Pharmacological modulation of fish-induced depth selection in D. magna: the role of cholinergic and GABAergic signalling

Animal behaviour is closely related to individual fitness, which allows animals to choose suitable mates or avoid predation. The central nervous system regulates many aspects of animal behaviour responses. Therefore, behavioural responses can be especially sensitive to compounds with a neurodevelopmental or neurofunctional mode of action. Phototactic behavioural changes against fish in the freshwater crustacean Daphnia magna have been the subject of many ecological investigations. The aim of this study was to identify which neurotransmitter systems modulate phototactic behaviour to fish kairomones. We used a positive phototactic D. magna clone (P132,85) that shows marked negative phototactism after exposure to fish kairomones. Treatments included up to 16 known agonists and antagonists of the serotonergic, cholinergic, dopaminergic, histaminergic, glutamatergic and GABAergic systems. It was hypothesized that many neurological signalling pathways may modulate D. magna phototactic behaviour to fish kairomones. A new custom-designed device with vertically oriented chambers was used, and changes in the preferred areas (bottom, middle, and upper areas) were analysed using groups of animals after 24 h of exposure to the selected substance(s). The results indicated that agonists of the muscarinic acetylcholine and GABAA receptors and their equi-effective mixture ameliorated the negative phototactic response to fish kairomones, whereas antagonists and their mixtures increased the negative phototactism to fish kairomones. Interestingly, inhibition of the muscarinic acetylcholine receptor abolished positive phototaxis, thus inducing the phototactic response to fish kairomones. Analysis of the profile of neurotransmitters and their related metabolites showed that the D. magna behavioural responses induced by fish depend on changes in the levels of acetylcholine, dopamine and GABA.

The impact of diel vertical migration on fatty acid patterns and allocation in Daphnia magna

In freshwater zooplankton diel vertical migration (DVM) is a widespread predator-avoidance behavior that is induced by kairomones released from fish. Thereby zooplankton reduces predation by fish by staying in deep and dark colder strata during daytime and migrating into warmer layers during night, and thus experiences diel alterations in temperature. Constantly lower temperatures have been shown to increase the relative abundance of polyunsaturated fatty acids (PUFAs) in Daphnia sp. Furthermore, a low dietary supply of the ω3-PUFA eicosapentaenoic acid (EPA) has been shown to limit the induction of DVM in Daphnia magna and the performance of D. magna under fluctuating temperatures, as experienced during DVM. In nature DVM of D. magna in response to fish is accompanied by the presence of fish-borne kairomone and diel fluctuations of depth dependent-parameters like temperature, food, and oxygen supply. Here we investigated the effect of factors, which are differing between Daphnia that perform DVM and those which do not. We selected to examine the effect of changing temperature and light conditions and of the presence/absence of fish kairomones on D. magna. For this purpose, we conducted a full factorial experimental design in which we grew D. magna under constantly warm temperatures in a diel light-dark regime or under alternating temperatures in darkness crossed with the presence or absence of fish kairomones. We analyzed the fatty acid composition of mature animals and of their offspring in each treatment. Simulation of the light and temperature regime of migrating animals in presence of the fish kairomone resulted in an increased relative allocation of the ω3-PUFA EPA, from adult animals to their offspring, manifesting as decreased EPA concentrations in mothers and increased EPA concentrations in their offspring in response to simulated DVM (mothers). Additionally, EPA concentrations in the offspring were affected by the interaction of simulated DVM and the fish cue. The presence of the fish kairomone alone increased the EPA concentration in the offspring, that was not experiencing simulated DVM. These findings lead to the conclusion that the temperature and light regime associated with DVM alone, as well as in combination with the DVM-inducing fish kairomones, alter the allocation of fatty acids to the offspring in a manner, which is beneficial for the offspring under the decreased average temperatures, which migrating animals are exposed to. A low dietary supply of ω3-PUFAs may constrain D. magna’s amplitude of DVM, but our results suggest that the next generation of animals may be capable of regaining the full DVM amplitude due to the effect of the fish kairomone and the experienced temperature fluctuations (and darkness) on tissue fatty acid composition. These findings suggest that fatty acid limitation in DVM performing Daphnia may be more severe for the maternal than for the offspring generation.

Predator recognition and anti-predatory behaviour in a recent aquatic invader, the killer shrimp (Dikerogammarus villosus)

The killer shrimp (Dikerogammarus villosus) is one of the most recent, but also most damaging, aquatic invasive species in Europe, but information on how the species responds to novel predation pressures in recently invaded areas is very limited. We employed an open test arena to examine predator recognition and anti-predatory behaviour in killer shrimp exposed to either blank water or water conditioned with fish kairomones to simulate a predator threat. Within five years after their introduction, killer shrimp spent much more time hiding in the presence of fish kairomones than when they were exposed to blank water. However, no significant difference was found in aggregation behaviour, and killer shrimp were strongly attracted to the scent of conspecifics regardless of predator threat. Given the strong selective pressures that fish predators can exert on native and invasive gammarids, our findings highlight the need to consider prey-predator interactions to better predict the dispersal and likely impact of killer shrimp into invaded ecosystems.

Insights into the genetic basis of predator-induced response in Daphnia galeata

Phenotypic plastic responses allow organisms to rapidly adjust when facing environmental challenges - these responses comprise morphological, behavioral but also life-history changes. Alteration of life-history traits when exposed to predation risk have been reported often in the ecological and genomic model organism Daphnia. However, the molecular basis of this response is not well understood, especially in the context of fish predation. Here, we characterized the transcriptional profiles of two Daphnia galeata clonal lines with opposed life histories when exposed to fish kairomones. First, we conducted a differential gene expression, identifying a total of 125 candidate transcripts involved in the predator-induced response, uncovering substantial intra-specific variation. Second, we applied a gene co-expression network analysis to find clusters of tightly linked transcripts revealing the functional relations of transcripts underlying the predator-induced response. Our results showed that transcripts involved in remodeling of the cuticle, growth and digestion correlated with the response to environmental change in D. galeata. Furthermore, we used an orthology-based approach to gain functional information for transcripts lacking gene ontology (GO) information, as well as insights into the evolutionary conservation of transcripts. We could show that our candidate transcripts have orthologs in other Daphnia species but almost none in other arthropods. The unique combination of methods allowed us to identify candidate transcripts, their putative functions and evolutionary history associated with predator-induced responses in Daphnia. Our study opens up to the question as to whether the same molecular signature is associated fish kairomones-mediated life-history changes in other Daphnia species.

Intraspecific phenotypic variation in life history traits ofDaphnia galeatapopulations in response to fish kairomones

Phenotypic plasticity is the ability of a genotype to produce different phenotypes depending on the environment. It has an influence on the adaptive potential to environmental change and the capability to adapt locally. Adaptation to environmental change happens at the population level, thereby contributing to genotypic and phenotypic variation within a species. Predation is an important ecological factor structuring communities and maintaining species diversity. Prey developed different strategies to reduce their vulnerability to predators by changing their behaviour, their morphology or their life history. Predator-induced life history responses inDaphniahave been investigated for decades, but intra-and inter-population variability was rarely addressed explicitly. We addressed this issue by conducting a common garden experiment with 24 clonal lines of EuropeanDaphnia galeataoriginating from four populations, each represented by six clonal lines. We recorded life history traits in the absence and presence of fish kairomones. Additionally, we looked at the shape of experimental individuals by conducting a geometric morphometric analysis, thus assessing predator-induced morphometric changes. Our data revealed high intraspecific phenotypic variation within and between fourD. galeatapopulations, the potential to locally adapt to a vertebrate predator regime as well as an effect of the fish kairomones on morphology ofD. galeata.