Despite the lack of endogenous synthesis and relevant nuclear receptors, several papers have been published over the decades claiming that the physiology of mollusks is affected by natural and synthetic sex steroids. With scant evidence for the existence of functional steroid nuclear receptors in mollusks, some scientists have speculated that the effects of steroids might be mediated via membrane receptors (i.e. via non-genomic/non-classical actions) - a mechanism that has been well-characterized in vertebrates. However, no study has yet investigated the ligand-binding ability of such receptor candidates in mollusks. The aim of the present study was to further trace the evolution of the endocrine system by investigating the presence of functional membrane sex steroid receptors in a mollusk, the great pond snail (Lymnaea stagnalis). We detected sequences homologous to the known vertebrate membrane sex steroid receptors in the Lymnaea transcriptome and genome data: G protein-coupled estrogen receptor-1 (GPER1); membrane progestin receptors (mPRs); G protein-coupled receptor family C group 6 member A (GPRC6A); and Zrt- and Irt-like protein 9 (ZIP9). Sequence analyses, including conserved domain analysis, phylogenetics, and transmembrane domain prediction, indicated that the mPR and ZIP9 candidates appeared to be homologs, while the GPER1 and GPRC6A candidates seemed to be non-orthologous receptors. All candidates transiently transfected into HEK293MSR cells were found to be localized at the plasma membrane, confirming that they function as membrane receptors. However, the signaling assays revealed that none of the candidates interacted with the main vertebrate steroid ligands. Our findings strongly suggest that functional membrane sex steroid receptors which would be homologous to the vertebrate ones are not present in Lymnaea. Although further experiments are required on other molluscan model species as well, we propose that both classical and non-classical sex steroid signaling for endocrine responses are specific to chordates, confirming that molluscan and vertebrate endocrine systems are fundamentally different.

Microplastics (MP) are now present in all ecosystems and undergo weathering processes, including physical or chemical degradation. Although most studies have been carried out on MP toxicity in the marine ecosystem, interest is growing for the terrestrial and entire aquatic compartments. However, the interface between both environments, also known as the soil/water continuum, is given little consideration in MP toxicity studies. Only a few studies considered the toxicity of artificially aged or soil field-collected MP on species living at this interface. The present study evaluates the impact of artificial and field aging polyethylene (PE) MP on the bivalve Scrobicularia plana, a key organism of the estuarine compartment, living at the soil/water interface. Clams were exposed for 21 days to environmental concentrations (0.008, 10 and 100 μg L-1) of unaged as well as artificially and field aged PE MP. Toxicity was assessed from individual to molecular levels including condition index, clearance rate, burrowing behavior, energy reserves, enzyme activities and DNA damage. Results showed differential effects at all biological levels depending on the type and the concentration of the MP tested. Indeed, a decrease in burrowing behavior was observed in S. plana exposed to aged and field PE at low concentration (0.008 μg L-1). In the gills of clams, exposures to aged PE (0.008 and 100 μg L-1), virgin PE (10 μg L-1) and field PE (all tested concentrations) decreased CAT activity while DNA damage increased after exposure to virgin PE (0.008 μg L-1 and 10 μg L-1) and field PE (0.008 μg L-1). Our findings suggest that aging modifies the toxicity profile of PE polymer on S. plana and considering plastic from field at environmental concentrations is important when performing ecotoxicological studies.

Allatotropin (AT) has been identified in many insects and plays important roles in the regulation of their intestinal contraction, heart rate, ion transport, and digestive enzyme secretion. However, information on AT-related bioinformatics in other animal phyla is scarce. In this study, we cloned a full-length cDNA encoding the AT-related peptide receptor (ATRPR) of the abalone Haliotis discus hannai (Hdh) and further characterized Hdh-ATRPR with its potential ligands, Hdh-ATRPs. In luciferase reporter and Ca2+ mobilization assays, Hdh-ATRPs, including a D-type Phe at the second amino acid position, Hdh-D2-ATRP, activated Hdh-ATRPR in a dose-dependent manner, whereas all-L-type Hdh-ATRP was a more potent ligand than Hdh-D2-ATRP. Furthermore, Hdh-ATRPs induced ERK1/2 phosphorylation in Hdh-ATRPR-expressing HEK293 cells, which was dose-dependently abolished by the PKC inhibitor Gö6983. The heart rate decreased significantly within 10 min when Hdh-D2-ATRP was injected into the adduct muscle sinus of abalone (0.2 or 1.0 µg/g body weight), while the abalone injected with a high concentration of Hdh-D2-ATRP (1.5 μg/g body weight) were sublethal within 5 h. Thus, Hdh-ATRP signaling is primarily linked to the Gαq/PKC and is possibly associated with heart rate regulation in abalone.

The presence of the organotin compound tributyltin (TBT) in aquatic ecosystems has been a serious environmental problem for decades. Although a number of studies described the negative impact of TBT on mollusks at different levels, investigations connected to its potential effects during embryogenesis have been neglected. For a better understanding of the impact of TBT on mollusks, in the present study, embryos of previously TBT-treated or not treated specimens of the great pond snail (Lymnaea stagnalis) were exposed to 100 ng L-1 TBT from egg-laying (single-cell stage) until hatching. According to our results, TBT significantly delayed hatching and caused shell malformation. TBT transiently decreased the locomotion (gliding) and also reduced the feeding activity, demonstrating for the first time that this compound can alter the behavioral patterns of molluscan embryos. The heart rate was also significantly reduced, providing further support that cardiac activity is an excellent indicator of metal pollution in molluscan species. At the histochemical level, tin was demonstrated for the first time in TBT-treated hatchlings with intensive reaction in the central nervous system, kidney, and hepatopancreas. Overall, the most notable effects were observed in treated embryos derived from TBT treated snails. Our findings indicate that TBT has detrimental effects on the development and physiological functions of Lymnaea embryos even at a sub-lethal concentration, potentially influencing their survival and fitness. Highlighting our observations, we have demonstrated previously unknown physiological changes (altered heart rate, locomotion, and feeding activity) caused by TBT, as well as visualized tin at the histochemical level in a molluscan species for the first time following TBT exposure. Further studies are in progress to reveal the cellular and molecular mechanisms underlying the physiological and behavioral changes described in the present study.

Tissue clearing is an old-fashioned method developed in the 1900\'s and used to turn an opaque biological object into a 3D visualizable transparent structure. Developed and diversified over the last decade, this method is most of the time applied to mammals\' tissues, and especially mouse and human tissues for cytological, histological and pathophysiological studies. Through autofluorescence, immunofluorescence, in situ hybridization, intercalating agents, fluorescent transfection markers or fluorescent particle uptake, optically cleared samples can be monitored to discover new biological structures and cellular interactions through 3D-visualization, which can be more challenging in some extend through classical histological methods. Most of the tissue clearing procedures have been developed for specific applications like endogenous fluorescence visualization, immunolabeling or for revealing specific organs. Thus, choosing the adapted protocol may be empirical for non-model species, especially for mollusks for which very little related literature is available. Herein, we suggest an effective optical tissue clearing procedure for the freshwater snail Biomphalaria glabrata, known as the intermediate host of the human parasite Schistosoma mansoni. This clearing procedure involves solvents with a minimal toxicity, preserves the endogenous fluorescence of labeled parasites inside snail tissues and is compatible with an immunolabeling procedure.

Comprising about 60 % of gastropod diversity, caenogastropods display almost all kinds of shell forms and include many commercially important marine groups. Although the monophyly of Caenogastropoda has been widely accepted, thier internal phylogenetic relationships remain unclear. In the present study, a total of 27 caenogastropods belonging to eight superfamilies were sequenced and used for phylogenetic reconstruction. All newly sequenced mitogenomes adhered to the consensus gene order of caenogastropods, except for those of Vanikoroidea, Vermetoidea and Cerithioidea, which involved protein-coding genes. The reconstructed mitogenomic phylogeny suggested the monophylies of Architaenioglossa, Sorbeoconcha, Hypsogastropoda and the siphonate clade. The present study also identified a close affinity among Cypraeoidea, Ficoidea, Tonnoidea, and Neogastropoda, supported by the presence of a pleurembolic proboscis. The monophyly of Neogastropoda was not supported, as Cancellariidae was found to be sister to the limpet-shaped group Calyptraeoidea, and (Tonooidea + Ficoidea) were sister to the remaining neogastropods. This study provides important information for better understanding the evolution of caenogastropods, as well as for the protection and utilization of these diverse and economically significant marine resources.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan \"GnRH\" should be classified as \"CRZ\".

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.