Background. Caffeine is a widespread pharmaceutically active compound that enters surface waters mainly with municipal wastewater and may alter the behavior of aquatic organisms even at sublethal concentrations. Behavioral endpoints are especially important in ecotoxicology because changes in movement, shelter use, social interactions, and aggression can affect survival, dispersal, and population-level responses before mortality occurs. Crayfish are suitable model organisms for such studies because they demonstrate distinct spatial, social, and agonistic behavior that can be recorded and quantified under laboratory conditions. The marbled crayfish Procambarus virginalis is of particular interest because it is a parthenogenetic, highly plastic, and invasive species capable of rapidly establishing new populations from aquarium releases. For invasive hydrobionts, pollutant-induced behavioral changes may influence both individual stress tolerance and the ecological success of populations in transformed freshwater ecosystems. Previous studies have shown that psychoactive pollutants can modify locomotor activity, shelter-related behavior, and risk-taking in aquatic invertebrates, including crayfish. However, the behavioral effects of caffeine on P. virginalis remain insufficiently described. Therefore, short- term laboratory exposure combined with ethogram-based video analysis can provide useful preliminary data on how this invasive crayfish responds to caffeine contamination.
Aim. The aim of this study was to evaluate the effects of caffeine exposure on the behavioral structure of the invasive marbled crayfish Procambarus virginalis and to describe the temporal dynamics of changes in locomotor, anxiety-related, shelter- related, and aggressive activity over 24 hours.
Materials and methods. The study was carried out under laboratory conditions using video recording followed by ethogram analysis in BORIS software. Marbled crayfish were kept in an open 20 L container. In the control series, baseline behavior was recorded without the addition of any substance. In the experimental series, behavior was recorded after a single addition of caffeine at a dose of 200 mg per 20 L of water, corresponding to 10 mg/L. Behavior was assessed at 0, 6, 12, and 24 h after exposure. The duration and frequency of the following behavioral categories were analyzed: Move, Rest, Swim, Shelter, Wall, Field, Aggression, Attacking pose, and Ball of crayfish. The control and experimental observations were compared to identify changes in the distribution of behavioral forms and in the temporal structure of activity.
Results. In the control group, the behavioral profile remained relatively stable throughout the observation period: Rest predominated (60‒68% of the time), locomotor activity remained moderate (15‒22%), and aggressive or anxiety-related reactions were rare. During the first 10 min after caffeine addition, crayfish demonstrated pronounced hyperactivity and stress-related orientation: Move increased to 33.5%, Wall to 12.5%, Ball of crayfish to 18.9%, and Attacking pose to 18.7%. After 6 h, the behavioral pattern changed sharply: Rest reached 86.3%, whereas Aggression increased to 25.0% and Attacking pose to 25.7%, indicating motor exhaustion combined with persistent social tension. After 12 h, locomotor activity almost disappeared (Move 0.6%), while Attacking pose remained high (24.8%), suggesting postural tension under conditions of minimal mobility. After 24 h, Rest again predominated (73.9%), Aggression decreased to 6.6%, Attacking pose decreased to 4.2%, and episodic shelter use appeared.
Conclusions. Caffeine exposure caused a phase restructuring of the behavioral pattern of P. virginalis: from acute hyperactivity and anxiety through aggressive exhaustion to a relatively stable state of low-mobility adaptation. The results indicate that the behavior of marbled crayfish may be used as a sensitive indicator of caffeine and other pharmaceutical pollutants in aquatic environments. This approach can be applied in further studies of behavioral ecotoxicology of invasive hydrobionts and in the assessment of ecological risks in transformed freshwater ecosystems.
Keywords: marbled crayfish • Procambarus virginalis • caffeine • behavioral responses • invasive species • ecotoxicology.
