Aggression repeatability in stressed fish in response to an environmental concentration of sertraline and lunar cycle as evidenced by brain metabolomics.

Sertraline is an environmental pollutant which received magnified scientific attention due to its global presence in waters. Adverse effects on feeding, reproduction and other traits were observed mostly in unstressed aquatic organisms. Chronic stress, however, induces significant physiological changes, and the effects of sertraline in stressed fish may differ from those observed in non-stressed individuals. The current laboratory study addresses this gap by repeatedly monitoring the individual aggression of chronically stressed juvenile chub (Squalius cephalus L.) using the non-reversing mirror test at an environmental sertraline concentration of 0.022 g/L every three to four days for a period of 39 days. Specifically, it was hypothesized that the level and repeatability of aggressiveness would be (i) correlated with the concentration of sertraline/norsertraline in the fish brain; (ii) linked to the individual brain metabolomic profile described by LC-HRMS analyses; (iii) related to the lunar cycle. Sertraline led to an increase in fish aggression and more repeatable/consistent behaviour compared to control fish. While the level of sertraline in the brain did not correlate with aggressiveness, aggressive responses increased with higher norsertraline concentration. The observed aggressive behaviour also varied depending on the individual metabolomic profile of the brain. The behavioural outcome and metabolic change in fish brain may indicate that sertraline has demonstrated neuroprotective effects by reducing cortisol release. It is possible that fish exposed to sertraline could suffer a blunted stress response under the chronic stressors in the wild. Aggressiveness of both treatments evolved in time, revealing a sinusoid-like pattern corresponding to a lunar cycle with a peak of the aggressiveness during the new moon. There is a need for future studies to focus on this relationship to reveal its details and general validity. Our results emphasize that long-term behavioural variability should generally be taken into account in laboratory behavioural studies.

From metabolism to behaviour - Multilevel effects of environmental methamphetamine concentrations on fish.

Methamphetamine (METH) is a concerning drug of abuse that produces strong psychostimulant effects. The use of this substance, along with the insufficient removal in the sewage treatment plants, leads to its occurrence in the environment at low concentrations. In this study, brown trout (Salmo trutta fario) were exposed to 1 µg/L of METH as environmental relevant concentration for 28 days in order to elucidate the complex effects resulting from the drug, including behaviour, energetics, brain and gonad histology, brain metabolomics, and their relations. Trout exposed to METH displayed lowered activity as well as metabolic rate (MR), an altered morphology of brain and gonads as well as changes in brain metabolome when compared to controls. Increased activity and MR were correlated to an increased incidence of histopathology in gonads (females - vascular fluid and gonad staging; males - apoptotic spermatozoa and peritubular cells) in exposed trout compared to controls. Higher amounts of melatonin in brain were detected in exposed fish compared to controls. Tyrosine hydroxylase expression in locus coeruleus was related to the MR in exposed fish, but not in the control. Brain metabolomics indicated significant differences in 115 brain signals between control and METH exposed individuals, described by the coordinates within the principal component analyses (PCA) axes. These coordinates were subsequently used as indicators of a direct link between brain metabolomics, physiology, and behaviour - as activity and MR varied according to their values. Exposed fish showed an increased MR correlated with the metabolite position in PC1 axes, whereas the control had proportionately lower MR and PC1 coordinates. Our findings emphasize the possible complex disturbances in aquatic fauna on multiple interconnected levels (metabolism, physiology, behaviour) as a result of the presence of METH in aquatic environments. Thus, these outcomes can be useful in the development of AOP's (Adverse Outcome Pathways).

Fish self-awareness: limits of current knowledge and theoretical expectations.

Animal self-awareness is divided into three levels: bodily, social, and introspective self-awareness. Research has focused mainly on the introspection of so-called higher organisms such as mammals. Herein, we turn our attention to fish and provide opinions on their self-awareness based on a review of the scientific literature. Our specific aims are to discuss whether fish (A) could have a neural substrate supporting self-awareness and whether they display signs of (B) social and (C) introspective self-awareness. The present knowledge does not exclude the possibility that fish could have a simple neocortex or other structures that support certain higher cognitive processes, as the function of the primate cerebral cortex can be replaced by other neurological structures. Fish are known to display winner, loser, and audience effects, which could be interpreted as signs of social self-awareness. The audience effect may be explained not only by ethological cost and benefit theory but also by the concept of public self-awareness, which comes from human studies. The behavioural and neural manifestations of depression may be induced in fish under social subordination and may be viewed as certain awareness of a social status. The current findings on fish introspective self-awareness have been debated in the scientific community and, therefore, demand replication to provide more evidence. Further research is needed to verify the outlined ideas; however, the current knowledge indicates that fish are capable of certain higher cognitive processes, which raises questions and implications regarding ethics and welfare in fish-related research and husbandry.

Prescribed aggression of fishes: Pharmaceuticals modify aggression in environmentally relevant concentrations.

Traces of psychoactive substances have been found in freshwaters globally. Fish are chronically exposed to pollution at low concentrations. The changes of aggressive behaviour of chub (Squalius cephalus) were determined under the exposure to four psychoactive compounds (sertraline, citalopram, tramadol, methamphetamine) at environmentally relevant concentrations of 1 µg/L for 42 days. We tested whether (A) the behavioural effect of compounds varies within a single species; (B) there is a correlation between the individual brain concentration of the tested pollutants and fish aggression using the novel analysis of pollutants in brain; and (C) there is detectable threshold to effective pollutant concentration in brain. Behaviour and pollutant concentrations in brain were determined repeatedly (1st, 7th, 21st, 42nd and 56th days), including a two-week-long depuration period. The effect of particular compounds varied. Citalopram and methamphetamine generally increased the fish aggression, while no such effect was found after exposure to tramadol or sertraline. The longitudinal analysis showed an aggression increase after depuration, indicating the presence of withdrawal effects in methamphetamine- and tramadol-exposed fish. The analysis of pollutant concentration in brain revealed a positive linear relationship of citalopram concentration and aggression, while no such effect was detected for other compounds and/or their metabolites. Structural break analyses detected concentration thresholds of citalopram (1 and 3 ng/g) and sertraline (1000 ng/g) in brain tissue, from which a significant effect on behaviour was manifested. While the effect of sertraline was not detected using traditional approaches, there was a reduction in aggression after considering its threshold concentration in the brain. Our results suggest that pursuing the concentration threshold of psychoactive compounds can help to reduce false negative results and provide more realistic predictions on behavioural outcomes in freshwater environments, especially in the case of compounds with bioaccumulation potential such as sertraline.

Methamphetamine pollution elicits addiction in wild fish.

Illicit drug abuse presents pervasive adverse consequences for human societies around the world. Illicit drug consumption also plays an unexpected role in contamination of aquatic ecosystems that receive wastewater discharges. Here, we show that methamphetamine, considered as one of the most important global health threats, causes addiction and behavior alteration of brown trout Salmo trutta at environmentally relevant concentrations (1 µg l-1). Altered movement behavior and preference for methamphetamine during withdrawal were linked to drug residues in fish brain tissues and accompanied by brain metabolome changes. Our results suggest that emission of illicit drugs into freshwater ecosystems causes addiction in fish and modifies habitat preferences with unexpected adverse consequences of relevance at the individual and population levels. As such, our study identifies transmission of human societal problems to aquatic ecosystems.

Traces of tramadol in water impact behaviour in a native European fish.

Tramadol is a widely used analgesic with additional antidepressant and anxiolytic effects. This compound has been reported in continental waters reaching concentrations of µg/L as a consequence of its inefficient removal in sewage treatment plants and increasing use over time. In this study, European chubs (Squalius cephalus) were exposed to 1 µg/L of tramadol in water for 42 days with a subsequent 14 days of depuration. Our results revealed that chubs exposed to this analgesic underwent changes in their behaviour as compared to the control group. The behavioural outcome was also influenced by the individual concentration of tramadol in brain tissue. In particular, experimental fish presented anxiolytic-like effects, characterized by less bold and less social individuals. Exposed animals were less frequently out of the shelter and moved a shorter distance, indicating that they explored the new environment less during the boldness test. In the novel object recognition experiment, although they distinguished the new item, they examined it less and displayed a reduced activity. Shoal cohesion was disrupted as observed in an increased distance between individuals. After the depuration phase, this alteration remained whereas the boldness effect disappeared. Moreover, the degree of behavioural changes was correlated with the concentration of the substance in brain. According to our findings, chronic presence of tramadol in the environment can impact the fitness of exposed aquatic fauna by altering evolutionary crucial behaviours.

Environmentally relevant levels of four psychoactive compounds vary in their effects on freshwater fish condition: a brain concentration evidence approach.

BACKGROUND: The aquatic environment has been contaminated with various anthropogenic pollutants, including psychoactive compounds that may alter the physiology and behavior of free-living organisms. The present study focused on the condition and related mortality of the juvenile chub (Squalius cephalus). The aim of the study was to test whether the adverse effects of the antidepressants sertraline and citalopram, the analgesic tramadol and the illicit drug methamphetamine, on fish condition exist under environmentally relevant concentrations and whether these effects persist after a depuration period. Innovative analyses of the fish brain concentrations of these compounds were performed with the aim to show relationship between compound brain tissue concentration and fish condition. METHODS: The laboratory experiment consisted of 42 days of exposure and a subsequent 14-day depuration period with regular monitoring of the condition and mortality of exposed and control fish. Identical methodology, including individual brain concentration analyses for the tested compounds, was applied for all substances. Additional study on feeding under sertraline exposure was also conducted. The feeding was measured from the 28th day of the exposure, three times in a week, by observation of food intake during 15 minutes in social environment. RESULTS: The effects of particular psychoactive compounds on chub condition varied. While sertraline induced a lower condition and increased mortality, the effects of methamphetamine were inverse, and tramadol and citalopram had no significant effect at all. Individual brain concentrations of the tested compounds showed that the effects of sertraline and methamphetamine on fish condition were increased with brain concentration increases. Additionally, the food intake was reduced in case of sertraline. In contrast, there was no relationship between tramadol and citalopram brain tissue concentration and fish condition, suggesting that the concentration-dependent effect is strongly compound-specific. Methamphetamine was the only compound with a persistent effect after the depuration period. Our results demonstrate the suitability of the brain concentration evidence approach and suggest that changes in fish condition and other related parameters can be expected in freshwater ecosystems polluted with specific psychoactive compounds.

Test-dependent expression of behavioral syndromes: A study of aggressiveness, activity, and stress of chub.

Aggressiveness has been one of the behavioral traits most examined with various standard testing methods. We used two distinct methods (the mirror and the real opponent tests) to evaluate individual aggression and relate it to the activity and individual stress of chub (Squalius cephalus L.). Three hypotheses were formulated and tested: (a) there is a significant positive relationship between the aggressiveness of individuals measured with the mirror and the real opponent tests, indicating their convergent validity; (b) the irregularities in response to the aggressiveness and activity tests lead to the context-specific expression of the behavioral syndromes; and (c) there is a significant positive relationship between the stress induced in individuals by both tests of aggressiveness, demonstrating individually consistent stress-coping strategies. The first and the second hypothesis were confirmed, while the third hypothesis was rejected. Our results suggest that particular tests of aggressiveness could act as a situation with high strength, leaving little variation between individual responses. Thus, we propose that for the proper interpretation of various studies using different tests to study identical behavioral traits, it is important to consider the convergent validity of not only the tested behavioral traits but also the individual stress responses. The chub also showed stress relieve through aggressiveness, suggesting the species as a prospective animal model to the study interaction between the stress and the aggressiveness. A detailed aggression ethogram of chub was provided to facilitate the use of this specie in future studies.