Molecular basis for the effects of SSRIs in non-target aquatic invertebrates: A case study with Mytilus galloprovincialis early larvae.

Selective Serotonin Reuptake Inhibitors (SSRIs) are among the most prescribed antidepressants, whose increasing consumption results in a continuous discharge into aquatic compartments, where they are detected at ng-µg/L levels. Whilst designed to modulate endogenous levels of circulating Serotonin (5-HT) in humans by selectively interfering with serotonin reuptake transporters (SERTs), SSRIs have been shown to induce a variety of adverse effects in non-target species, including aquatic invertebrates. In bivalve molluscs, adult exposure to environmental concentrations of SSRIs results in tissue bioaccumulation and induces different biomarker responses. However, the effects were not related to the mechanisms of action of SSRIs, due to poor knowledge of their direct molecular targets, SERT in particular. Much less information is available in embryo-larval stages. In this work, the effects of different SSRIs (Fluoxetine, Citalopram, Sertraline, 1-100 µg/L) were compared in the model of Mytilus galloprovincialis embryo-larval development. SSRIs showed small or no effects on normal larval development at 48 h post fertilization (hpf). The possible direct or indirect molecular targets of SSRIs were thus investigated in mussel larvae. Two conserved SERT sequences, SERT1-like and SERT2-like, were identified in M. galloprovincialis genome: their developmental expression showed increased transcription only from 44 and 20 hpf, respectively. A much higher and earlier expression (from 12 hpf) was observed for TPH (Tryptophan Hydroxylase), the rate limiting enzyme in 5-HT synthesis. Double in situ Hybridization Chain Reaction (HCR) showed partial colocalisation of TPH with SERT1-like and SERT2-like transcripts in 48 hpf larvae. At this stage, SSRIs induced a small but significant decrease in the number of TPH-positive cells. Finally, 19 Nose Resistance to Fluoxetine (nrf) sequences were identified, that were highly expressed across all early stages (0-48 hpf). At 48 hpf, nrf expression was associated with the digestive system. The results represent the first data on the establishment of the serotonergic system in mussel early larvae, representing the molecular basis for understanding the effects of SSRIs and their mechanisms of action in model non-target marine invertebrates.