metabolism, oxidative stress and mercury concentrations in marine and estuarine European glass eels (Anguilla anguilla).

The relationships between the migratory behavior, methylmercury (MeHg) concentrations, oxidative stress response and detoxification processes were investigated in glass eels collected in marine (Molliets) and estuarine (Urt) waters (Adour estuary, South West France) at the end of the fishing season (April). Glass eel migratory behavior was investigated in an experimental flume according to their response to dusk. Fish responding to the decrease in light intensity by ascending in the water column and moving with or against the flow were considered as having a high propensity to migrate (migrant). Glass eels still sheltering at the end of the 24h catching period were considered as having a low propensity to migrate and were called non-migrant. Our results provide some evidence that estuarine glass eels were bigger, presented a higher propensity to migrate and a lower oxidative stress response than marine glass eels. This might reflect a selection process, some marine glass eels progressively settling or dying before reaching Urt and/or a change in feeding behavior. In April, glass eels restart feeding in the Adour estuary which might decrease the oxidative stress possibly related to starvation, and enhance migration. MeHg concentrations was significantly higher in non-migrant than in migrant glass eels and it is suggested that non-migrant glass eels might present a higher vulnerability to stress (at least contamination and/or starvation), although the underlying mechanisms remain to be elucidated.

Modeling of autophagy-related gene expression dynamics during long term fasting in European eel (Anguilla anguilla).

Autophagy is an evolutionary conserved cellular self-degradation process considered as a major energy mobilizing system in eukaryotes. It has long been considered as a post-translationally regulated event, and the importance of transcriptional regulation of autophagy-related genes (atg) for somatic maintenance and homeostasis during long period of stress emerged only recently. In this regard, large changes in atg transcription have been documented in several species under diverse types of prolonged catabolic situations. However, the available data primarily concern atg mRNA levels at specific times and fail to capture the dynamic relationship between transcript production over time and integrated phenotypes. Here, we present the development of a statistical model describing the dynamics of expression of several atg and lysosomal genes in European glass eel (Anguilla anguilla) during long-term fasting at two temperatures (9 °C and 12 °C) and make use of this model to infer the effect of transcripts dynamics on an integrated phenotype - here weight loss. Our analysis shows long-term non-random fluctuating atg expression dynamics and reveals for the first time a significant contribution of atg transcripts production over time to weight loss. The proposed approach thus offers a new perspective on the long-term transcriptional control of autophagy and its physiological role.

Kleptoparasitism and aggressiveness are influenced by standard metabolic rate in eels.

Kleptoparasitism refers to either interspecific or intraspecific stealing of food already procured by other species or individuals. Within a given species, individuals might differ in their propensity to use such a tactic, in a similar manner to which they differ in their general level of aggressiveness. Standard metabolic rate is often viewed as a proxy for energy requirements. For this reason, it should directly impact on both kleptoparasitism and aggressiveness when individuals have to share the same food source. In the present study we first assessed the standard metabolic rate (SMR) of 128 juvenile European eels (Anguilla anguilla) by the determination of oxygen consumption. We then tested how the SMR could influence agonistic behavior of individuals competing for food in three distinct trials evenly distributed over three months. We demonstrate that SMR positively correlates with attacks (sum of bite and push events) in all trials. Similarly SMR correlated positively with kleptoparasitism (food theft), but this was significant only for the third trial (month 3). To our knowledge, the present study is the first reporting a link between kleptoparasitism and SMR in a fish species. This has ecological implications owing to the fact that this species is characterized by an environmental sex determination linked to early growth rate. We discuss theses findings in the light of the producer-scrounger foraging game.

No early gender effects on energetic status and life history in a salmonid.

Throughout an organism's early development, variations in physiology and behaviours may have long lasting consequences on individual life histories. While a large part of variation in critical life-history transitions remains unexplained, a significant proportion may be caused by early gender effects as part of gender-specific life histories shaped by sexual selection. In this study, we investigated the presence of early gender effects on the timing of emergence from gravel and the energetic status of brown trout (Salmo trutta) early stages. To investigate this question, individual measures of emergence timing, metabolic rate and energetic content were coupled for the first time with the use of a recent genetic marker for sdY (sexually dimorphic on the Y-chromosome), a master sex-determining gene. Our results show that gender does not influence the energetic content of emerging juveniles or their emergence timing. These findings suggest that gender differences may appear later throughout salmonid life history and that selective pressures associated with the critical period of emergence from gravel may shape early life-history traits similarly in both males and females.

Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): an experimental study using isotopic tracers.

The effect of methylmercury (MeHg) on glass eels' propensity to migrate, mitochondrial activity and antioxidative defence systems was investigated. Marine glass eels were first sorted in an experimental flume according to their response to dusk. Fish responding to the decrease in light intensity by ascending in the water column and moving with or against the flow were considered as having a high propensity to migrate (migrant). Glass eels still sheltering at the end of the 24 h catching period were considered as having a low propensity to migrate and were called non-migrant. Migrant and non-migrant glass eels were then individually tagged and exposed to isotopically enriched (201)MeHg (50 ng L(-1)) for 11 days. The effect of contamination was studied on muscle fibre structure, and the expression level of genes involved in mitochondrial activity and antioxidative defence systems. To investigate the effect of MeHg on glass eel behaviour, migrant and non-migrant glass eels were sorted again and the bioaccumulation of (201)MeHg and its demethylation product ((201)Hg(II)) were determined for each individual. MeHg exposure increased activity in non-migrant glass eels but not migratory behaviour. Contamination affected mitochondrial structure and metabolism and suggests a higher oxidative stress and activation of antioxidative defence systems in non-migrant glass eels. Overall, our results suggest that exposure to MeHg might induce an increase in energy expenditure and a higher vulnerability to predation in non-migrant glass eels in the wild.

Spatial and seasonal variations of methylmercury in European glass eels (Anguilla anguilla) in the Adour estuary (France) and relation to their migratory behaviour.

Overall recruitment of European glass eels (Anguilla anguilla) has decreased significantly since the early 1980s. Due to their long life cycle, benthic/demersal habits and high lipid content, eels might accumulate high concentrations of contaminants, but data concerning glass eels are still scarce. This study provides original data on methylmercury (MeHg) concentrations in glass eels at spatial (marine and estuarine), annual and seasonal scales. The relationship between MeHg concentrations in glass eels and their propensity to migrate up estuaries was also investigated. MeHg data were individually related to the eels' energetic condition which was estimated by dry weight. Glass eel migratory behaviour was investigated in an experimental flume and related to the MeHg concentration and dry weight at the individual scale. Marine and estuarine glass eels were caught from 2004 to 2011. There was a strong inverse correlation between MeHg concentrations and dry weight. MeHg concentrations increased in marine and estuarine glass eels from 2004 to 2009 and from 2004 to 2010, respectively, and then, both groups decreased in 2011. On a seasonal time scale, MeHg concentrations were higher at the end of the fishing season (April). MeHg bioaccumulation is likely to result from different sources, but the lack of significant differences between marine and estuarine glass eels suggests that direct contamination during estuarine migration is low. Other sources such as maternal transfer or oceanic contamination are discussed.

Female effects on offspring energetic status and consequences on early development in yolk feeding brown trout (Salmo trutta).

Energetic status can be defined as the interaction between energy stores and metabolic rate. In salmonids, it is variable and influences the timing of emergence, and therefore may have strong effects on both juvenile and maternal fitness. The aim of this study is to (i) describe the ontogeny of energy use for different brown trout clutches to understand how such a variability of energetic status is developed at the end of incubation and (ii) to estimate maternal influences over offspring physiological processes. Using individual measures of total mass and metabolism throughout ontogeny combined with a hierarchical Bayesian modeling approach, we successfully described clutch-specific (i) metabolic trajectories, (ii) use of yolk resources and the building of new tissues throughout ontogeny. Our results show that females laying large eggs have offspring with lower metabolic costs and higher yolk conversion efficiencies. Females also influence within clutch variance of metabolic and yolk consumption rates leading to potential developmental variations. These results are discussed with regard to their consequences on early life history through the critical period of emergence.

Assessing maternal effects on metabolic rate dynamics along early development in brown trout (Salmo trutta): an individual-based approach.

Routine metabolic rate (oxygen consumption) of individual eggs and larvae of brown trout (Salmo trutta) originating from different families were monitored from fertilisation to the onset of emergence by means of flow through micro-respirometry. This measuring system revealed an accurate tool to measure oxygen consumption on small organisms at the individual level, and daily consumption proved to be very stable. The mass-specific metabolic rate remained low from fertilisation to hatching, and then increased quickly until the age of emergence. A Bayesian modelling approach was used to adequately infer maternal effects on metabolic rate dynamics all along the development period. Substantial differences were found between families, affecting average metabolic rate as well as intra-family variance. That is, offspring originating from different females may have different energetic needs at emergence from gravel. Moreover, between siblings, variability in metabolic rate is also under the influence of maternal effects. Implications of this metabolic rate variability are discussed with regard to life history strategies and early behaviours.

Endocrine control of Anguilla anguilla glass eel dispersal: effect of thyroid hormones on locomotor activity and rheotactic behavior.

Dispersal, one of the most important processes in population ecology, is an issue linking physiological and behavioral features. However, the endocrine control of animal dispersal remains poorly understood. Here, we tested whether and how thyroid hormones may influence dispersal in glass eels of Anguilla anguilla, by testing their influence on locomotor activity and rheotactic behavior. Glass eels were caught during their estuarine migration and treated by immersion in either a l-thyroxine (T(4)) or a thiourea (TU) solution. As measured by radioimmunoassay, T(4) and TU treatments induced, respectively, increased and decreased whole-body thyroid hormone levels relative to untreated controls. We tested a total of 960 glass eels distributed into control, and T(4) and TU treatment groups, on their swimming behavior in experimental flume tanks equipped with upstream and downstream traps that allowed us to concurrently measure both the locomotor activity and the rheotactic behavior. Compared to controls, locomotor activity significantly increased among the hyperthyroid, T(4)-treated eels, but significantly decreased among the hypothyroid, TU-treated eels. The results on rheotactic behavior suggested a more complex regulatory mechanism, since TU but not T(4) treatment significantly affected rheotactic behavior. The influence of thyroid hormones on locomotor activity suggests a central role for these hormones in the regulation of mechanisms leading to the colonization of continental habitats by glass eels. Thyroid hormones are also implicated in the control of locomotor activity in mammals and migratory behavior in birds, suggesting that these hormones represent conserved, proximate mediators of dispersal in vertebrates.