Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans

Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans Réka Svigruha, Luis Alfonso Yañez-Guerra, Víctor Hugo Caña-Bozada, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8193722/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Ecdysone-controlled pathways regulate ecdysozoan development through both a nuclear (nEcR) and a membrane receptor (mEcR). Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids. Nevertheless, there is still no clear consensus regarding the presence or functional role of ecdysone-like signaling in mollusks. This study aimed to advance our understanding of the evolution of ecdysone signaling by investigating the potential synthesis and physiological role of 20-hydroxyecdysone (20HE) in the great pond snail ( Lymnaea stagnalis ) by applying a complex experimental approach. Exposure to different concentrations (10 ng/L, 100 ng/L, and 1 µg/L) of 20HE had no effect on hatching, heart rate, locomotion (gliding), or feeding in Lymnaea embryos. Furthermore, the treatments did not alter the expression of Lymnaea homologs of Drosophila nEcR and mEcR. Using cluster analysis and phylogenetics to resolve the evolution of Halloween genes involved in ecdysone synthesis, we found no clear homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species, while Phantom and Shade appear to have a many to many homologues in lophotrochozoans. Although mass spectrometric analysis detected trace concentrations of 20HE in various tissues of adult Lymnaea specimens, we suggest that its presence is of dietary origin. Our findings clearly indicate that an endogenous ecdysone synthesis pathway is not present in Lymnaea and that 20HE has no effect on the physiology of Lymnaea embryos. Although further experiments are required on other molluscan models as well, we propose that molluscan sequences homologous to Drosophila nEcR and mEcR are unlikely to function as ecdysone receptors, and that ecdysone-like signaling is absent in lophotrochozoans. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Evolution Biological sciences/Molecular biology Biological sciences/Zoology ecdysone mollusk Lymnaea stagnalis embryogenesis synthesis receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Ecdysone signaling is a central endocrine pathway regulating development (e.g., molting, metamorphosis) and reproduction in ecdysozoans. The discovery of ecdysone and its active metabolite, 20-hydroxyecdysone (20E), in insects provided the first evidence of steroid hormones directing major developmental transitions in invertebrates 1 . Subsequent molecular studies in Drosophila identified the Halloween genes, which encode cytochrome P450 enzymes responsible for converting dietary cholesterol into ecdysteroids, revealing the canonical ecdysone synthesis pathway (reviewed by 2 ). Ecdysone and 20E primarily act via the ecdysone receptor (nEcR) and its partner ultraspiracle (USP/RXR), a heterodimeric nuclear receptor complex that activates early-response genes 3 . Moreover, ecdysteroids can also induce rapid, non-genomic effects via a GPCR (mEcR), called the dopamine/ecdysteroid receptor in Drosophila 4 . Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids 5-10 . Previous studies also demonstrated the upregulation of the nEcR homolog, as well as the increase of serum concentration of ecdysone, during mussel embryonic development 8,9 . Nevertheless, functional studies in mollusks are lacking and there is still no clear consensus regarding the presence of ecdysone-like signaling in mollusks (reviewed by 11,12 ). An older study by the Lafont lab, using radiolabeled ecdysteroids, suggested that certain steps of the ecdysone synthesis pathway are absent in various land snail species 13 . To the best of our knowledge, however, no study has investigated the presence of molluscan homologs of Halloween genes in details. Clearly, there is a need of further and more detailed analysis to clarify the functional role of ecdysone-like signaling in mollusks. The aim of the present study was to advance our understanding of the evolution of ecdysone signaling by applying a complex experimental approach in Lymnaea , a widely used molluscan model in neuroscience and neuroendocrinology (reviewed by 14-17 ). To accomplish our aim, we first exposed embryos of Lymnaea to different concentrations (10 ng/L, 100 ng/L, and 1 µg/L) of 20HE and investigated potential changes in embryogenesis (e.g., hatching, feeding) and in the expression of nEcR and mEcR homologs. Next, using cluster analysis and phylogenetics, we mapped the presence of homologs of Halloween genes in Lymnaea and lophotrochozoans in general. We also investigated whether 20HE is present in the tissues of embryos and adult specimens of Lymnaea using mass spectrometry. Our results strongly suggest that ecdysone-like signaling is absent in mollusks. 2. Results and Discussion No effect of 20HE exposure on hatching, heart rate, and behaviors of embryos Although previous studies investigated the expression of nEcR homolog during the development of mussel embryos 8,9 , there was no study providing functional evidence of 20HE on molluscan embryogenesis (reviewed by 11 ). Hereby we investigated the effect of 20HE on hatching, heart rate, and well-defined behaviors of Lymnaea embryos. No significant mortality was observed during the experiment ( Supplementary Table 1 ). No statistically significant differences were observed between the control, solvent control, and 20HE-exposed groups in any endpoints examined ( Figure 1 ). Although minor variations among treatments were detected, these changes remained within the range of natural biological variability. As shown in Figure 1a , the number of hatched embryos increased steadily from day 11 to day 13 in all experimental groups. The two-way repeated-measures Scheirer-Ray-Hare test revealed a significant effect of time (observation days) [H(2, 30) = 35.165, P ≤ 0.001], but no effect of treatment [H(4, 30) = 2.066, P > 0.05] or time x treatment interaction [H(8, 40) = 0.45, P > 0.05]. Subsequent analysis with the Kruskal-Vallis test showed no significant difference between the groups at any time point (observation day), indicating that embryonic development proceeded normally regardless of 20HE concentration. In the case of heart rate ( Figure 1b ), the two-way repeated-measures Scheirer-Ray-Hare test also revealed significant effects of time [H(4, 682) = 398.67, P ≤ 0.001], but no effect of treatment [H(4, 682) = 13.87, P > 0.05] or time x treatment interaction [H(16, 682) = 5.74, P > 0.05]. Similar to hatching, the heart rate showed a similar pattern across treatments, reflecting normal cardiac activity even in the presence of 20HE. Embryos of Lymnaea are known to show a lower gliding activity before hatching 18,19 , and indeed, animals in all experimental groups exhibited this gradual decreasing trend during the observation period ( Figure 1c ). Statistical analysis showed significant effects of time [H(3, 40) = 40.87, P ≤ 0.001], but no effect of treatment [H(4, 40) = 1.402, P > 0.05] or time x treatment interaction [H(12, 40) = 1.502, P > 0.05]. Subsequent analysis the between experimental groups found no significant effect of 20HE at any time point. Regarding the feeding behavior of embryos (E80%) ( Figure 1d ), the two-way repeated-measures Scheirer-Ray-Hare revealed significant effects of time [H(2, 405) = 175.758, P ≤ 0.001], but no significant effect of treatment [H(4, 405) = 0.653, P > 0.05] or time x treatment interaction [H(8, 405) = 7.937, P > 0.05]. In contrast to gliding, embryos are known to show a higher feeding activity before hatching 18,19 , this pattern was observed in all groups without any significant difference, indicating normal feeding activity even in the presence of 20HE. Altogether, continuous 20HE exposure during the entire embryogenesis did not cause detectable effects on development, physiology, and behaviors of Lymnaea embryos under the applied concentrations. No effect of 20HE exposure on the expression of nEcR and mEcR homologs Since 2010, studies have reported the presence of an orthologue of the nEcR/NR1H receptor in mollusks 5-9 . In our recent work, we also identified homologous sequences to both nEcR and mEcR in Lymnaea 10 . Previous studies further demonstrated the upregulation of the nEcR homolog during the embryonic development of mussels 8,9 ; however, no study has investigated the potential effect of 20HE on the expression of nEcR and mEcR homologs. In the present study, we examined whether the expression of Lymnaea nEcR and mEcR homologs changes during embryonic development following exposure to 20HE ( Figure 2 ). Gene expression analysis revealed that the expression levels of both nEcR ( Figure 2a, b ) and mEcR ( Figure 2a, c ) did not differ significantly between 20HE-treated and control groups before metamorphosis or after metamorphosis (p > 0.05). The lack of significant changes in Lymnaea nEcR and mEcR expression following 20HE exposure suggests that these receptors may not be directly responsive to exogenous ecdysteroids during embryonic development. A previous critical evaluation questioned the usefulness of expression of the so-called “molluscan nuclear estrogen receptor” as a biomarker for estrogen exposure in mollusks 20 . Similarly, we propose that nuclear receptors termed “molluscan nEcR” are not suitable biomarkers for ecdysone/20HE exposure in mollusks and should be called NR1H receptor orthologs rather ecdysone receptor homologs. Although further investigations focusing on receptor localization and ligand-binding assays are required to unequivocally clarify the functional relevance of these receptors in Lymnaea and in mollusks in general (already in progress in our laboratory), we propose that these molluscan sequences are not functional ecdysone receptors. This proposal is supported by previous assays showing no interaction between “nEcR” and RXR in a mussel 9 . Although investigation of the changes in the basal expression of the identified Lymnaea nuclear receptor during embryonic development was not the subject of the present study, we propose that the receptor might play an important role in molluscan embryogenesis. Ecdysone has no endogenous origin in Lymnaea As mentioned in the Introduction, previous studies, using radiolabeled precursors, demonstrated that although some enzymes required for the ecdysone biosynthetic pathway were present in land snails and slugs, at least two key enzyme activity could not be detected 13,21 . The Authors concluded that an endogenous origin would imply that snails use a biosynthetic pathway different from that of insects, but also raised the idea of a dietary origin. However, no study has investigated the presence of molluscan homologs of Halloween genes at the molecular level. Using Hidden Markov Models, we made thorough searches in the available genome and/or transcriptome data of deuterostomes, protostomes, and cnidarians to reveal homologs of Halloween genes of Drosophila . Then, using all-vs-all cluster-based methodologies ( Supplementary Figure 2 ) and phylogenetic analysis ( Figure 3 ; Figure 4 ; Supplementary Figure 3; Supplementary Figure 4 ), we clearly demonstrated that 1) there are no homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species and that 2) Phantom and Shade appear to have a many to many homologues in lophotrochozoans. In Lymnaea , specifically, we identified two homologs of Phantom and seven homologs of Shade ( Supplementary information ). Based on these findings, we propose that there is no ecdysone synthesis pathway in Lymnaea that would be equivalent to that of Drosophila ( Figure 5 ). Our results are in alignment with the earlier findings of the Lafont lab, which reported 3β-hydroxysteroid dehydrogenase (3ß-HSD), 25-hydroxylase (i.e. Phantom), and 20-hydroxylase (i.e. Shade) activity, but found no 22-hydroxylase (i.e. Disembodied) or 2-hydroxylase (i.e. Shadow) activity in land snails and slugs 13 . 3ß-HSD activity and the presence of 3ß-HSD gene in molluscan genomes, including Lymnaea 22 , are well-known (reviewed by 20,23 ). However, despite the presence of these enzymes in Lymnaea and lophotrochozoans in general, we propose that the corresponding reactions in these species are not part of an ecdysteroid biosynthetic pathway equivalent to that of ecdysozoans. Finally, our mass spectrometric investigation revealed only a very low amount of 20HE in different tissues of adults Lymnaea specimens, including the stomach (0.76 ± 0.09 ng/g), the hepatopancreas (0.37 ± 0.03 ng/g), the kidney (2.5 ± 0.2 ng/g), and the central nervous system (10.1 ± 0.37 ng/g) ( Figure 6; Supplementary information ). 20HE was not detected in early-stage or hatched embryos ( Figure 6; Supplementary information ). 20HE is well-known to be a major phytoecdysteroid in leafy plants (e.g., spinach) and to play a defensive role against insect pests 24 . Our analysis also showed the presence of 20HE in the lettuce (90.2 ± 2.57 ng/g) that the animals were being fed on ( Figure 6; Supplementary information ). These results, complemented with our sequence data, clearly indicate a dietary origin for 20HE in Lymnaea . The Lafont lab previously demonstrated that land snails metabolize ecdysone and 20HE in the same way whether ingested or injected and that only small amounts of dietary ecdysteroids can reach the tissues from the digestive tract 13 ; the results of the present study support this. Interestingly, the central nervous system contained the highest concentration of 20HE from the tissues investigated. Speculatively, the lack of 20HE in early-stage and hatched embryos is likely the result of poor maternal transfer from reproductive tissues. The reason for the observed tissue distribution still needs to be determined, but it is beyond the scope of the present study. Our findings also highlight the critical importance of carefully evaluating the dietary sources of lab-bred animals, or the environmental conditions (e.g., presence of ecdysteroid-producing plants) of field-collected animals, in endocrine- and ecotoxicological-related studies, as these factors can significantly influence the interpretation of results – including the origin of ecdysone in tissues. Specifically, in Lymnaea , our results show that if animals were fed a different diet in the laboratory, one would naturally expect that ecdysteroids would be undetectable in their tissues. Conclusion This study provides comprehensive experimental and bioinformatic evidence that the ecdysone signaling system, as characterized in ecdysozoans, is absent in the great pond snail, Lymnaea stagnalis . Continuous exposure of embryos to a range of 20HE concentrations induced no detectable effects on developmental, physiological, or behavioral endpoints (e.g., heart rate and feeding), suggesting low/no sensitivity of Lymnaea embryos to 20HE. Similarly, the expression of Lymnaea homologs of Drosophila ecdysone receptors remained unchanged following 20HE treatment, indicating an absence of transcriptional responsiveness to ecdysteroids at least during embryogenesis. Although additional studies focusing on receptor localization and ligand-binding properties are needed, we propose that molluscan sequences homologous to Drosophila nEcR and mEcR are unlikely to function as ecdysone receptors. Our cluster and phylogenetic analyses revealed that key components of the canonical ecdysone biosynthetic pathway – Spook, Disembodied, and Shadow – are absent in lophotrochozoans, including Lymnaea . Although homologs of Phantom and Shade were identified, there is no evidence that these reactions in mollusks are part of an ecdysteroid biosynthetic pathway which is equivalent to that of ecdysozoans. Mass spectrometric detection of trace 20HE levels in Lymnaea tissues, together with its presence in the lettuce diet, strongly supports a dietary origin of this compound. These findings reconcile earlier biochemical observations with new analytical and molecular evidence and collectively argue against the presence of ecdysone biosynthesis in mollusks. Taken together, our data demonstrate that Lymnaea lacks an ecdysone synthesis pathway and does not respond physiologically to 20HE during embryogenesis. We therefore propose that ecdysone-like signaling did not evolve in mollusks. Our study clarifies a long-standing ambiguity regarding the presence of ecdysone signaling in lophotrochozoans and refines our understanding of steroid hormone evolution across protostomes. Materials and Methods Chemicals for exposure 20HE (#H5142, Merck, Germany) standard was used for the treatments. From this, a 1 mg/mL stock solution was prepared in ethanol. From this stock solution, 5 µg/mL, 500 ng/mL, and 50 ng/mL working solutions were prepared weekly for the exposure. Ethics statement All procedures, methods, and experiments on Lymnaea embryos were carried out in accordance with the relevant guidelines and regulations approved by the Scientific Committee of Animal Experimentation of the HUN-REN Balaton Limnological Research Institute (VE/21/00706-4/2024). Experimental animals For 20HE exposure, embryos originating from 5-month-old adult Lymnaea specimens were obtained from our laboratory-bred stock. The adult snails were maintained in large plastic tanks containing 10L oxygenated artificial snail water (composition in mM: 0.1309 NaHCO 3 , 0.0378 K 2 SO 4 , 0.4013 CaCl 2 . 2H 2 O, 0.0390 Mg(NO 3 )2 . 6H 2 O; pH=7.6) at 20 °C (±1 °C) on a 12:12 h dark:light regime with light of natural wavelength. The snails were fed on lettuce ad libitum three times a week. The egg masses were collected within 2 hours after egg laying (single-cell stage 18 ) and assessed under a stereomicroscope to check their quality. Following established methodologies for Lymnaea embryo research 19,25-28 , individual eggs were isolated from the egg masses to allow precise observation of developmental progression and behavioral activity. Lymnaea embryos develop within transparent eggs clustered in a translucent gelatinous mass, their progression to be readily followed using a stereomicroscope. Embryogenesis is staged according to a specific set of morphological and behavioral features 18,29,30 . Similar to our previous study 19 , developmental stages - including the anatomical differentiation, the appearance of different organs, and the onset of behaviors - are given as a percentage of development (E0%-E100%). Here, E0% denotes the single-cell stage and E100% corresponds to hatching (see in 30 ). The exact time of embryogenesis strongly depends on a number of conditions of the location where the embryos are being raised 14 . In our laboratory, the embryonic development lasts approximately 13 days ( Supplementary Figure 1 ). 20HE exposure Separated individual E0% embryos were divided into 5 experimental groups as follows: a) control, b) solvent control, c) 10 ng/L 20HE-treated, d) 100 ng/L 20HE-treated, and e) 1 μg/L 20HE-treated. Two experimental set up was made. In Experiment 1 (investigation of hatching, heart rate, gliding, and radula), following the methods of our previous studies 19,27 , embryos in the control group were placed in a 6-well plate (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water without either 20HE or ethanol. Embryos in the solvent control group were also put into a 6-well plate (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water with 0.05% ethanol. Finally, embryos in the 20HE-treated groups were also placed in 6-well plates (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water and 10 ng/L, 100 ng/L, or 1 µg/L 20HE (<0.05% final ethanol concentration in all groups). Each experimental group included three replicates (i.e. a total of 30 animals per group; 150 animals in total in Experiment 1 ). In Experiment 2 (investigation of gene expression), the experimental setup was identical to that of Experiment 1 , except that a higher number of embryos was used (n = 20 embryos/well). The gene expression measurements were performed from two developmental stages (n = 20 embryos/stage) – before metamorphosis and after metamorphosis – in three independent replicates for each stage (i.e. a total of 120 animals per group; 600 animals in total in Experiment 2 ). The artificial water was completely changed every second day and 20HE was added again to maintain the exposure concentrations. Observation of embryonic development, heart rate, gliding, and feeding Embryonic development, heartbeat, locomotor activity (i.e., sliding along the inner surface of the egg), and feeding behavior (i.e., radula protrusion) were examined following established protocols 19,25-27 . Embryonic development (E0%-E100%) was monitored daily until hatching. The heartbeat was observed for 5 days following from the appearance of the heart (E60%). The gliding activity was assessed for 4 days from the start of gliding (E65%). The feeding activity was monitored for 3 days from the start of radula protrusion (E80%). The heartbeat and the radula protrusion (considered as fast actions) were counted over two-minute intervals on each observation day, while the number of full circles around the inner surface of the egg performed by gliding embryos (considered as a slow action) was recorded over four-minute intervals on each observation day. All observations were performed under a Leica M205c stereomicroscope equipped with a DFC3000G digital camera. qRT-PCR measurement of nEcR and mEcR expression Embryos in different developmental stages (before metamorphosis and after metamorphosis; n = 20 animals/stages/replicate) from all experimental groups were collected and pooled in each replicate. The procedure of RNA isolation, reverse transcription, and qRT-PCR was according to the description of previous studies 31-34 . Briefly, the samples were homogenized in TRI reagent (#93289, Merck) with a TissueLyser LT device (Qiagen, Germany). RNA was isolated with the Direct-zol TM RNA MiniPrep kit (#R2050, Zymo Research) and quantified by a NanoDrop One device (#ND-ONE-W, Thermo Fisher Scientific). For reverse transcription, the RevertAid H Minus First Strand cDNA Synthesis Kit (#K1631, Thermo Fisher Scientific) was used, applying random hexamer primers and 200 ng from all RNA samples. For qRT-PCR, each 20 μL reaction volume contained 10 μL 2x SYBR Green PCR Master Mix (#208252, Qiagen), 6 μL distilled water, 2 μL cDNA sample (20 ng), and 1 μL for each primer (500 nM final concentration). Lymnaea elongation factor 1α (EF1α) and β-tubulin (βTUB) were used as housekeeping genes. The applied primer sets were as follows: forward for nEcR: 5′ – AGG AGC TGT GTA GGA TTT GTG GAG – 3′; reverse primer for nEcR: 5′ – GAT TGG CAC TTT CGT CGC ATC C – 3′; forward for mEcR: 5′ – GTG CCC TTT GTT ATC ATC AAC CTT GT – 3′; reverse primer for mEcR: 5′ – CGC TCA GCA GCA GCA GTA TGA A– 3′; forward for EF1α: 5′ – GTG TAA GCA GCC CTC GAA CT – 3′; reverse primer for EF1α: 5′ – TTC GCT CAT CAA TAC CAC CA – 3′; forward for βTUB: 5′ – GAA ATA GCA CCG CCA TCC – 3′; reverse for βTUB: 5′ – CGC CTC TGT GAA CTC CAT CT – 3′ (Integrated DNA Technologies). Each preparation of nEcR, mEcR, EF1α, and βTUB was added to 96-well plates in triplicates. The reaction was performed at 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s using the QIAquant 96 2plex device (Qiagen) with the Q-Rex software (v. 2.0). At the end of the measurement, a melting curve analysis was performed (plate read every 0.3 °C from 60 to 95 °C) to determine the formation of the specific products. The amplification efficiency of the primer pairs calculated from the standard curves was within the desired range of 95%–105% (nEcR: 98%, mEcR: 101%; EF1α: 97%, βTUB: 100%). Changes in nEcR and mEcR mRNA expression, normalized to the arithmetic mean of the two reference genes, were calculated with the 2 -ΔΔCt method. Identification of Cytochrome P450 proteins Cytochrome P450 proteins were identified in Lymnaea through a search for homology to Disembodied, Shadow, and Shade proteins of D. melanogaster (UniProt IDs: Q9NGX9, Q9VGH1, Q9VUF8) using BLASTP 2.12 with e-values < 1e −10 35 . To remove redundant sequences, proteins with hits to P450 proteins were clustered with a 95% identity threshold using the CD-HIT v4.8.1 36 . Cytochrome P450 proteins were also identified in 14 other species, using the same methodology previously described, to improve the annotation. The 15 species analyzed corresponding to two Cnidaria, five Ecdysozoa, two Lophotrochozoa, two Ambulacraria, three Chordata ( Supplementary Table 1 ). Among the reference proteins it was included those of Homo sapiens and D. melanogaster . The amino acid sequences of the species are available in https://github.com/Imnotabioinformatician/iGluEvolutionChannels/tree/main. Highly divergent proteins, truncated sequences, or possible false positives were filtered through clustering analyses using the CLANS software 37 . The clustering level was assessed using P cutoff values of 1e -45 , 1e -50 , 1e -55 , 1e -75 , and 1e -80 . After this evaluation, a P cutoff value of 1e -50 was selected according on their connection to the CLAN containing the three reference proteins (Disembodied, Shadow, and Shade proteins). Only the sequences with at least one connection to this CLAN were keep for the posterior phylogenetic analysis. For the phylogenetic analyses, the two main CLANS were selected: one that contains Disembodied, Shadow, and Shade proteins; and other that contains Spook and Phantom proteins (UniProt ID: Q9VRM7and Q9VWR5, respectively). The software MAFFT 7.49 38 was used to align the sequences of main CLAN using the iterative refinement method L-INS-i (parameters: --localpair and --maxiterate 1000). Trimal 39 was used to remove gaps using the gappyout mode. IQ-TREE2 2.0.7 40 , was used to the construction of the phylogenetic tree using with the Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) branch support (1,000 replicates). To select the best model evolutive, the algorithm of ModelFinder 41 incorporated in IQ-TREE2 was activated (-mset), and the best model was selected among WAG, LG, Blosum62, Dayhoff, JTT, and Poisson, based on the Bayesian information criterion. The best model determined for the CLANS containing disembodied and shadow proteins was LG+F+R10 while for spook and phantom proteins was LG+R7. The visualization and annotation of the trees was performed in FigTree v1.4.4 (available from http://tree.bio.ed.ac.uk/software/figtree/). The FASTA files used in each phylogenetic analysis are available in https://github.com/victorcana/P450_Lymnaea-stagnalis. HPLC-MS measurement of 20HE in different tissues The central nervous system (n=10), stomach (n=10), kidney (n=10), and hepatopancreas (n=5) were dissected from naïve adult (5-month-old) snails, pooled per tissues in 1.5 mL low-bind Eppendorf tubes, and weighted. Moreover, 100 naïve early-stage and hatched embryos were also pooled in 1.5 mL low-bind Eppendorf tubes and weigthed. First, 200 µL of water was added to each sample. In the first stage, the samples were manually homogenized using plastic pestles. In the second stage, they were individually sonicated on ice using a Sonics & Materials, Inc. VCX 130 ultrasonic homogenizer (53 Church Hill Road, USA) for 1 min at 50% amplitude with a 50% duty cycle. After homogenization, 1 mL of ice-cold methanol was added, and the samples were vortexed for 1 min. Next, the samples were centrifuged at 20,000 × g for 10 min at 7 °C using an Eppendorf 5430 R centrifuge (Hamburg, Germany). After centrifugation, the supernatants were carefully collected and completely removed from the tubes. Three independent replicates were made for all samples. For lettuce samples, 200 mg of lyophilized lettuce was transferred into 50 mL Falcon tubes, and 20 mL of methanol–water (70:30, v/v) was added to the sample. Extraction was made using the same ultrasonic homogenizer with the same parameters, except that the ultrasonication time was increased to 2 min. After that, the samples were centrifuged at 2,000 × g for 10 min at room temperature, and the supernatants were collected and filtered through 0.2 µm Acrodisc Premium GxF/GHP 25 mm syringe filters (Pall Corporation, Port Washington, NY, USA). Three independent replicates were made. Solid-phase extraction was performed using Strata-XL 100 µm polymeric reversed-phase cartridges (200 mg/6 mL; Phenomenex, Torrance, CA, USA). Initially, the columns were conditioned with 3 mL of methanol and equilibrated with 3 mL of water. The samples were diluted with water to reduce the residual organic solvent content to below 5% (v/v) and were subsequently loaded slowly onto the columns. In the washing step, 3 mL of water–methanol (90:10, v/v) was used, and the analytes were eluted with 1.5 mL of methanol–water (70:30, v/v) in the final phase. The eluted samples were concentrated with a SpeedVac Concentrator Plus (Eppendorf AG, Hamburg, Germany) vacuum concentrator device. Finally, the samples were reconstituted with 100 μL methanol/water/formic acid (60:40:0.1, v/v/v), induced by vortex mixing and ultrasonication. LC-MS analysis was performed using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Thermo Ultimate 3000 UPLC™ system. Chromatographic separation was performed on a Luna Omega PS C18 reversed-phase column (1.6 µm, 2.1 mm × 150 mm i.d.) (Phenomenex, USA). The mobile phase consisted of two solvents: solvent A was water/formic acid (99.9:0.1, v/v), and solvent B was methanol/isopropanol/formic acid (60:40:0.1, v/v/v). Both eluents contained 10 mM ammonium formate. The flow rate was 150 µL min⁻¹, and the column temperature was maintained at 45 °C. The gradient program included the following steps: from 0.0 to 10.0 minutes, the composition of solvent B increased from 0.0% to 80.0%; from 10.0 to 14.0 minutes, it increased from 80.0% to 100.0%; from 14.0 to 22.0 minutes, it was held at 100.0%; from 22.0 to 25.0 minutes, it decreased from 100.0% to 0.0%; and from 25.0 to 34.9 minutes, it was held at 0.0% for re-equilibration. The mass spectrometer was operated in positive ion mode using parallel reaction monitoring for data acquisition. HESI source conditions were: capillary temperature 300 °C, S-Lens RF level 30, spray voltage 3.5 kV, sheath gas flow 25 (arbitrary units), auxiliary gas flow 8 (arbitrary units). The resolution was set to 35,000, and the quadrupole isolation window was set to 2 Da. For molecular ion fragmentation, a normalized collision energy of 15 was used. The most intense precursor-to-fragment transitions were used for analysis of 20HE: 481.31 m/z → 445.29 m/z and 371.22 m/z. The transitions and the optimal ionization condition were derived from our recorded mass spectra using a 10 ng/mL standard solution prepared from 20HE analytical standard (#H5142, Merck). Peak detection and quantification were achieved using the Xcalibur 4.2 software. The ions under observation were verified based on specific criteria: accurate MS1 mass, retention time, MS2 masses, and the pattern of fragmentation. The production was used for the quantitation. A six-point calibration curve was generated for the quantitative analysis using 0, 0.5, 1, 5, 10, 50, and 100 ng/mL standard solutions. The correlation coefficient (R 2 ) for the calibration curve was 0.99 (not shown). Statistical analysis Statistical analysis was made with the OriginPro 2018 software (OriginLab Corp., USA). The normality of the datasets was investigated using the Shapiro-Wilk test and the homogeneity of variances between groups was investigated using the Levene-test. In the case of hatching, heart rate, locomotion, and feeding, the nonparametric Scheirer-Ray-Hare test were applied to study the effect of time, treatment, and time x treatment interaction. This analysis was followed by ANOVA and Scheffe’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test to identify significant differences between control and treatment groups at a given time point. The qRT-PCR data were analyzed with ANOVA and Scheffe’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test. Declarations Author Statement The authors declare that the recommendations of the ARRIVE 2.0. guideline were considered and followed during the experiments, analysis, and data reporting. Declaration of conflict of interest The authors declare that there is no conflict of interest. Funding This work was supported by the National Brain Project (#NAP2022-I-10/2022, Z.P.), the Hungarian Scientific Research Fund (#138039, Z.P.; #146787, I.F.), the Biotechnology and Biological Sciences Research Council (#BB/W010305/2, L.A.Y.-G.), the Royal Society (#RG\R1\241397, L.A.Y.-G.), and the Thematic Excellence Program (#TKP2021-EGA-17, J.S.). Author Contribution RS: Investigation, Methodology, Writing – review & editing, Data curation, Visualization; LAYG: Methodology, Investigation, Writing – review & editing, Visualization, Funding acquisition; VHCB: Methodology, Investigation, Writing – review & editing, Visualization; JS: Methodology, Investigation, Writing – review & editing, Data curation, Visualization, Funding acquisition; ZP: Writing – review & editing, Funding acquisition; IF: Conceptualization, Methodology, Investigation, Writing – original draft, Data curation, Visualization, Funding acquisition. 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16:33:01","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129898,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/3f371d4acfcbc5ab907f4295.html"},{"id":98423974,"identity":"d38cbcce-18fe-471a-892f-80251273a45d","added_by":"auto","created_at":"2025-12-17 16:32:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14267696,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 20HE on hatching (\u003cstrong\u003ea\u003c/strong\u003e), heart rate (\u003cstrong\u003eb\u003c/strong\u003e), gliding activity (\u003cstrong\u003ec)\u003c/strong\u003e, and (\u003cstrong\u003ed\u003c/strong\u003e) feeding activity in the different experimental groups of \u003cem\u003eLymnaea \u003c/em\u003eembryos\u003cem\u003e \u003c/em\u003e(control, solvent control, 10 ng/L 20HE-treated; 100 ng/L 20HE-treated, and 1 μg/L 20HE-treated). The heartbeat, gliding, and feeding were observed from the E60%, E65%, and E80% stages, respectively. Each data point represents the mean ± SEM (n = 10 animals/replicate/group). No statistically significant differences were observed between the experimental groups at any endpoints investigated.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/43b3414393b6a3a5e63cb4fa.png"},{"id":97981020,"identity":"c467f549-0d7b-4348-b160-fc8bd4839259","added_by":"auto","created_at":"2025-12-11 12:47:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18530235,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 20HE on the expression of nEcR (\u003cstrong\u003ea,\u003c/strong\u003e \u003cstrong\u003eb\u003c/strong\u003e) and mEcR (\u003cstrong\u003ec,\u003c/strong\u003e \u003cstrong\u003ed\u003c/strong\u003e) before and after metamorphosis in the different experimental groups of \u003cem\u003eLymnaea \u003c/em\u003eembryos\u003cem\u003e \u003c/em\u003e(control, solvent control, 10 ng/L 20HE-treated; 100 ng/L 20HE-treated, and 1 μg/L 20HE-treated). Each column represents the mean ± SEM (n = 10 animals/replicate/group). No statistically significant differences were observed in the expression of nEcR or mEcR between the experimental groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/b73db66eacd492bc67edb254.png"},{"id":98424482,"identity":"deb31b12-fbe6-4c2e-bafb-3c88d2cb6084","added_by":"auto","created_at":"2025-12-17 16:33:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6051809,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree showing the occurrence and relationships of Disembodied, Shadow, and Shade. The tree shows that lophotrochozoans has no clear homologues of Disembodied and Shadow but appear to have many to many homologues to Shade. Branch support values are indicated at nodes in the format aLRT/UFBoot (e.g., 100/100). The raw tree is presented in Supplementary Figure 3.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/b46003078837f95e7ab4bc16.png"},{"id":98423931,"identity":"8343c5bc-fedf-4d2d-aa40-d02f8b26d15c","added_by":"auto","created_at":"2025-12-17 16:32:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19624052,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree showing the occurrence and relationships of Spook and Phantom. The tree shows that lophotrochozoans has no clear homologues of Spook but appear to have many to many homologues to Phantom. Branch support values are indicated at nodes in the format aLRT/UFBoot (e.g., 100/100). The raw tree is presented in Supplementary Figure 4.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/9234cec83e898cd645e78113.png"},{"id":98424761,"identity":"a15623fa-84d4-48d5-ad6b-f1863d9773e4","added_by":"auto","created_at":"2025-12-17 16:33:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22383114,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the canonical ecdysone synthesis pathway in \u003cem\u003eDrosophila\u003c/em\u003e with the potential ecdysone synthesis pathway in \u003cem\u003eLymnaea\u003c/em\u003e. Based on our cluster and phylogenetics analyses, there are no homologues of Spook, Disembodied, or Shadow in \u003cem\u003eLymnaea\u003c/em\u003e(marked by red X symbols), indicating that \u003cem\u003eLymnaea\u003c/em\u003e does not possess an ecdysone synthesis pathway in \u003cem\u003eLymnaea\u003c/em\u003e that would be equivalent to that of \u003cem\u003eDrosophila\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/ee7810c4c3ece731553e0975.png"},{"id":97981004,"identity":"54063145-baa3-47a9-906a-e1a988f75dc3","added_by":"auto","created_at":"2025-12-11 12:47:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":529663,"visible":true,"origin":"","legend":"\u003cp\u003ePresence of 20HE in different tissues of adult \u003cem\u003eLymnaea\u003c/em\u003e, hatched \u003cem\u003eLymnaea\u003c/em\u003e embryos, and in the lettuce that the animals were being fed on. Detected values were as follows: stomach - 0.76 ± 0.09 ng/g, hepatopancreas (Hep) - 0.37 ± 0.03 ng/g, kidney - 2.5 ± 0.2 ng/g, central nervous system (CNS) - 10.1 ± 0.37 ng/g, embryo – not detected (n.d.), lettuce – 90.2 ± 2.57 ng/g. Each bar represents the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/62430c93ba0f28ceed90d6f4.png"},{"id":97981007,"identity":"4b07a609-4731-48e7-b2c8-ea8e3ecf30e7","added_by":"auto","created_at":"2025-12-11 12:47:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1269282,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationmerged.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8193722/v1/8a78ddb72f7c08b3d47f3259.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEcdysone signaling is a central endocrine pathway regulating development (e.g., molting, metamorphosis) and reproduction in ecdysozoans. The discovery of ecdysone and its active metabolite, 20-hydroxyecdysone (20E), in insects provided the first evidence of steroid hormones directing major developmental transitions in invertebrates\u003csup\u003e1\u003c/sup\u003e. Subsequent molecular studies in \u003cem\u003eDrosophila\u003c/em\u003e identified the Halloween genes, which encode cytochrome P450 enzymes responsible for converting dietary cholesterol into ecdysteroids, revealing the canonical ecdysone synthesis pathway (reviewed by \u003csup\u003e2\u003c/sup\u003e). Ecdysone and 20E primarily act via the ecdysone receptor (nEcR) and its partner ultraspiracle (USP/RXR), a heterodimeric nuclear receptor complex that activates early-response genes\u003csup\u003e3\u003c/sup\u003e. Moreover, ecdysteroids can also induce rapid, non-genomic effects via a GPCR (mEcR), called the dopamine/ecdysteroid receptor in \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids\u003csup\u003e5-10\u003c/sup\u003e. Previous studies also demonstrated the upregulation of the nEcR homolog, as well as the increase of serum concentration of ecdysone, during mussel embryonic development\u003csup\u003e8,9\u003c/sup\u003e. Nevertheless, functional studies in mollusks are lacking and there is still no clear consensus regarding the presence of ecdysone-like signaling in mollusks (reviewed by \u003csup\u003e11,12\u003c/sup\u003e). An older study by the Lafont lab, using radiolabeled ecdysteroids, suggested that certain steps of the ecdysone synthesis pathway are absent in various land snail species\u003csup\u003e13\u003c/sup\u003e. To the best of our knowledge, however, no study has investigated the presence of molluscan homologs of Halloween genes in details. Clearly, there is a need of further and more detailed analysis to clarify the functional role of ecdysone-like signaling in mollusks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aim of the present study was to advance our understanding of the evolution of ecdysone signaling by applying a complex experimental approach in \u003cem\u003eLymnaea\u003c/em\u003e, a widely used molluscan model in neuroscience and neuroendocrinology (reviewed by \u003csup\u003e14-17\u003c/sup\u003e). To accomplish our aim, we first exposed embryos of \u003cem\u003eLymnaea\u003c/em\u003e to different concentrations (10 ng/L, 100 ng/L, and 1 µg/L) of 20HE and investigated potential changes in embryogenesis (e.g., hatching, feeding) and in the expression of nEcR and mEcR homologs. Next, using cluster analysis and phylogenetics, we mapped the presence of homologs of Halloween genes in \u003cem\u003eLymnaea\u003c/em\u003e and lophotrochozoans in general. We also investigated whether 20HE is present in the tissues of embryos and adult specimens of \u003cem\u003eLymnaea\u003c/em\u003e using mass spectrometry. Our results strongly suggest that ecdysone-like signaling is absent in mollusks.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cp\u003e\u003cem\u003eNo effect of 20HE exposure on hatching, heart rate, and behaviors of embryos\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAlthough previous studies investigated the expression of nEcR homolog during the development of mussel embryos\u003csup\u003e8,9\u003c/sup\u003e, there was no study providing functional evidence of 20HE on molluscan embryogenesis (reviewed by \u003csup\u003e11\u003c/sup\u003e). Hereby we investigated the effect of 20HE on hatching, heart rate, and well-defined behaviors of \u003cem\u003eLymnaea\u003c/em\u003e embryos.\u003c/p\u003e\n\u003cp\u003eNo significant mortality was observed during the experiment (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e). No statistically significant differences were observed between the control, solvent control, and 20HE-exposed groups in any endpoints examined (\u003cstrong\u003eFigure 1\u003c/strong\u003e). Although minor variations among treatments were detected, these changes remained within the range of natural biological variability. As shown in \u003cstrong\u003eFigure 1a\u003c/strong\u003e, the number of hatched embryos increased steadily from day 11 to day 13 in all experimental groups. The two-way repeated-measures Scheirer-Ray-Hare test revealed a significant effect of time (observation days) [H(2, 30) = 35.165, P ≤ 0.001], but no effect of treatment [H(4, 30) = 2.066, P \u0026gt; 0.05] or time x treatment interaction [H(8, 40) = 0.45, P \u0026gt; 0.05]. Subsequent analysis with the Kruskal-Vallis test showed no significant difference between the groups at any time point (observation day), indicating that embryonic development proceeded normally regardless of 20HE concentration. In the case of heart rate (\u003cstrong\u003eFigure 1b\u003c/strong\u003e), the two-way repeated-measures Scheirer-Ray-Hare test also revealed significant effects of time [H(4, 682) = 398.67, P ≤ 0.001], but no effect of treatment [H(4, 682) = 13.87, P \u0026gt; 0.05] or time x treatment interaction [H(16, 682) = 5.74, P \u0026gt; 0.05]. Similar to hatching, the heart rate showed a similar pattern across treatments, reflecting normal cardiac activity even in the presence of 20HE. Embryos of \u003cem\u003eLymnaea\u003c/em\u003e are known to show a lower gliding activity before hatching \u003csup\u003e18,19\u003c/sup\u003e, and indeed, animals in all experimental groups exhibited this gradual decreasing trend during the observation period (\u003cstrong\u003eFigure 1c\u003c/strong\u003e). Statistical analysis showed significant effects of time [H(3, 40) = 40.87, P ≤ 0.001], but no effect of treatment [H(4, 40) = 1.402, P \u0026gt; 0.05] or time x treatment interaction [H(12, 40) = 1.502, P \u0026gt; 0.05]. Subsequent analysis the between experimental groups found no significant effect of 20HE at any time point. Regarding the feeding behavior of embryos (E80%) (\u003cstrong\u003eFigure 1d\u003c/strong\u003e), the two-way repeated-measures Scheirer-Ray-Hare revealed significant effects of time [H(2, 405) = 175.758, P ≤ 0.001], but no significant effect of treatment [H(4, 405) = 0.653, P \u0026gt; 0.05] or time x treatment interaction [H(8, 405) = 7.937, P \u0026gt; 0.05]. In contrast to gliding, embryos are known to show a higher feeding activity before hatching\u003csup\u003e18,19\u003c/sup\u003e, this pattern was observed in all groups without any significant difference, indicating normal feeding activity even in the presence of 20HE.\u003c/p\u003e\n\u003cp\u003eAltogether, continuous 20HE exposure during the entire embryogenesis did not cause detectable effects on development, physiology, and behaviors of \u003cem\u003eLymnaea\u003c/em\u003e embryos under the applied concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNo effect of 20HE exposure on the expression of nEcR and mEcR homologs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSince 2010, studies have reported the presence of an orthologue of the nEcR/NR1H receptor in mollusks\u003csup\u003e5-9\u003c/sup\u003e. In our recent work, we also identified homologous sequences to both nEcR and mEcR in \u003cem\u003eLymnaea\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e. Previous studies further demonstrated the upregulation of the nEcR homolog during the embryonic development of mussels\u003csup\u003e8,9\u003c/sup\u003e; however, no study has investigated the potential effect of 20HE on the expression of nEcR and mEcR homologs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In the present study, we examined whether the expression of \u003cem\u003eLymnaea\u003c/em\u003e nEcR and mEcR homologs changes during embryonic development following exposure to 20HE (\u003cstrong\u003eFigure 2\u003c/strong\u003e). Gene expression analysis revealed that the expression levels of both nEcR (\u003cstrong\u003eFigure 2a,\u003c/strong\u003e \u003cstrong\u003eb\u003c/strong\u003e) and mEcR (\u003cstrong\u003eFigure 2a, c\u003c/strong\u003e) did not differ significantly between 20HE-treated and control groups before metamorphosis or after metamorphosis (p \u0026gt; 0.05). The lack of significant changes in \u003cem\u003eLymnaea\u003c/em\u003e nEcR and mEcR expression following 20HE exposure suggests that these receptors may not be directly responsive to exogenous ecdysteroids during embryonic development. A previous critical evaluation questioned the usefulness of expression of the so-called “molluscan nuclear estrogen receptor” as a biomarker for estrogen exposure in mollusks\u003csup\u003e20\u003c/sup\u003e. Similarly, we propose that nuclear receptors termed “molluscan nEcR” are not suitable biomarkers for ecdysone/20HE exposure in mollusks and should be called NR1H receptor orthologs rather ecdysone receptor homologs. Although further investigations focusing on receptor localization and ligand-binding assays are required to unequivocally clarify the functional relevance of these receptors in \u003cem\u003eLymnaea\u003c/em\u003e and in mollusks in general (already in progress in our laboratory), we propose that these molluscan sequences are not functional ecdysone receptors. This proposal is supported by previous assays showing no interaction between “nEcR” and RXR in a mussel\u003csup\u003e9\u003c/sup\u003e. Although investigation of the changes in the basal expression of the identified \u003cem\u003eLymnaea\u003c/em\u003e nuclear receptor during embryonic development was not the subject of the present study, we propose that the receptor might play an important role in molluscan embryogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEcdysone has no endogenous origin in Lymnaea\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs mentioned in the Introduction, previous studies, using radiolabeled precursors, demonstrated that although some enzymes required for the ecdysone biosynthetic pathway were present in land snails and slugs, at least two key enzyme activity could not be detected\u003csup\u003e13,21\u003c/sup\u003e. The Authors concluded that an endogenous origin would imply that snails use a biosynthetic pathway different from that of insects, but also raised the idea of a dietary origin. However, no study has investigated the presence of molluscan homologs of Halloween genes at the molecular level.\u003c/p\u003e\n\u003cp\u003eUsing Hidden Markov Models, we made thorough searches in the available genome and/or transcriptome data of deuterostomes, protostomes, and cnidarians to reveal homologs of Halloween genes of \u003cem\u003eDrosophila\u003c/em\u003e. Then, using all-vs-all cluster-based methodologies (\u003cstrong\u003eSupplementary Figure 2\u003c/strong\u003e) and phylogenetic analysis (\u003cstrong\u003eFigure\u003c/strong\u003e \u003cstrong\u003e3\u003c/strong\u003e; \u003cstrong\u003eFigure\u003c/strong\u003e \u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e; Supplementary Figure 3; Supplementary Figure 4\u003c/strong\u003e), we clearly demonstrated that 1) there are no homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species and that 2) Phantom and Shade appear to have a many to many homologues in lophotrochozoans. In \u003cem\u003eLymnaea\u003c/em\u003e, specifically, we identified two homologs of Phantom and seven homologs of Shade (\u003cstrong\u003eSupplementary information\u003c/strong\u003e). Based on these findings, we propose that there is no ecdysone synthesis pathway in \u003cem\u003eLymnaea\u003c/em\u003e that would be equivalent to that of \u003cem\u003eDrosophila\u003c/em\u003e (\u003cstrong\u003eFigure 5\u003c/strong\u003e). Our results are in alignment with the earlier findings of the Lafont lab, which reported 3β-hydroxysteroid dehydrogenase (3ß-HSD), 25-hydroxylase (i.e. Phantom), and 20-hydroxylase (i.e. Shade) activity, but found no 22-hydroxylase (i.e. Disembodied) or 2-hydroxylase (i.e. Shadow) activity in land snails and slugs\u003csup\u003e13\u003c/sup\u003e. 3ß-HSD activity and the presence of 3ß-HSD gene in molluscan genomes, including \u003cem\u003eLymnaea\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e, are well-known (reviewed by \u003csup\u003e20,23\u003c/sup\u003e). However, despite the presence of these enzymes in \u003cem\u003eLymnaea\u0026nbsp;\u003c/em\u003eand lophotrochozoans in general, we propose that the corresponding reactions in these species are not part of an ecdysteroid biosynthetic pathway equivalent to that of ecdysozoans.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, our mass spectrometric investigation revealed only a very low amount of 20HE in different tissues of adults \u003cem\u003eLymnaea\u003c/em\u003e specimens, including the stomach (0.76 ± 0.09 ng/g), the hepatopancreas (0.37 ± 0.03 ng/g), the kidney (2.5 ± 0.2 ng/g), and the central nervous system (10.1 ± 0.37 ng/g) (\u003cstrong\u003eFigure 6; Supplementary information\u003c/strong\u003e). 20HE was not detected in early-stage or hatched embryos (\u003cstrong\u003eFigure 6; Supplementary information\u003c/strong\u003e). 20HE is well-known to be a major phytoecdysteroid in leafy plants (e.g., spinach) and to play a defensive role against insect pests\u003csup\u003e24\u003c/sup\u003e. Our analysis also showed the presence of 20HE in the lettuce (90.2 ± 2.57 ng/g) that the animals were being fed on (\u003cstrong\u003eFigure 6; Supplementary information\u003c/strong\u003e). These results, complemented with our sequence data, clearly indicate a dietary origin for 20HE in \u003cem\u003eLymnaea\u003c/em\u003e. The Lafont lab previously demonstrated that land snails metabolize ecdysone and 20HE in the same way whether ingested or injected and that only small amounts of dietary ecdysteroids can reach the tissues from the digestive tract\u003csup\u003e13\u003c/sup\u003e; the results of the present study support this. Interestingly, the central nervous system contained the highest concentration of 20HE from the tissues investigated. Speculatively, the lack of 20HE in early-stage and hatched embryos is likely the result of poor maternal transfer from reproductive tissues. The reason for the observed tissue distribution still needs to be determined, but it is beyond the scope of the present study. Our findings also highlight the critical importance of carefully evaluating the dietary sources of lab-bred animals, or the environmental conditions (e.g., presence of ecdysteroid-producing plants) of field-collected animals, in endocrine- and ecotoxicological-related studies, as these factors can significantly influence the interpretation of results – including the origin of ecdysone in tissues. Specifically, in \u003cem\u003eLymnaea\u003c/em\u003e, our results show that if animals were fed a different diet in the laboratory, one would naturally expect that ecdysteroids would be undetectable in their tissues.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides comprehensive experimental and bioinformatic evidence that the ecdysone signaling system, as characterized in ecdysozoans, is absent in the great pond snail,\u0026nbsp;\u003cem\u003eLymnaea\u003c/em\u003e \u003cem\u003estagnalis\u003c/em\u003e. Continuous exposure of embryos to a range of 20HE concentrations induced no detectable effects on developmental, physiological, or behavioral endpoints (e.g., heart rate and feeding), suggesting low/no sensitivity of \u003cem\u003eLymnaea\u003c/em\u003e embryos to 20HE. Similarly, the expression of \u003cem\u003eLymnaea\u003c/em\u003e homologs of \u003cem\u003eDrosophila\u003c/em\u003e ecdysone receptors remained unchanged following 20HE treatment, indicating an absence of transcriptional responsiveness to ecdysteroids at least during embryogenesis. Although additional studies focusing on receptor localization and ligand-binding properties are needed, we propose that molluscan sequences homologous to \u003cem\u003eDrosophila\u003c/em\u003e nEcR and mEcR are unlikely to function as ecdysone receptors.\u003c/p\u003e\n\u003cp\u003eOur cluster and phylogenetic analyses revealed that key components of the canonical ecdysone biosynthetic pathway – Spook, Disembodied, and Shadow – are absent in lophotrochozoans, including \u003cem\u003eLymnaea\u003c/em\u003e. Although homologs of Phantom and Shade were identified, there is no evidence that these reactions in mollusks are part of an ecdysteroid biosynthetic pathway which is equivalent to that of ecdysozoans. Mass spectrometric detection of trace 20HE levels in \u003cem\u003eLymnaea\u003c/em\u003e tissues, together with its presence in the lettuce diet, strongly supports a dietary origin of this compound. These findings reconcile earlier biochemical observations with new analytical and molecular evidence and collectively argue against the presence of ecdysone biosynthesis in mollusks.\u003c/p\u003e\n\u003cp\u003eTaken together, our data demonstrate that \u003cem\u003eLymnaea\u0026nbsp;\u003c/em\u003elacks an ecdysone synthesis pathway and does not respond physiologically to 20HE during embryogenesis. We therefore propose that ecdysone-like signaling did not evolve in mollusks. Our study clarifies a long-standing ambiguity regarding the presence of ecdysone signaling in lophotrochozoans and refines our understanding of steroid hormone evolution across protostomes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eChemicals for exposure\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e20HE (#H5142, Merck, Germany) standard was used for the treatments. From this, a 1 mg/mL stock solution was prepared in ethanol.\u0026nbsp;From this stock solution, 5 µg/mL, 500 ng/mL, and 50 ng/mL working solutions were prepared weekly for the exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures, methods, and experiments on \u003cem\u003eLymnaea\u003c/em\u003e embryos were carried out in accordance with the relevant guidelines and regulations approved by the Scientific Committee of Animal Experimentation of the HUN-REN Balaton Limnological Research Institute (VE/21/00706-4/2024).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperimental animals\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor 20HE exposure, embryos originating from 5-month-old adult \u003cem\u003eLymnaea\u003c/em\u003e specimens were obtained from our laboratory-bred stock. The adult snails were maintained in large plastic tanks containing 10L oxygenated artificial snail water (composition in mM: 0.1309 NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.0378 K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.4013 CaCl\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.\u003c/sup\u003e2H\u003csub\u003e2\u003c/sub\u003eO, 0.0390 Mg(NO\u003csub\u003e3\u003c/sub\u003e)2\u003csup\u003e.\u003c/sup\u003e6H\u003csub\u003e2\u003c/sub\u003eO; pH=7.6) at\u0026nbsp;20 °C (±1 °C) on a 12:12 h dark:light regime with light of natural wavelength. The snails were fed on lettuce \u003cem\u003ead libitum\u003c/em\u003e three times a week.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe egg masses were collected within 2 hours after egg laying (single-cell stage\u003csup\u003e18\u003c/sup\u003e) and assessed under a stereomicroscope to check their quality.\u0026nbsp;Following established methodologies for \u003cem\u003eLymnaea\u003c/em\u003e embryo research\u003csup\u003e19,25-28\u003c/sup\u003e, individual eggs were isolated from the egg masses to allow precise observation of developmental progression and behavioral activity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLymnaea\u003c/em\u003e embryos develop within transparent eggs clustered in a translucent gelatinous mass, their progression to be readily followed using a stereomicroscope. Embryogenesis is staged according to a specific set of morphological and behavioral features\u003csup\u003e18,29,30\u003c/sup\u003e. Similar to our previous study\u003csup\u003e19\u003c/sup\u003e, developmental stages - including the anatomical differentiation, the appearance of different organs, and the onset of behaviors - are given as a percentage of development (E0%-E100%). Here, E0% denotes the single-cell stage and E100% corresponds to hatching (see in \u003csup\u003e30\u003c/sup\u003e). The exact time of embryogenesis strongly depends on a number of conditions of the location where the embryos are being raised\u003csup\u003e14\u003c/sup\u003e. In our laboratory, the embryonic development lasts approximately 13 days (\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e20HE exposure\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSeparated individual E0% embryos were divided into\u0026nbsp;5 experimental groups as follows: a) control, b) solvent control, c) 10 ng/L 20HE-treated, d) 100 ng/L 20HE-treated, and e) 1 μg/L 20HE-treated. Two experimental set up was made.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eExperiment 1\u003c/em\u003e (investigation of hatching, heart rate, gliding, and radula), following the methods of our previous studies\u003csup\u003e19,27\u003c/sup\u003e, embryos in the control group were\u0026nbsp;placed in a 6-well plate (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water without either 20HE or ethanol. Embryos in the solvent control group were also put into a 6-well plate (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water with 0.05% ethanol. Finally, embryos in the 20HE-treated groups were also placed in 6-well plates (n = 10 embryos/well) containing 10 mL pre-oxygenated artificial snail water and 10 ng/L, 100 ng/L, or 1 µg/L 20HE (\u0026lt;0.05% final ethanol concentration in all groups). Each experimental group included three replicates (i.e. a total of 30 animals per group; 150 animals in total in\u003cem\u003e\u0026nbsp;Experiment 1\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eExperiment 2\u003c/em\u003e (investigation of gene expression),\u0026nbsp;the experimental setup was identical to that of \u003cem\u003eExperiment 1\u003c/em\u003e, except that a higher number of embryos was used (n = 20 embryos/well).\u0026nbsp;The gene expression measurements were performed from two developmental stages (n = 20 embryos/stage) – before metamorphosis and after metamorphosis – in three independent replicates for each stage (i.e. a total of 120 animals per group; 600 animals in total in\u003cem\u003e\u0026nbsp;Experiment 2\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe artificial water was completely changed every second day and 20HE was added again to maintain the exposure concentrations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eObservation of embryonic development, heart rate, gliding, and feeding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEmbryonic development, heartbeat, locomotor activity (i.e., sliding along the inner surface of the egg), and feeding behavior (i.e., radula protrusion) were examined following established protocols\u003csup\u003e19,25-27\u003c/sup\u003e. Embryonic development (E0%-E100%) was monitored daily\u0026nbsp;until hatching. The heartbeat was observed for 5 days following from the appearance of the heart (E60%). The gliding activity was assessed for 4 days from the start of gliding (E65%). The feeding activity was monitored for 3 days from the start of radula protrusion (E80%). The heartbeat and the radula protrusion (considered as fast actions) were counted over two-minute intervals on each observation day, while the number of full circles around the inner surface of the egg performed by gliding embryos (considered as a slow action) was recorded over four-minute intervals on each observation day. All observations were performed under a\u0026nbsp;Leica M205c stereomicroscope equipped with a DFC3000G digital camera.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eqRT-PCR measurement of nEcR and mEcR expression\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEmbryos in different developmental stages (before metamorphosis and after metamorphosis; n = 20 animals/stages/replicate) from all experimental groups were collected and pooled in each replicate.\u0026nbsp;The procedure of RNA isolation, reverse transcription, and qRT-PCR was according to the description of previous studies\u003csup\u003e31-34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBriefly, the samples were homogenized in TRI reagent (#93289, Merck) with a TissueLyser LT device (Qiagen, Germany). RNA was isolated with the Direct-zol\u003csup\u003eTM\u003c/sup\u003e RNA MiniPrep kit (#R2050, Zymo Research) and\u0026nbsp;quantified by a NanoDrop One device (#ND-ONE-W, Thermo Fisher Scientific). For reverse transcription, the RevertAid H Minus First Strand cDNA Synthesis Kit (#K1631, Thermo Fisher Scientific) was used, applying random hexamer primers and 200 ng from all RNA samples.\u003c/p\u003e\n\u003cp\u003eFor qRT-PCR, each 20 μL reaction volume contained 10 μL 2x SYBR Green PCR Master Mix (#208252, Qiagen), 6 μL distilled water, 2 μL cDNA sample (20 ng), and 1 μL for each primer (500 nM final concentration). \u003cem\u003eLymnaea\u003c/em\u003e elongation factor 1α (EF1α) and β-tubulin (βTUB) were used as housekeeping genes. The applied primer sets were as follows: forward for nEcR: 5′ – AGG AGC TGT GTA GGA TTT GTG GAG – 3′; reverse primer for nEcR: 5′ – GAT TGG CAC TTT CGT CGC ATC C – 3′; forward for mEcR: 5′ – GTG CCC TTT GTT ATC ATC AAC CTT GT – 3′; reverse primer for mEcR: 5′ – CGC TCA GCA GCA GCA GTA TGA A– 3′; forward for EF1α: 5′ – GTG TAA GCA GCC CTC GAA CT – 3′; reverse primer for EF1α: 5′ – TTC GCT CAT CAA TAC CAC CA – 3′; forward for βTUB: 5′ – GAA ATA GCA CCG CCA TCC – 3′; reverse for βTUB: 5′ – CGC CTC TGT GAA CTC CAT CT – 3′ (Integrated DNA Technologies). Each preparation of nEcR, mEcR, EF1α, and βTUB was added to 96-well plates in triplicates. The reaction was performed at 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s using the QIAquant 96 2plex device (Qiagen) with the Q-Rex software (v. 2.0). At the end of the measurement, a melting curve analysis was performed (plate read every 0.3 °C from 60 to 95 °C) to determine the formation of the specific products. The amplification efficiency of the primer pairs calculated from the standard curves was within the desired range of 95%–105% (nEcR: 98%, mEcR: 101%; EF1α: 97%, βTUB: 100%). Changes in nEcR and mEcR mRNA expression, normalized to the arithmetic mean of the two reference genes, were calculated with the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIdentification of Cytochrome P450 proteins\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCytochrome P450 proteins were identified in \u003cem\u003eLymnaea\u003c/em\u003e through a search for homology to Disembodied, Shadow, and Shade proteins of \u003cem\u003eD. melanogaster\u003c/em\u003e (UniProt IDs: Q9NGX9, Q9VGH1, Q9VUF8) using BLASTP 2.12 with e-values \u0026lt; 1e\u003csup\u003e−10\u003c/sup\u003e \u003csup\u003e35\u003c/sup\u003e. To remove redundant sequences, proteins with hits to P450 proteins were clustered with a 95% identity threshold using the CD-HIT v4.8.1\u003csup\u003e36\u003c/sup\u003e. Cytochrome P450 proteins were also identified in 14 other species, using the same methodology previously described, to improve the annotation. The 15 species analyzed corresponding to two Cnidaria, five Ecdysozoa, two Lophotrochozoa, two Ambulacraria, three Chordata (\u003cstrong\u003eSupplementary Table\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e). Among the reference proteins it was included those of\u0026nbsp;\u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eD. melanogaster\u003c/em\u003e.\u0026nbsp;The amino acid sequences of the species are available in https://github.com/Imnotabioinformatician/iGluEvolutionChannels/tree/main.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHighly divergent proteins, truncated sequences, or possible false positives were filtered through clustering analyses using the CLANS software\u003csup\u003e37\u003c/sup\u003e. The clustering level was assessed using P cutoff values of 1e\u003csup\u003e-45\u003c/sup\u003e, 1e\u003csup\u003e-50\u003c/sup\u003e, 1e\u003csup\u003e-55\u003c/sup\u003e, 1e\u003csup\u003e-75\u003c/sup\u003e, and 1e\u003csup\u003e-80\u003c/sup\u003e. After this evaluation, a P cutoff value of 1e\u003csup\u003e-50\u003c/sup\u003e was selected according on their connection to the CLAN containing the three reference proteins (Disembodied, Shadow, and Shade proteins). Only the sequences with at least one connection to this CLAN were keep for the posterior phylogenetic analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the phylogenetic analyses, the two main CLANS were selected: one that contains Disembodied, Shadow, and Shade proteins; and other that contains Spook and Phantom proteins (UniProt ID: Q9VRM7and Q9VWR5, respectively). The software MAFFT 7.49\u003csup\u003e38\u003c/sup\u003e was used to align the sequences of main CLAN using the iterative refinement method L-INS-i (parameters: --localpair and --maxiterate 1000). Trimal\u003csup\u003e39\u003c/sup\u003e was used to remove gaps using the gappyout mode. IQ-TREE2 2.0.7\u003csup\u003e40\u003c/sup\u003e, was used to the construction of the phylogenetic tree using with the Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) branch support (1,000 replicates). To select the best model evolutive, the algorithm of ModelFinder\u003csup\u003e41\u003c/sup\u003e incorporated in IQ-TREE2 was activated (-mset), and the best model was selected among WAG, LG, Blosum62, Dayhoff, JTT, and Poisson, based on the Bayesian information criterion. The best model determined for the CLANS containing disembodied and shadow proteins was LG+F+R10 while for spook and phantom proteins was LG+R7. The visualization and annotation of the trees was performed in FigTree v1.4.4 (available from http://tree.bio.ed.ac.uk/software/figtree/). The FASTA files used in each phylogenetic analysis are available in https://github.com/victorcana/P450_Lymnaea-stagnalis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHPLC-MS measurement of 20HE in different tissues\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe central nervous system (n=10), stomach (n=10), kidney (n=10), and hepatopancreas (n=5) were dissected from naïve adult (5-month-old) snails, pooled per tissues in 1.5 mL low-bind Eppendorf tubes, and weighted. Moreover, 100 naïve early-stage and hatched embryos were also pooled in 1.5 mL low-bind Eppendorf tubes and weigthed. First, 200 µL of water was added to each sample. In the first stage, the samples were manually homogenized using plastic pestles. In the second stage, they were individually sonicated on ice using a Sonics \u0026amp; Materials, Inc. VCX 130 ultrasonic homogenizer (53 Church Hill Road, USA) for 1 min at 50% amplitude with a 50% duty cycle. After homogenization, 1 mL of ice-cold methanol was added, and the samples were vortexed for 1 min. Next, the samples were centrifuged at 20,000 × g for 10 min at 7 °C using an Eppendorf 5430 R centrifuge (Hamburg, Germany). After centrifugation, the supernatants were carefully collected and completely removed from the tubes. Three independent replicates were made for all samples.\u003c/p\u003e\n\u003cp\u003eFor lettuce samples, 200 mg of lyophilized lettuce was transferred into 50 mL Falcon tubes, and 20 mL of methanol–water (70:30, v/v) was added to the sample. Extraction was made using the same ultrasonic homogenizer with the same parameters, except that the ultrasonication time was increased to 2 min. After that, the samples were centrifuged at 2,000 × g for 10 min at room temperature, and the supernatants were collected and filtered through 0.2 µm Acrodisc Premium GxF/GHP 25 mm syringe filters (Pall Corporation, Port Washington, NY, USA). Three independent replicates were made.\u003c/p\u003e\n\u003cp\u003eSolid-phase extraction was performed using Strata-XL 100 µm polymeric reversed-phase cartridges (200 mg/6 mL; Phenomenex, Torrance, CA, USA). Initially, the columns were conditioned with 3 mL of methanol and equilibrated with 3 mL of water. The samples were diluted with water to reduce the residual organic solvent content to below 5% (v/v) and were subsequently loaded slowly onto the columns. In the washing step, 3 mL of water–methanol (90:10, v/v) was used, and the analytes were eluted with 1.5 mL of methanol–water (70:30, v/v) in the final phase. The eluted samples were concentrated with a SpeedVac Concentrator Plus (Eppendorf AG, Hamburg, Germany) vacuum concentrator device. Finally, the samples were reconstituted with 100 μL methanol/water/formic acid (60:40:0.1, v/v/v), induced by vortex mixing and ultrasonication.\u003c/p\u003e\n\u003cp\u003eLC-MS analysis was performed using a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Thermo Ultimate 3000 UPLC™ system. Chromatographic separation was performed on a Luna Omega PS C18 reversed-phase column (1.6 µm, 2.1 mm × 150 mm i.d.) (Phenomenex, USA). The mobile phase consisted of two solvents: solvent A was water/formic acid (99.9:0.1, v/v), and solvent B was methanol/isopropanol/formic acid (60:40:0.1, v/v/v). Both eluents contained 10 mM ammonium formate. The flow rate was 150 µL min⁻¹, and the column temperature was maintained at 45 °C. The gradient program included the following steps: from 0.0 to 10.0 minutes, the composition of solvent B increased from 0.0% to 80.0%; from 10.0 to 14.0 minutes, it increased from 80.0% to 100.0%; from 14.0 to 22.0 minutes, it was held at 100.0%; from 22.0 to 25.0 minutes, it decreased from 100.0% to 0.0%; and from 25.0 to 34.9 minutes, it was held at 0.0% for re-equilibration. The mass spectrometer was operated in positive ion mode using parallel reaction monitoring for data acquisition. HESI source conditions were: capillary temperature 300 °C, S-Lens RF level 30, spray voltage 3.5 kV, sheath gas flow 25 (arbitrary units), auxiliary gas flow 8 (arbitrary units). The resolution was set to 35,000, and the quadrupole isolation window was set to 2 Da. For molecular ion fragmentation, a normalized collision energy of 15 was used. The most intense precursor-to-fragment transitions were used for analysis of 20HE: 481.31 m/z → 445.29 m/z and 371.22 m/z. The\u0026nbsp;transitions and the optimal ionization condition were derived from our recorded mass spectra using a 10 ng/mL standard solution prepared from 20HE analytical standard (#H5142, Merck).\u003c/p\u003e\n\u003cp\u003ePeak detection and quantification were achieved using the Xcalibur 4.2 software. The ions under observation were verified based on specific criteria: accurate MS1 mass, retention time, MS2 masses, and the pattern of fragmentation. The production was used for the quantitation. A six-point calibration curve was generated for the quantitative analysis using 0, 0.5, 1, 5, 10, 50, and 100 ng/mL standard solutions. The correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) for the calibration curve was 0.99 (not shown).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was made with the OriginPro 2018 software (OriginLab Corp., USA). The normality of the datasets was investigated using the Shapiro-Wilk test and the homogeneity of variances between groups was investigated using the Levene-test. In the case of hatching, heart rate, locomotion, and feeding, the nonparametric Scheirer-Ray-Hare test were applied to study the effect of time, treatment, and time x treatment interaction. This analysis was followed by ANOVA and Scheffe’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test to identify significant differences between control and treatment groups at a given time point. The qRT-PCR data were analyzed with ANOVA and Scheffe’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the recommendations of the ARRIVE 2.0. guideline were considered and followed during the experiments, analysis, and data reporting.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of conflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Brain Project (#NAP2022-I-10/2022, Z.P.), the Hungarian Scientific Research Fund (#138039, Z.P.; #146787, I.F.), the Biotechnology and Biological Sciences Research Council (#BB/W010305/2, L.A.Y.-G.), the Royal Society (#RG\\R1\\241397, L.A.Y.-G.), and the Thematic Excellence Program (#TKP2021-EGA-17, J.S.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRS: Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing, Data curation, Visualization; LAYG: Methodology, Investigation, Writing \u0026ndash; review \u0026amp; editing, Visualization, Funding acquisition; VHCB: Methodology, Investigation, Writing \u0026ndash; review \u0026amp; editing, Visualization; JS: Methodology, Investigation, Writing \u0026ndash; review \u0026amp; editing, Data curation, Visualization, Funding acquisition; ZP: Writing \u0026ndash; review \u0026amp; editing, Funding acquisition; IF: Conceptualization, Methodology, Investigation, Writing \u0026ndash; original draft, Data curation, Visualization, Funding acquisition. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Dr. K\u0026aacute;lm\u0026aacute;n Tapolczai (HUN-REN Balaton Limnological Research Institute) for his help in the statistical analysis. Open access funding was provided by the Hungarian Research Network (HUN-REN).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSupplementary Tables, Supplementary Figures, and Lymnaea sequence data, chromatograms and MS/MS spectra are available in the Supplementary information file. The FASTA files used in each phylogenetic analysis are available in https://github.com/victorcana/P450_Lymnaea-stagnalis. The amino acid sequences of the 15 species are available in https://github.com/Imnotabioinformatician/iGluEvolutionChannels/tree/main. 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Met.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 587\u0026ndash;589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nmeth.4285\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.4285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ecdysone, mollusk, Lymnaea stagnalis, embryogenesis, synthesis, receptor","lastPublishedDoi":"10.21203/rs.3.rs-8193722/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8193722/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEcdysone-controlled pathways regulate ecdysozoan development through both a nuclear (nEcR) and a membrane receptor (mEcR). Ecdysone signaling was long thought to be exclusive to ecdysozoans; however, studies since 2010 have demonstrated the presence of an orthologue of the nEcR/NR1H receptor in other protostome taxa, including mollusks and annelids. Nevertheless, there is still no clear consensus regarding the presence or functional role of ecdysone-like signaling in mollusks. This study aimed to advance our understanding of the evolution of ecdysone signaling by investigating the potential synthesis and physiological role of 20-hydroxyecdysone (20HE) in the great pond snail (\u003cem\u003eLymnaea stagnalis\u003c/em\u003e) by applying a complex experimental approach. Exposure to different concentrations (10 ng/L, 100 ng/L, and 1 \u0026micro;g/L) of 20HE had no effect on hatching, heart rate, locomotion (gliding), or feeding in \u003cem\u003eLymnaea\u003c/em\u003e embryos. Furthermore, the treatments did not alter the expression of \u003cem\u003eLymnaea\u003c/em\u003e homologs of \u003cem\u003eDrosophila\u003c/em\u003e nEcR and mEcR. Using cluster analysis and phylogenetics to resolve the evolution of Halloween genes involved in ecdysone synthesis, we found no clear homologues of Spook, Disembodied, or Shadow in any lophotrochozoan species, while Phantom and Shade appear to have a many to many homologues in lophotrochozoans. Although mass spectrometric analysis detected trace concentrations of 20HE in various tissues of adult \u003cem\u003eLymnaea\u003c/em\u003e specimens, we suggest that its presence is of dietary origin. Our findings clearly indicate that an endogenous ecdysone synthesis pathway is not present in \u003cem\u003eLymnaea\u003c/em\u003e and that 20HE has no effect on the physiology of \u003cem\u003eLymnaea\u003c/em\u003e embryos. Although further experiments are required on other molluscan models as well, we propose that molluscan sequences homologous to \u003cem\u003eDrosophila\u003c/em\u003e nEcR and mEcR are unlikely to function as ecdysone receptors, and that ecdysone-like signaling is absent in lophotrochozoans.\u003c/p\u003e","manuscriptTitle":"Functional and evolutionary evidence from a widely used molluscan model reveals the absence of ecdysone signaling in lophotrochozoans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 12:47:36","doi":"10.21203/rs.3.rs-8193722/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-12-09T00:32:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T00:11:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-08T16:54:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-05T12:09:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-26T19:08:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ccce1e3-7fd3-4cc8-a3f2-773bd29490b1","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59343576,"name":"Biological sciences/Ecology"},{"id":59343577,"name":"Earth and environmental sciences/Ecology"},{"id":59343578,"name":"Biological sciences/Evolution"},{"id":59343579,"name":"Biological sciences/Molecular biology"},{"id":59343580,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-12-11T12:47:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 12:47:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8193722","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8193722","identity":"rs-8193722","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}