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.

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.

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.

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.

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.

Altered thermoregulation as a driver of host behaviour in glochidia-parasitised fish.

Parasites alter their host behaviour and vice versa as a result of mutual adaptations in the evolutionary arms race. One of these adaptations involves changes in host thermoregulation, which has the potential to harm the parasite and thereby act as a defence mechanism. We used a model of the brown trout (Salmo trutta) experimentally parasitised with glochidia ectoparasitic larvae from the endangered freshwater pearl mussel (Margaritifera margaritifera) to reveal whether parasitisation alters fish behavioural thermoregulation. A study using radiotelemetry temperature sensors was performed during almost one year of the M. margaritifera parasitic stage. Glochidia-infested S. trutta altered their thermoregulation through active searching for habitats with different thermal regimes. The general preference for temperatures in infested fish varied and was either above or below the temperature preferred by uninfested individuals. Infested fish also preferred different temperatures across localities, whereas uninfested fish maintained their thermal preference no matter which stream they inhabited. Glochidia further induced the expression of a behavioural syndrome among S. trutta personality traits, suggesting that it might increase the probability that the fish host would occur in the glochidia temperature optimum. Our findings present the first evidence that thermoregulation plays a fundamental role in the relationship of affiliated mussels and their fish hosts. Incorporating thermoregulation as a factor in the study of this relationship can help to interpret results from previous behavioural studies, as well as to optimise management measures related to endangered mussels.

Improved river continuity facilitates fishes' abilities to track future environmental changes.

Barriers represent one of the largest anthropogenic impacts on the ecological status of rivers, and they also potentially restrict fishes' ability to respond to future environmental changes. Thus, river management aims to restore the longitudinal connectivity of rivers to allow continuous migration and movement of water, sediments and biota. However, it is often unclear whether the targeted barriers are also those most relevant for fish species, particularly to track future habitat shifts caused by environmental change. In this study, we applied species distribution models and the GIS-based fish dispersal model FIDIMO to evaluate the impacts of barriers (e.g. weirs and dams) on the dispersal of 17 native fish species in the European River Elbe with a particular focus on climate- and land use-induced habitat shifts. Specifically, we compared three scenarios of longitudinal connectivity: (i) current longitudinal connectivity, (ii) connectivity improvements as planned by river managers for 2021 and (iii) a reference with full longitudinal connectivity. The models indicated that barriers restricted the movement of two modeled fish species on average, thus impeding fishes' abilities to track future habitat shifts. Moreover, the number of species affected by barriers increased downstream. For the River Elbe, our results suggest that river management has most likely identified the most relevant barriers in respect to the modeled species and future environmental change. We emphasize that river management and barrier prioritization must thoroughly consider species-specific movement and dispersal abilities, as well as the specific spatial arrangement of barriers in the river system in relation to the spatial distribution of species' populations and suitable habitats.

The future distribution of river fish: The complex interplay of climate and land use changes, species dispersal and movement barriers.

The future distribution of river fishes will be jointly affected by climate and land use changes forcing species to move in space. However, little is known whether fish species will be able to keep pace with predicted climate and land use-driven habitat shifts, in particular in fragmented river networks. In this study, we coupled species distribution models (stepwise boosted regression trees) of 17 fish species with species-specific models of their dispersal (fish dispersal model FIDIMO) in the European River Elbe catchment. We quantified (i) the extent and direction (up- vs. downstream) of predicted habitat shifts under coupled "moderate" and "severe" climate and land use change scenarios for 2050, and (ii) the dispersal abilities of fishes to track predicted habitat shifts while explicitly considering movement barriers (e.g., weirs, dams). Our results revealed median net losses of suitable habitats of 24 and 94 river kilometers per species for the moderate and severe future scenarios, respectively. Predicted habitat gains and losses and the direction of habitat shifts were highly variable among species. Habitat gains were negatively related to fish body size, i.e., suitable habitats were projected to expand for smaller-bodied fishes and to contract for larger-bodied fishes. Moreover, habitats of lowland fish species were predicted to shift downstream, whereas those of headwater species showed upstream shifts. The dispersal model indicated that suitable habitats are likely to shift faster than species might disperse. In particular, smaller-bodied fish (<200 mm) seem most vulnerable and least able to track future environmental change as their habitat shifted most and they are typically weaker dispersers. Furthermore, fishes and particularly larger-bodied species might substantially be restricted by movement barriers to respond to predicted climate and land use changes, while smaller-bodied species are rather restricted by their specific dispersal ability.

Synergistic and antagonistic interactions of future land use and climate change on river fish assemblages.

River ecosystems are threatened by future changes in land use and climatic conditions. However, little is known of the influence of interactions of these two dominant global drivers of change on ecosystems. Does the interaction amplify (synergistic interaction) or buffer (antagonistic interaction) the impacts and does their interaction effect differ in magnitude, direction and spatial extent compared to single independent pressures. In this study, we model the impact of single and interacting effects of land use and climate change on the spatial distribution of 33 fish species in the Elbe River. The varying effects were modeled using step-wise boosted regression trees based on 250 m raster grid cells. Species-specific models were built for both 'moderate' and 'extreme' future land use and climate change scenarios to assess synergistic, additive and antagonistic interaction effects on species losses, species gains and diversity indices and to quantify their spatial distribution within the Elbe River network. Our results revealed species richness is predicted to increase by 0.7-2.9 species by 2050 across the entire river network. Changes in species richness are likely to be spatially variable with significant changes predicted for 56-85% of the river network. Antagonistic interactions would dominate species losses and gains in up to 75% of the river network. In contrast, synergistic and additive effects would occur in only 20% and 16% of the river network, respectively. The magnitude of the interaction was negatively correlated with the magnitudes of the single independent effects of land use and climate change. Evidence is provided to show that future land use and climate change effects are highly interactive resulting in species range shifts that would be spatially variable in size and characteristic. These findings emphasize the importance of adaptive river management and the design of spatially connected conservation areas to compensate for these high species turnovers and range shifts.

The effect of parasitism on boldness and sheltering behaviour in albino and pigmented European catfish (Silurus glanis).

Parasites can change the behaviour of their hosts, but little attention has been given to the relationship between parasite effects on host behaviour and colouration. The correlation between disrupted melanin production and alterations in various physiological and behavioural traits, e.g., aggression, shoaling behaviour, stress responsiveness and sensitivity to brood parasitism, has been reported in albino fish. We hypothesized that parasitism would affect the behaviour of albino and pigmented conspecifics differently. In laboratory conditions, we infested a group of pigmented and a group of albino individuals of European catfish Silurus glanis with glochidia of two Uninoidea species, namely, the native species Anodonta anatina and the invasive species Sinanodonta woodiana, and investigated the effect of parasitization on the boldness and sheltering behaviour of the hosts. The behaviour of albino individuals differed from that of pigmented conspecifics both before and after parasitization. Parasitization with glochidia did not affect sheltering behaviour, but it increased boldness in pigmented individuals, whereas albino individuals did not exhibit any changes in behaviour. Sheltering results were consistent in both binomial and continuous variable analyses, whereas boldness was significant only in the binomial analyses. Our results demonstrate the reduced susceptibility of the albino phenotype to glochidia infestation, together with questions of the choice of analyses.

Differential effects of antidepressant sertraline in glochidia-fish interactions involving drug transfer from parasite to host.

This study examined the impact of sertraline, an antidepressant common in treated wastewater, on the host-parasite dynamics between parasitic freshwater mussel (Unio tumidus, Unionidae) larvae (glochidia) and their host fish (Squalius cephalus, Cyprinidae). Employing a full-factorial design, both fish and glochidia were subjected to sertraline at the combinations of 0 µg L-1 (control), 0.2 µg L-1 (environmentally relevant concentration), and 4 µg L-1 (elevated concentration, short-term exposure of the parasite). The results showed that long-term host exposure (involving intensive sertraline accumulation in the fish brain) marginally increased subsequent glochidia attachment success by 2 %, while parasite exposure at the same environmentally relevant concentrations had no detectable effect. There was also no effect of exposure of glochidia to 0.2 µg L-1 of sertraline on their viability and encapsulation success during the initial parasitic stage. However, a significant alteration in attachment behavior, marked by a 3.3 % increase in attachment success and changes in the glochidia spatial distribution on the host body, was noted after 24 h of glochidia exposure to 4 µg L-1 of sertraline. Importantly, this study provides the first evidence of sertraline transfer from exposed glochidia to nonexposed host fish, as indicated by elevated levels of sertraline (12.8 ng g-1) in the brain tissue of nonexposed hosts. These findings highlight the subtle yet significant effects of pharmaceutical pollutants on freshwater ecosystems but also underscore the importance of understanding the unexpected dynamics of such contamination to predict and address future ecological changes.

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).

Foreign stingers: South American freshwater river stingrays Potamotrygon spp. established in Indonesia.

The pet trade is known to be one of the most important pathways of aquatic non-native species introduction and Indonesia is a significant trade partner. Popular ornamental South American river stingrays (Potamotrygon spp.) were introduced to Indonesia in the 1980s and the culture was established. Here we present a detailed Indonesian market and aquaculture survey, the volume of trade between January 2020 and June 2022, and the list of customer countries with the total amount of imported stingrays. Climate similarities between the native range of P. motoro and P. jabuti and Indonesia were analysed. A significant number of areas of Indonesian islands were identified as suitable for the establishment of this species. This was confirmed by the first record of probably established populations in the Brantas River (Java). In total 13 individuals including newborns were captured. The culture of potamotrygonid stingrays is unregulated in Indonesia, and the risk of the establishment of this predator and its potential spread is alarming for wildlife. Moreover, the first case of envenomation caused by Potamotrygon spp. in the wild outside of South America was recorded. The current condition is predicted as the 'tip of the iceberg' and continuous monitoring and mitigation of risks are strongly recommended.

Comparative study of stress responses, laterality and familiarity recognition between albino and pigmented fish.

Oculocutaneous albinism is the result of a combination of homozygous recessive mutations that block the synthesis of the tyrosine and melatonin hormones. This disability is associated with physiological limitations, e.g., visual impairment expressed by lower visual acuity and movement perception, and eventually leads to acrophobia and/or photophobia, suggesting a potentially higher stress level associated with the behavioral responses of individuals with albinism to external stimuli compared to their pigmented conspecifics. However, in fish, differences in behavioral and/or physiological responses and stress levels between these phenotypes have been poorly documented. While acoustic perception of albino individuals is well known, the use of olfactory sensors for social communication, e.g., for the preference for familiar conspecifics, remains poorly understood. We performed two laboratory experiments with albino and pigmented European catfish Silurus glanis to observe: i) their behavioral and physiological responses to short-term stress induced by a combination of air exposure and novel environmental stressors and ii) their ability to use odor keys to recognize of familiar conspecifics and the influence of lateralization on this preference. In response to stress stimuli, albino fish showed higher movement activities and ventilatory frequencies and more often changed their swimming directions compared to their pigmented conspecifics. Blood plasma analysis showed significantly higher values of stress-, deprivation-, and emotional arousal-associated substances, e.g., glucose and lactate, as well as of substances released during intensive muscle activity of hyperventilation and tissue hypoxia, e.g., hemoglobin, mean corpuscular hemoglobin, erythrocytes, and neutrophil granulocytes. A preference test between environments with and without scented water showed the preference by both albino and pigmented catfish for environments with scent of familiar conspecifics, and both groups of fish displayed left-side lateralization associated with the observation of conspecifics and group coordination. The results tended to show higher physiological and behavioral responses of albinos to stress stimuli compared to the responses of their pigmented conspecifics, but the uses of olfactory sensors and lateralization were not differentiated between the two groups.

Effects of physical parameters on fish migration between a reservoir and its tributaries.

Reservoirs interrupt natural riverine continuity, reduce the overall diversity of the environment, and enhance the spread of non-native fish species through suitable environments. Under favourable conditions, invasive species migrate to tributaries to benefit from local resource supplies. However, the changes in physical conditions in reservoirs that motivate fish species to migrate remain poorly understood. We analysed migration between a reservoir and its tributary in three non-native (asp Leuciscus aspius, ide Leuciscus idus, and bream Abramis brama) and two native (chub Squalius cephalus and pike Esox lucius) species equipped with radio tags. This 5-year study revealed that an increasing day length was the most general predictor of migration into the tributary in all observed species except E. lucius. Only L. aspius responded to the substantially increasing water level in the reservoir, while the migration of L. idus and S. cephalus was attenuated. Abramis brama and S. cephalus occurred more frequently in tributaries with an increase in temperature in the reservoir and vice versa, but if the difference in temperature between the reservoir and its tributary was small, then A. brama did not migrate. Our results showed that migration from the reservoir mainly followed the alterations of daylight, while responses to other parameters were species specific. The interindividual heterogeneity within the species was significant and was not caused by differences in length or sex. Our results contribute to the knowledge of how reservoirs can affect the spread of non-native species that adapt to rapid human-induced environmental changes.

Spatial distribution of native fish species in tributaries is altered by the dispersal of non-native species from reservoirs.

Reservoirs are known to alter temperature and flow regimes, shift nutrient cycles, reduce downstream species diversity and enable a predominantly upstream spread of non-native species. However, information about the seasonal dynamics of the spread of non-natives from a reservoir to its tributaries and the further consequences regarding the spatial distribution of native species is rare. We observed the occurrence of fish in the Vltava River and its tributaries (Elbe catchment area, central Europe) upstream of the Lipno Reservoir for five consecutive years. We radio-tagged two non-native and four native species. To detect assemblage spatial variability, we sampled sites in the study area by electrofishing twice per year (spring and autumn). We expected seasonal trends in non-native species appearance in upstream reservoir tributaries and, conversely, low motivation of native fishes to descend to the reservoir. By analysing nearly 3000 individuals of 21 species from the longitudinal profile of the study area, we observed an effect of reservoir distance on the native species ratio in the upper Vltava catchment area, i.e., an increase in distance increased the native species proportion, and the opposite was observed for non-native species. Analyses of 3798 tracking positions of 193 tagged individuals showed massive spring dispersal of non-native species from the reservoir to the main tributary, the Vltava River, and their return to the reservoir for wintering. Their upstream movement positively correlated with an increase in flow rate. Native Salmo trutta showed a specific shift from the Vltava River to smaller streams during the summer, when the presence of non-native species in the Vltava River was most significant. These findings indicate that non-native species repeatedly spread from the reservoir to the upstream river stretch and its tributaries and potentially compete with native species for resources.

Crack Propagation Analysis of Compression Loaded Rolling Elements.

The problem of crack propagation from internal defects in thermoplastic cylindrical bearing elements is addressed in this paper. The crack propagation in these elements takes place under mixed-mode conditions-i.e., all three possible loading modes (tensile opening mode I and shear opening modes II and III) of the crack are combined together. Moreover, their mutual relation changes during the rotation of the element. The dependency of the stress intensity factors on the crack length was described by general parametric equations. The model was then modified by adding a void to simulate the presence of a manufacturing defect. It was found that the influence of the void on the stress intensity factor values is quite high, but it fades with crack propagating further from the void. The effect of the friction between the crack faces was find negligible on stress intensity factor values. The results presented in this paper can be directly used for the calculation of bearing elements lifetime without complicated finite element simulations.

Pupil size variation as a response to stress in European catfish and its application for social stress detection in albino conspecifics.

Hormonal changes such as increased cortisol level in blood plasma in response to stress and social environmental stimuli are common among vertebrates including humans and typically accompanied by other physiological processes, such as changes in body pigmentation and/or pupil dilatation. The role of pupil size variation (PSV) as a response to stress have yet to be investigated in fish. We exposed albino and pigmented European catfish to short-term stress and measured changes in pupil size and cortisol level. Albinos showed lower pupil dilatation and higher cortisol levels than did pigmented conspecifics. A clear positive relationship between pupil dilatation and cortisol concentrations was observed for both pigmented and albino specimens, suggesting that PSV can be used as a stress indicator in fish, irrespective of albino's inability to express social communication by coloring. During the follow-up, we investigated whether a penultimate contest between albino individuals would impact contestants' social stress during subsequent contact. We observed PSV during the contact of unfamiliar albino catfish with different penultimate experiences (winner (W) and/or loser (L)). Then, the following treatment combinations were tested: WW, WL and LL. Twenty-four-hour contact of two unfamiliar catfish resulted in higher pupil dilatation among individuals with previous winner experience. Among treatment combinations, a WL contest displayed the highest pupil dilatation for winners. PSV reflected socially induced stress in individuals that was accompanied by the "winner" experience and dominancy in albinos. To conclude, the present study validates pupil dilatation as a non-invasive method to evaluate stress level in pigmented as well as albino fish in various contexts.

In situ and low-cost monitoring of particles falling from freshwater animals: from microplastics to parasites.

A simple and low-cost method of monitoring and collecting particulate matter detaching from (or interacting with) aquatic animals is described using a novel device based on an airlift pump principle applied to floating cages. The efficiency of the technique in particle collection is demonstrated using polyethylene microspheres interacting with a cyprinid fish (Carassius carassius) and a temporarily parasitic stage (glochidia) of an endangered freshwater mussel (Margaritifera margaritifera) dropping from experimentally infested host fish (Salmo trutta). The technique enables the monitoring of temporal dynamics of particle detachment and their continuous collection both in the laboratory and in situ, allowing the experimental animals to be kept under natural water quality regimes and reducing the need for handling and transport. The technique can improve the representativeness of current experimental methods used in the fields of environmental parasitology, animal feeding ecology and microplastic pathway studies in aquatic environments. In particular, it makes it accessible to study the physiological compatibility of glochidia and their hosts, which is an essential but understudied autecological feature in mussel conservation programs worldwide. Field placement of the technique can also aid in outreach programs with pay-offs in the increase of scientific literacy of citizens concerning neglected issues such as the importance of fish hosts for the conservation of freshwater mussels.