Today, freshwaters, such as lakes and rivers, are subject to controlled pollution. Steroid hormones are chemically very stable highly lipophilic molecules. Their biological properties have a strong impact on the endocrine regulation of species. Steroids have estrogenic, androgenic, thyroidogenic or progestogenic effects and based on them, they could disturb the physiological mechanisms of freshwater species. We focused on progestins as they are the main active ingredients of contraceptive pharmaceuticals. Progestins have been shown to impair reproduction in fish, amphibians, and mollusks at low ng/L concentrations. Certain progestins, such as levonorgestrel (LNG) have androgenic properties also. We selected the most used active substances drospirenone (DRO), LNG, and progesterone (PRG) and then developed and optimized a liquid chromatographic-mass spectrometric method with solid-phase extraction to measure them. Using our sensitive method (LOQ 0.03-0.11 ng/L) we could measure steroids even between 0.1 and 1 ng/L. Analyzing freshwater samples from the Lake Balaton catchment area, we found influents where the concentration of these hormones was 0.26-4.30 (DRO), 0.85-3.40 (LNG), and 0.23-13.67 (PRG) ng/L. Out of 53 collecting places, 21 contained measurable progestin levels, which clearly demonstrates the applicability of our method, legitimates toxicology experiments with effected species, and indicates monitoring efforts.

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In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.