Environmental modulation of methyl farnesoate release in two clones of Daphnia pulex differing in the sensitivity to exogenous stimuli

Daphnia is a widely recognised model organism in ecotoxicological studies, exhibiting environmental sex determination. Environmental cues such as temperature, photoperiod and crowding are known to trigger transitions from parthenogenetic to sexual reproduction. Experimental studies based on exogenous exposure suggest that methyl farnesoate (MF), a juvenile hormone-like compound, is associated with male production and predator-induced defensive traits in Daphnia; however, direct evidence of endogenous MF production and environmental dynamics remains limited. In this study, we investigated the release of endogenous MF into the rearing water of Daphnia pulex. Two D. pulex clonal lineages differing in propensity for male, ephippia and neckteeth production were selected. Experimental conditions simulating crowding and different habitat suitability, including variations in population density, food availability, photoperiod and temperature, were investigated. MF was quantified at ultra-trace levels using solid-phase microextraction coupled to gas chromatography-tandem mass spectrometry. In the first generation, MF concentrations decreased from approximately 18.5-20.0 ng/L in controls to 11.0-14.5 ng/L under crowding conditions and 6.7-7.2 ng/L under crowding combined with MK-801. In the second generation, concentrations were approximately 10.5 ng/L in control offspring and 12.0-12.3 ng/L after recovery from crowding. Exposure to crowding over two generations resulted in clone-specific concentrations, whereas MF remained below the limit of quantification of 0.08 ng/L, when parental females had been exposed to MK-801. In the density and seasonal experiments, MF released in the medium was higher release per female under early-spring conditions and in clone S. These variations occurred under conditions promoting male production, ephippia formation and antipredator responses. While not demonstrating a causal endocrine role of MF, our findings provide quantitative and environmentally relevant evidence that D. pulex releases MF into the aquatic environment and this release is modulated by both environmental conditions and genetic background.