Low extent of sex-based venom variation in Earth′s most widespread viper, the common adder (Vipera berus)

proteomics, cytotoxicity, coagulotoxicity, bioactivity, snake bite, snake venom (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint 2 Introduction Snake venom s ar e complex mixtures of several bioactive molecules, mainly proteins and peptides, able to alter the physiological balance of the organism into which they are injected (Casewell et al., 2013). Extensive evidence suggests that the main selective driver of snake venom evolution and variation is predation (Daltry et al., 1996b; Barlow et al., 2009; Holding et al., 2021). Furthermore, various factors have been shown to affect the type and abundance of snake venom compounds, thus determining remarkable compositional and functional variation at all taxonomic levels . Indeed, while interspecific venom variation has long been acknowledged, intraspecific variation has recently started to be recognized and investigated (Casewell et al., 2020). Among the drivers of intraspecific venom variation identified so far are differences in geographic origin (Zancolli et al., 2019; Avella et al., 2023), life history stages (Cipriani et al. , 2017; Avella et al. , 2022; Ferreira -Rodrigues et al. , 2024) , and sex of the specimens considered (Menezes et al., 2006; Ferreira -Rodrigues et al., 2024). Sex-related venom variation is expected in taxa that employ their venom for intraspecific competition and/or displaying sexually dimorphic feeding or dispersal ecologies (Schendel et al., 2019; Lüddecke et al., 2022). Considering snakes, evidence of venom being used for intraspecific competition is currently lacking, and male and female conspecifics typically present virtually identical diets (Madsen and Shine, 1994; Bonnet et al., 1998; Avolio et al., 2006; Do et al., 2023). Therefore, while intraspecific venom variation can often be explained by differences in diet and/or prey availability (e.g., between snakes from different localities or age), the occurrence of sex-related venom variation remains a conundrum. To date, a limited number of studies have investigated this question within members of the family Viperida e, with pit vipers ( subfamily Crotalinae) being the major model group . For instance, Menezes et al. (2006) detected variation in venom composition and activities between male and female Bothrops jararaca siblings, suggesting it to be genetically inherited and imposed by evolutionary forces. Similarly, Zelanis et al. (2016) demonstrated the presence of sex -related compositional and functional variation in the same species. Furthermore, Furtado et al. (2006) proposed that the observed differences in composition and bioactivity between females and males might be attributed to sexual dimorphism associated with different diets. Studies in Bothrops moojeni also indicate a pronounced sex -based venom variation within this species (Hatakeyama et al. , 2021; Ferreira -Rodrigues et al. , 2024) , alongsid e documented differences in diet where males and juveniles feed mainly on ectotherms, while the considerably larger adult females prefer mammalian prey (Nogueira et al., 2003). Similar claims have been made across a number of other members of the genus Bothrops (Machado Braga et al., 2020; Hatakeyama et al., 2020; Kallel et al., 2024). However, studies in Bothrops jararacussu (Aguiar et al., 2020) and Bothrops asper (Gómez et al., 2021) found no significant differences between male and female venoms, despite marked sexual dimorphism . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Furthermore, proteomics on the venom of Tropidolaemus wagleri , a species of pit viper presenting remarkable sexual size dimorphism, did not reveal noteworthy differences in venom composition and lethality between sexes (Tan et al., 2017). Besides pit vipers, venom variation has been sporadically investigated in other viperid snakes. For instance, several SDS-PAGE profiling studies on Old World vipers (subfamily Viperinae) showed at least subtle differences between male and female venoms (Marsh and Glatston, 1974; Mebs and Kornalik, 1984) . Recent proteomic studies on the genus Vipera (Petras et al., 2019; Avella et al., 2023; Lakušić et al. , 2025a) have identified varying degrees of sex -based differences. However, these differences were generally considered to have an insignificant impact on venom composition, although bioactivity assessments remain lacking. Given this, it is clear that the underlying drivers and the extent of these differences require further investigation. The common adder (Vipera berus) represents a promising model species to investigate sex - based snake venom variation. It is a medically important snake with the widest distribution in the world, ranging from the United Kingdom to North Korea and southeastern Russia (Figure 1, A), where it occurs in a variety of habitats (Speybroeck et al., 2016, 2020; Geniez, 2018; World Health Organisation , 2024). As in several other vipers, V. berus presents a marked ontogenetic shift in diet, with juveniles mostly feeding on ectotherm prey (e.g., reptiles and amphibians), and gradually incorporating more endotherms (e.g., small mammals and birds) in their diet as they grow (Otte et al., 2020; Samsonov et al., 2022). Furthermore, V. berus exhibits sexual dimorphism in color and size (Figure 1, B), with females being generally longer and bulkier than males, likely to facilitate reproduction (Forsman, 1991; Madsen and Shine, 1994). To date, whether this sexual dimorphism is associated with differences in diet composition between male and female common adders rem ains inconclusive (Forsman, 1991). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Figure 1 – The common adder, Vipera berus. Geographic distribution of V. berus (green), based on data from the International Union for Conservation of Nature (IUCN; accessed 20 March 2024). (B) Representative male (left) and female (right) V. berus specimens, illustrating sexual dimorphism in coloration and tail morphology. The male exhibits a whitish body with a distinct dark zigzag dorsal pattern, whereas the female displays a pale brownish body with a less contrasting brownish zigzag pattern. Tail morphology differences are highlighted. The distribution was plotted using R version 4.2.2 (R Core Team, 2024). Credits: Harry Wölfel Due to its wide distribution, V. berus is frequently involved in snakebite incidents and is thus listed among the snakes of the highest medical relevance in Europe (Hermansen et al., 2019; Di Nicola et al., 2021). Proteomic studies identified phospholipases A2 (PLA2s), snake venom metalloproteinases (svMPs), and snake venom serine proteases (svSPs) as major enzymatic components within the venom of this species (Ramazanova et al., 2008; Latinović et al., 2016; Bocian et al., 2016; Al-Shekhadat et al., 2019; Damm et al., 2021). Intriguingly, several studies have highlighted the presence of considerable variation in the diversity and abundance of venom compounds within V. berus, indicating differences related to several factors, such as regional, ontogenetic, and individual variation (Mebs and Langelüddeke, 1992; Nedospasov Mongolia China Kazakhstan Russia Ukraine PolandGermany France United Kingdom Norway Sweden Finland Romania North Korea Japan Belarus Türkiye Italy B) A) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint and Rodina, 1992; Malina et al., 2017). Similar patterns were observed in venoms of V. berus subspecies, such as V. berus barani (Damm et al., 2024) and V. berus nikolskii (Kovalchuk et al., 2016), as well as closely related species e.g. Vipera seoanei (Avella et al., 2023), Vipera kaznakovi (Petras et al. , 2019) , and Vipera ammodytes (Lakušić et al. , 2025a; b) . Taken together, this suggests that the extent of intraspecific venom variation within V. berus is likely underestimated, and the underlying factors are yet to be linked to conclusive patterns. This particularly concerns sex-based venom variation, which may occur within this species as per its sexual dimorphism. Here, we present the first in-depth investigation of sex-based variation in V. berus venom. We tested for qualitative differences betwe en the p ooled venoms of male and female adult specimens of the same region by comparing SDS-PAGE, RP-HPLC profiles, and shotgun proteomes. Furthermore, we screened the bioactivity of the venoms based on previously described dominant enzymatic toxin families (Latinović et al., 2016; Bocian et al., 2016; Al - Shekhadat et al., 2019; Damm et al., 2024; Nicolaysen et al., 2024), their modes of action, and clinical reports of V. berus envenomation (Persson, 2014; Hermansen et al., 2019; Di Nicola et al., 2021; Siigur and Siigur, 2022). Therefore, we performed bioassays aiming to elucidate protease and PLA2 activities of the venoms, as well as the role they play in activation of Factor Xa (FXa)-like, thrombin-like, and plasmin-like blood-coagulation-related enzymes. Finally, we assessed the venoms impact on the cell viability of two different mammalian cell lines (i.e., MDCK II and Calu-3), their effect on the coagulation time of human plasma, and their hemolytic effect on equine erythrocytes. 3 Materials and Methods 3.1 Venom Venoms were gifted by members of the “Terrarienclub Bayreuth und Umgebung e.V.”, and were sourced from captive-bred, adult V. berus specimens originating from northern Bavaria (Germany). The venoms were non-invasively collected by letting each viper bite a parafilm - covered microtube without applying any pressure to the venom glands. The obtained venom samples were collected individually and stored on dry ice until lyophilization. Individual venoms of six male and six female vipers were weighed and then redissolved in double-distilled water (ddH2O) prior pooling by sex. Aliquots were lyophilized and stored at –20 °C until analysis. Specimens’ sex, body size, and dry venom amount are reported in Supplementary Table 1. 3.2 Compositional profiling of the venoms 3.2.1 Reducing- and non-reducing SDS-PAGE Venom profiling based on sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS - PAGE) was carried out following the procedure previously applied by our group (see Schulte (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint et al. , 2023) . Therefore, r educing and non -reducing SDS-PAGE with 5 µg of venom was carried out for both venom pools. Raw gel image is provided in Supplementary Figure 1. 3.2.2 RP-HPLC profiling For reverse-phase high-performance liquid chromatography (RP-HPLC), the protocol applied by Schulte et al., 2023 was adjusted as follows: 200 µg of lyophilized venom was reconstituted in ddH2O with 5% (v/v) acetonitrile ( MeCN) and 1% (v/v) formic acid (HFo) to a final concentration of 10 mg/ml. After centrifugation for 10 min at 12,000 × g, the supernatant (20 µL) was measured on a reverse -phase Discovery BIO wide Pore C18 -3 column (4.6 × 150 mm, 3 µm particle size; Supelco) operated by a n HPLC Agilent 1100 (Agilent Technologies) chromatography system. The column was heated to 40 °C and the following gradient with solvent A (ddH2O with 0.1% (v/v) HFo ) and solvent B (MeCN with 0.1% (v/v) HFo ) at a flow rate of 1 ml/min was used, given at min (B%): 0-5 (5% const.), 5-65 (5 to 45%), 65-75 (40 to 70%), 75-80 (95% const.), and 10 min re -equilibration at 5% B. The chromatograms were detected by a diode array detector (DAD) at λ = 214 nm wavelength (360 nm reference). A previous blank run , injected with 20 µl of ddH2O with 5% (v/v) MeCN and 1% (v/v) HFo, centrifuged for 10 min at 12,000 × g, was measured under identical parameters and subtracted from the venom profile s. Raw measurements at λ = 214 nm are provided in Supplementary Table 2. 3.2.3 Shotgun proteomics In preparation for the shotgun mass spectrometry (MS) approach, 50 µg of lyophilized venoms were dissolved to a final concentration of 1.7 µg/µl in an aqueous solution of 6 M Guanidinium hydrochloride (GdnHC l) and 100 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris/HCl, pH 8.5) for complete denaturation. Further, the venoms were transferred to Protein LoBind tubes ( 0030108116, Eppendorf), incubated for 30 min at 37 °C with 10 mM Dithiothreitol (DTT) for disulfide reduction, and followed by incubation for 30 min at 22 °C in the dark with 40 mM chloroacetamide for alkylation of free thiols. Afterwards, Trypsin/LysC mix (V5071, Promega) was added at a 1:50 protease -to-protein ratio and incubated for 1 hour at 37°C and 500 rounds per minute (RPM) shaking. The samples were then diluted 1:7 with 50 mM Tris/HCl (pH 8.0) to decrease the GdnHCl concentration below 1 M and to reactivate trypsin. The digestion was continued overnight at 37 °C and 500 RPM shaking and stopped the next day by adding 1.5 % trifluoroacetic acid . The resulting peptides were desalted and cleaned-up using C18-Chromabond columns (730011, Macherey-Nagel). The eluted peptides were dried using a vacuum -concentrator plus (Eppendorf) and redissolved in 20 μl of an aqueous solution with 5% (v/v) MeCN and 0.15% (v/v) HFo, by vortexing. The peptides were transferred to 96-well PCR plates (PCR-96-FS-C, Axygen), sonicated for 5 min in a water bath, and loaded for LC-MS/MS analysis. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Prior to MS, a coupled chromatographic separation of the peptides was performed on a n UltiMate 3000 RSLCnano device (Thermo Fisher Scientific). From the prepared venoms, we loaded 5 μl into a PepMap Neo Trap column (Thermo Fisher Scientific) for concentration and desalting, followed by separation using a 50 cm µPAC column (PharmaFluidics, Thermo Fisher Scientific) connected with a TriVersa NanoMate (Advion) robot for chip -based nano electrospray ionization. Throughout the analysis, the analytical column was kept in an oven at 35°C. Peptide elution was performed using a linear gradient of buffer A (HPLC-grade H2O with 0.1 % (v/v) HFo) and buffer B (MeCN with 0.1% (v/v) HFo) at flow rate of 0.7 µl/min, given at min (%B): 0-90 (5–35%), 90-100 (35-85%), 100-108 (cons. 85%), and equilibrated for 10 min at 5% buffer B. MS analysis of the peptides was carried out on an Orbitrap Eclipse Tribrid MS (Thermo Fisher Scientific) in positive mode. The NanoMate -assisted electrospray was established by applying 1.7 kV; the source temperature was set to 250 °C. MS2 spectra were obtained in data-dependent acquisition (DDA) with CID fragmentation and data-independent acquisition (DIA) with HCD fragmentation. We used Xcalibur v4.3.73.11. (Thermo Fisher Scientific) for data acquisition and analysis. Protein identification was performe d with PEAKS 12 (Bioinformatics Solution Inc.) and searched against the UniProt database (taxonomy: “serpentes”; status: “reviewed”; “canonical and isoforms”; accessed 11.02.2025). Analyzing the DDA data, precursor ion mass tolerance set to 15 ppm with 3 missed cleavage s allowed and carbamidomethylation set as fixed modification. Fragment ion mass tolerance in linear ion trap MS2 detection was set to 0.5 Da, and the false discovery rate was limited to 0.1. For the qualitative analysis, we only considered proteins that were identified with a –10lgP score of at least 15 and at least two unique peptides. For the analysis of the DIA data, the combined spectral library from male and female DDA analysis was used , applying the same database and settings as for the DDA analysis. The detailed list of parameters for data acquisition, analysis, and annotation is provided in Supplementary Table S 3. A comprehensive list of all annotated proteins is provided in Supplementary Table S4. 3.3 Enzymatic activity profiling Investigating the venom composition and bioactivity we adopted and modified previously published protocols for enzymatic activity assays on protease, PLA2, FXa-like, thrombin-like, and plasmin -like activit ies (Schulte et al., 2024; Avella et al. , 2024), as well as effect s on mammalian cell viability (Hurka et al., 2022) and hemolysis (Avella et al., 2024). All analyses were performed in at least triplicate. Unless otherwise stated , measured signals at specific wavelengths were averaged (x̅ ), subtracted by their respective negative control, and normalized against their respective positive control: (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = x̅ 𝑠𝑎𝑚𝑝𝑙𝑒 − x̅ 𝑛𝑒𝑔. 𝑐𝑡𝑟𝑙 x̅ 𝑝𝑜𝑠. 𝑐𝑡𝑟𝑙 − x̅ 𝑛𝑒𝑔. 𝑐𝑡𝑟𝑙 3.3.1 Protease activity assay The protease activity of the venoms was assessed using the non -specific Protease Activity Assay Kit (Calbiochem, cat. no. 539125) for the 96-well plate format. Venoms were redissolved (Reaction buffer 1:6 ddH 2O) and added to the substrate (final concentrations 400, 200, 100, 50, and 25 μg/ml), as well as the negative control (Reaction buffer 1:6 ddH 2O, 0%) and the positive control (166 µg/ml trypsin in Reaction buffer 1:6 ddH 2O, 100%). After incubation for 2 h at 37 °C and shaking at 120 rpm on a Multitron device (Infors HT), t he absorption was measured at λ = 492 nm on a Synergy H4 Hybrid Microplate Reader (BioTek) operated using Gen 5 v2.09 software (BioTek). Raw data from the protease activity assay is provided in Supplementary Table S5. 3.3.2 Phospholipase A2 assay Phospholipase A2 activity was assessed using the EnzChek Phospholipase A 2 Assay Kit (Invitrogen, cat. no. E10217) for the 96-well plate format. Venoms were redissolved in ddH2O and mixed with the substrate (final concentrations: 50, 25, 12.5 , 6.25, and 3.125 µg/ml), as well as the negative control (Reaction Buffer 1: 20 ddH2O, 0%) and the positive control (5 U/ml purified bee venom phospholipase A2 in Reaction buffer 1:20 ddH2O, 100%). After incubation for 1 h at room temperature, the plate s were measured on a Synergy H4 Hybrid Microplate Reader (BioTek) operated using Gen 5 v2.09 software (BioTek), with excitation set to λ = 470 nm and absorbance set to λ = 515 nm. Raw data from the phospholipase A2 activity assay is provided in Supplementary Table S6. 3.3.3 Factor Xa-like activity assay The Factor Xa (FXa)-like activity was assessed using the FXa Activity Fluorometric Assay Kit (MAK238-1KT, Sigma -Aldrich) for the 96 -well plate format. Lyophilized venoms were redissolved (Factor Xa Assay Buffer 20: 1 ddH 2O) and added to the substrate (final concentrations 50, 25, 12.5, 6.25, and 3.125 μg/ml), as well as the negative control (FXa Assay Buffer 1:20 ddH2O, 0%) and the positive controls (2 µg/ml FXa Enzyme Standard in FXa Assay Buffer 20 :1 ddH 2O, 100%). The assay was transferred to a Synergy H4 plate reader (H4MLFPTAD, BioTek) operated using Gen 5 v2.09 software (BioTek) for 15 min of incubation at 37 °C, protected from light. The FXa activation was measured, with excitation set to λ = 350 nm and fluorescence detection at λ = 450 nm. Raw data from the FXa activity assay is provided in Supplementary Table S7. 3.3.4 Thrombin-like activity assay The thrombin-like activity was assessed using the Thrombin Activity Fluorometric Assay Kit (Sigma-Aldrich, MAK242) for the 96 -well plate format . Venoms were redissolved (Thrombin (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Assay Buffer 1:20 ddH2O) and added to the substrate (final concentrations 50, 25, 12.5, 6.25, and 3.125 µg/ml ), as well as the negative control (Thrombin Assay Buffer 1 :20 ddH2O, 0%) and the positive control (0.3 µg/ml Thrombin Enzyme Standard in Thrombin Assay Buffer 20:1 ddH2O, 100%). In a standardized pre-measurement procedure, the assay was handled for 5 min at room temperature to define a replicable starting time point, before being transferred to a Synergy H4 plate reader (H4MLFPTAD, BioTek) operated using Gen 5 v2.09 software (BioTek). After 15 min of incubation at 37 °C, protected from light, the thrombin activation was measured, with excitation set to λ = 350 nm and fluorescence detection at λ = 450 nm. Raw data from the thrombin activation assay is provided in Supplementary Table S8. 3.3.5 Plasmin-like activity assay The plasmin-like activity was assessed using the Plasmin Activity Assay Kit (MAK244, Sigma- Aldrich) for the 96-well plate format. The lyophilized venoms were suspended in Plasmin Assay Buffer 20:1 ddH2O) and added to the substrate (final concentrations 50, 25, 12.5, 6.25, and 3.125 μg/ml), as well as the negative control (Plasmin Assay Buffer 1:20 ddH2O, 0%) and the positive controls (5 µg/ml Plasmin Enzyme Standard in Plasmin Assay Buffer 20:1 ddH 2O, 100%). The assay was transferred to Synergy H4 plate reader (H4MLFPTAD, BioTek) operated using Gen 5 v2.09 software (BioTek), for 15 min of incubation at 37 °C , protected from light. Plasmin activation was measured with excitation set to λ = 360 nm and fluorescence detection at λ = 450 nm. Raw data from the plasmin activation assay is provided in Supplementary Table S9. 3.4 Plasma coagulation assays The assessment of the venom induced coagulation time (VICT), the prothrombin time (PT) and the activated partial thromboplastin time (aPPT), was performed in a setup with platelet -poor plasma and a microplate reader (Pratt and Monroe, 1992) refined for snake venom -induced coagulation studies (Kerns et al., 1999; Banerjee et al., 2005; Barnwal et al., 2016). Human blood was obtained according to DIN 58905 from a healthy volunteer with no diagnosed circulatory or coagulation disease or medication intake before donation. To obtain platelet-poor plasma (PPP), the blood was added (9:1) to a sterile-filtered sodium citrate solution (0.11 mol/l) and centrifuged for 15 min at 2500 × g. The supernatant, essentially platelet-free plasma, was aliquoted and used immediately or flash -frozen in liquid nitrogen to be stored at -80 °C until further use. Preliminary to the assay, PPP aliquots were thawed and kept at room temperature at a maximum of 4 h. Setting up the assay, 25 µl of each PPP and lyophilized venom (4x final concentration of 10, 1, 0.1 , and 0.01 µg/ml) reconstituted in Tris-Buffered Saline (TBS) were added to a transparent, flat-bottom 96-well plate. Depending on the assessment, 25 µl of the following were added: (i) TBS for the VICT; (ii) TBS with 5% Dade®Innovin ( B4212-50, Siemens Healthcare) for the PT; (iii) DAPTTIN®TC (5035060, Technoclone) for aPPT. Plates (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint were transferred to a microplate reader (CLARIOstar Plus, BMG Labtech) with the thermostat set at 37 °C and absorbance monitored at λ = 405 nm in 5 s intervals for 2 min before 25 µl of CaCl2 (25 mM, final concentration: 6.25 mmol/l) were injected automa tically via the internal injection system (t = 0) and monitoring continued for 8 min. The controls included a venom - free sample for each assessment, a sample without Dade®Innovin for the VICT and PT, and a sample without CaCl 2 injection for the aPPT , substituted each by 25 µl TBS. T here is no universally accepted standardized method to determine optically measured coagulation time, so we used the time point when maximum fibrin deposition occurs. This is identical to the highest change of absorption signal. This was determined by calculating the first derivative of absorbance at λ = 405 over time: 𝑓(𝑥)′ = 𝛥𝐸/ΔT E = Absorbance (λ = 405 nm) T = Time (s) Raw and processed data on venom-induced coagulation-, prothrombin- and activated partial thromboplastin time assay are provided in Supplementary Table S10-S13. 3.5 Cytotoxic activity profiling 3.5.1 Cell viability assay The cytotoxicity of venoms was assessed in Madin -Darby canine kidney II (MDCK II; kindly provided by Prof. Dr. Eva Böttcher -Friebertshäuser, Institute of Virology, Philipps University, Marburg) and human epithelial lung adenocarcinoma (Calu-3; SCC438, Merck) cell lines using the CellTiter -Glo Luminescent Cell Viability Assay (G7570, Promega). The venoms were redissolved in cultivation medium and added to the plate (final concentrations 50, 25, 12.5, 6.25, and 3.125 µg/ml), as well as the negative control (cultivation medium, 100% cell viability) and the positive control (100 µ M ionomycin in DMSO, 0% cell viability). Confluent cells were treated with the venom/controls and incubated at 37 °C in a 5% CO 2 atmosphere. After 48 h, luminescence was measured according to the manufacturer's protocol on a Synergy H4 Hybrid Microplate Reader operated using Gen 5 v2.09 software (BioTek). Raw data from the cell viability assay is provided in Supplementary Table S14. 3.5.2 Hemolytic activity assay The hemolytic activity was assessed in Dulbecco’s phosphate-buffered saline (DPBS) and additional DBPS with supplemented Ca 2+ and Mg2+ (DPBS+), the latter to saturate potential cofactors. Lyophilized venom was redissolved in the respective buffer and added to the purified erythrocytes (final concentrations 50, 25, 12.5, 6.25, and 3.125 μg/ml), as well as the negative control (DPBS/DPBS+ 20:1 ddH 2O, 0% lysis) and the positive control ( 1% Triton X -100 in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint DPBS/DPBS+ 20:1 ddH2O, 100% lysis). After incubation at 37°C for 1 h, the absorbance was detected at λ = 405 nm with a Synergy H4 plate reader (H4MLFPTAD, BioTek) operated using Gen 5 v2.09 software (BioTek). Raw data and buffer receipt from the hemolytic assay are provided in Supplementary Table S15. 3.6 Data visualization The distribution shown in Figure 1 was generated in R R (R Core Team, 2024) using the following packages: tmap (Tennekes, 2018), sf (Pebesma, 2018; Pebesma and Bivand, 2023), dplyr (Wickham et al., 2023), magrittr (Bache et al., 2022), purr (Wickham et al., 2025), raster (Hijmans et al., 2025), rnaturalearth (Massicotte et al., 2023), rnaturalearthdata (South et al., 2024), rnaturalearthhires (South et al. , 2025) , and elevatr (Hollister et al. , 20 23). The chromatographic profiles, proteomic data, and bioactivity results were visualized using Origin 2020b. 4 Results 4.1 Venom yield Figure 2 – Body size positively correlates with venom yield in Vipera berus. The graph shows the correlation of the specimen’s size and amount of dry venom for male and female V. berus. The linear fitting curve is represented as a dashed line. The insets display the distribution, mean value, and standard distribution of measured dry venom (upper) and body size (lower) separated by sex. 50 52 54 56 58 60 0 2 4 6 8 10 Male Female Dry venom (mg) Body size (cm) 50 52 54 56 58 60Body size (cm) 0 2 4 6 8 10Dry Venom (mg) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint The sampled V. berus specimens measured from 50 to 59 cm in total length, and their venom yields ranged between 0.783 and 9.708 mg (dry weight) (Figure 2). Females were generally larger than males (average: male 53 cm vs. female 57 cm ) and usually provided higher amounts of venom (average: male 2.380 mg vs. female 4.436 mg). In general, the amount of retrieved dry venom tended to increase with the specimen's body size. 4.2 Compositional profiling of Vipera berus venoms To investigate compositional differences between the V. berus venoms from specimens of different sexes, we analyzed their electrophoretic profiles obtained by performing SDS-PAGE under reducing and non-reducing (Figure 3 A, B) conditions. We assigned the obtained bands to putative toxin families based on the information reported in previous works (Latinović et al., 2016; Bocian et al., 2016; Al-Shekhadat et al., 2019). The SDS-PAGE at reducing conditions (Figure 3 A) revealed that for both venom pools the protein mass ranges from 12 to 70 kDa. The most intense bands cluster at 13-15 kDa, 30 kDa, 35 kDa, and 55 -60 kDa. Less conspicuous bands are detectable at 12 kDa, 18 kDa, 23 -25 kDa, 35 -43 kDa, 48 -55 kDa, and 60 -70 kDa. While differences in band diversity are not apparent, the bands at 13 kDa and 23 kDa appear slightly more intense in the female sample. Under non-reducing conditions (Figure 3 B), the protein masses range from 12 to 115 kDa for both venoms. The most intense bands appear to be at 16 kDa, 23 kDa, 24 kDa, 32 kDa, and 50 kDa. Less intense bands are visible at 12 kDa, 13 -15 kDa, 19-22 kDa, 26-29 kDa, 55-60 kDa, 60-65 kDa, 80-85 kDa, 90-95 kDa, and 110-120 kDa. No dominant differences in band diversity are evident, but the bands at 25 kDa and 60 kDa appear slightly more intense in the female venom. The chromatograms of the venoms present 45 peaks in total. Both profiles appear largely similar, and only minor differences could be detected. For instance, peak 20 is seemingly absent in the male venom. Add itionally, peaks 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, and 16 are slightly higher in the male venom, whereas peaks 33, 34, 35, 36, 37, 41, and 44 are slightly higher in female venom. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Figure 3 – Venom profiling of adult male and female Vipera berus specimens. SDS-PAGE profiling under A) reduced and B) non-reduced conditions, and C) RP-HPLC profile (absorbance (mAU) at λ = 214 nm)) . The initial peak (0) corresponds to the sample injection. Abbreviations: 5N, 5′-nucleotidase; AP, aminopeptidase; CTL, C-type lectin, snaclec, and C-type lectin-related protein; HYAL, hyaluronidase; LAAO, L-amino acid oxidase; NGF, nerve-growth factor; PLA2, phospholipase A2; PLB, phospholipase B; PDE, phosphodiesterase; QC, glutaminyl cyclase; svMP, snake venom metalloproteinase; svSP, snake venom serine protease; VEGF, vascular endothelial growth factor. 250 150 100 75 50 37 25 20 15 10 kDa 250 150 100 75 50 37 25 20 15 10 kDa putative toxin families 5N, LAAO, HYAL, PDE, PLB, svMP P-II/ P-III, svSP AP, svMP P-II/ P-I, svSP, QC CRISP, svSP CTL, DI, NGF, PLA2, VEGF 5N, LAAO, PDE, svMP P-III AP, svMP P-I, svSP, QC CRISP, CTL, NGF, PLA2, svSP CTL, DI, PLA2, VEGF 5N, LAAO, HYAL, PLB, svMP P-II/ P-III, svSP putative toxin families A) B) C) 0 20 40 60 80 -50 0 50 100 150 200 250 300 350 Male Female Absorbance (mAU) Time (min) 0 25 50 75 100 MeCN MeCN (%) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Figure 4 – Comparative shotgun proteomics of the venom of adult male (A & D) and female (B & E) Vipera berus. Proteomic data was retrieved in data-dependent acquisition (DDA, A & B) and data-independent acquisition (DIA, D & E ) mass spectrometry mode, respectively . The Venn diagram C) displays the total annotated proteins, exclusively for each approach and shared between approaches. Furthermore, qualitative venom proteomes (B -E) are presented with the relative diversity of protein families for the respective approach, based on the total number of identified protein groups (n). Abbreviations: svSP, snake venom serine protease; PLA2, phospholipase A2; svMP, snake venom metalloproteinase; CTL, C-type lectin inclusive C-type lectin-like protein and snaclec; LAAO, L-amino acid oxidase. “Other” includes protein families that comprise less than 5% of total protein family diversity: CRISP, cysteine-rich secretory proteins; KUN, Kunitz -type inhibitor; DI, disintegrin; PDE, phosphodiesterase; AP, aminopeptidase; PLB, phospholipase B; VEGF, vascular endothelial growth factor F; 5N, 5’-nucleotidase; NGF, nerve growth factor; HYAL, hyaluronidase; QC, glutaminyl cyclase; C3, venom complement C3 homolog; NP, natriuretic peptide; UNSPEC, venom non-specific component. Furthermore, we assessed for qualitative differences in venom composition via shotgun proteomics using DDA and DIA mass spectrometry (Figure 4). In DDA, we annotated a total of 179 proteins, of which 106 were shared between both sexes. 36 were exclusively found in male venom and 37 in female venom. (Figure 4, C) Those proteins were further clustered into protein groups and annotated. 97 protein groups were identified in the male venom (Figure 4, A), and 99 in the female venom (Figure 4, B). In DIA mode, 265 total proteins were annotated, Other 21.6% LAAO 9.3% svMP 16.5% CTL 13.4% PLA2 13.4% svSP 25.8% n = 97 Other 16.5% LAAO 7.5% svMP 16.6% CTL 15.1% PLA2 16.6% svSP 27.8% n = 134 Other 17.7% LAAO 8.5% svMP 15.6% CTL 13.5% PLA2 17% svSP 27.7% n = 141 Other 16.2% LAAO 12.1% svMP 18.2% CTL 9.1% PLA2 17.2% svSP 27.3% n = 99 A) B) D) E) DDA DIA 804 8 17 15 47 35 2 147 13 13 8 7 11 C) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint with 153 shared between males and females. 60 were only found in male venom and 52 only found in female venom (Figure 4, C) . Clustering into protein groups resulted in 141 hits for male venom (Figure 4, D) and 134 for female venom (Figure 4, E). Subsequent classification of protein groups revealed that five protein families, svSPs, PLA2s, svMPs, C-Type lectins (CTLs, including C -type lectin related proteins and snaclecs) , and L- amino acid oxidases (LAAOs), contain more than 5% of total protein family diversity in both sexes. In all cases, svSPs show the highest diversity of venom components ( Male: DDA 25.8%, DIA 27.7%; Female: DDA 27.3%, DIA, 27.8%). In DDA, the non-enzymatic CTLs follow next in diversity for both sexes (male 16.5% and female 18.2%), continued in male venom by PLA2s and svMPs equally (13.4%), as well as LAAO s (9.3%), while in female venom PLA 2s (17.2%) and LAAOs (12.1%) precede svMPs (9.1%) in diversity. In DIA, regardless of the venom, svSPs are followed by PLA 2s (male 17.0% and female 16.6%), svMPs (male 15.6% and female 16.6%), CTLs (male 13.5% and female 15.1%), and LAAOs (male 8.5% and female 8.5%). The diversity dominating protein families make up 78.4% for DDA and 82.3% for DIA mode in male venom, and 83.8% for DDA and 83.5% for DIA in female venom. Protein families with generally less than 5% of the total diversity share (“Others”) made collectively the greatest variation between the sexes in DDA mode. In general, differences between sexes in protein family diversity showed a lesser extent in DIA mode compared to DDA mode , comparing the proportions of major protein families. Overall, our SDS -PAGE and RP -HPLC profiling as well as our proteomic analysis under different data acquisition modes, revealed that the venom profiles of male and female V. berus are almost identical. Small deviations occur but only manifest in minuscule differences or affect minor components. 4.3 Bioactivity profiling To investigate functional differences between male and female venom s, we compared their normalized enzymatic activity and effects on mammalian cell lines and erythrocytes (Figure 5). Venoms were tested at concentrations of 50, 25, 12.5, 6.25, and 3.125 µg/ml in every assay, except for the protease activity assay, where concentrations of 400, 200, 100, 50, and 25 µg/ml were applied. Protease activity of both venoms (Figure 5, A) showed a concentration -dependent effect, ranging between 44% and 3%. Measured activity between male and female venoms was highly similar, e. g. 44% (female) vs. 42% (male) at 400 µg/ml, or 28% (female) vs. 24% (male) at 200 µg/ml. Likewise, the measured PLA 2 activity (Figure 5, B) showed a concentration - dependent effect for both samples, ranging from 100% to 42%. Again, measured dif ferences were only marginal at every tested concentration, e.g.: 100% vs. 96 at 50 µg/ml, or 96% vs. 91% at 25 µg/ml in female and males, respectively. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Figure 5 – Comparison of venom bioactivities of adult male and female Vipera berus venoms. A) Protease activity; B) Phospholipase A2 activity; venom-induced effect on the cell viability of C) Madin-Darby canine kidney II (MDCK II) cell line, and D) human epithelial lung adenocarcinoma (Calu-3) cell line; venom-induced hemolysis on equine erythrocytes in E) Dulbecco’s phosphate-buffered saline (DPBS) and F) DBPS with supplemented Ca 2+ and Mg2+ (DPBS+). Displayed are the results at five concentrations . Data are means ± standard deviations of technical triplicates (n = 3). Reminiscent of the enzymatic activity spectrum, effects from male and female venoms on the viability of MDCK II cells (Figure 5, C) were comparable at all tested concentrations. No 50 25 12.5 6.25 3.125 -20 0 20 40 60 80 100 120Hemolytic activity in DPBS+ (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 -20 0 20 40 60 80 100 120Hemolytic activity in DPBS (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 -20 0 20 40 60 80 100 120MDCKII - Cell viability (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 -20 0 20 40 60 80 100 120Calu-3 - Cell viability (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 -20 0 20 40 60 80 100 120Phospholipase A2 activity (%) Concentration (µg/ml) Male Female 400 200 100 50 25 -20 0 20 40 60 80 100 120Protease activity (%) Concentration (µg/ml) A) B) D)C) F)E) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint cytotoxicity was recorded at lower concentrations. The effects on Calu-3 viability were similar at all concentrations (Figure 5, D). Both venoms were strongly cytotoxic to a concentration of 12.5 µg/ml (24-25% cell viability) with marginal cytotoxicity at 6.25 µg/ml (78-74% cell viability) and no effect was detected at lower concentrations . Relevant hemolytic activity could not be measured up to the maximal concentration of 50 µg/ml for neither male nor female venom against purified equine erythrocytes (Figure 5 E, F). Furthermore, we compared the effects of our venom samples regarding their ability to target the coagulation cascade (Figure 6, A-C). We tested FXa-like-, thrombin-like-, and plasmin-like activities, as well as the venoms’ general coagulation capability by venom-induced coagulation time, prothrombin time, and activated partial thromboplastin time. No relevant FXa-like activity (Figure 6, A) was observed for any of the two venom pools, regardless of the tested concentrations. The thrombin-like activity (Figure 6, B) of both venoms displayed a similar, potent concentration-dependent effect, ranging from 138% (Female 50 µg/ml) to 33% (Male 3.125 µg/ml), exceeding the positive control ≥ 12.5 µg/ml. Similarly, plasmin-like activity (Figure 6, C) also showed a highly potent , concentration-dependent effect . Again, b oth venoms exhibited similar activities with minor differences, exceeding the positive control at concentrations ≥ 6.25 µg/ml. In addition, we assessed for differences in the coagulation time of human PPP for VICT, PT, and aPPT (Figure 6, D -F). The VICT assay (Figure 6, D) displays similar coagulation times, occurring in a dose-dependent manner (10 µg/ml: male 57 s, female 60 s; 1 µg/ml: male 126 s, female 133 s; 0.1 µg/ml: male 260 s, female 279 s; 0.01 µg/ml: male 562 s, female 585 s). Assessing PT (Figure 6, E), a reference for coagulation -induction through the extrinsic coagulation pathway, neither male and nor female venom had a strong effect on the coagulation time compared to the control (10 µg/ml: male 48 s, female 50 s; 1 µg/ml: male 52 s, female 52 s; 0.1 µg/ml: male 39 s, female 37 s; 0.01 µg/ml: male 35 s, female 36 s). In the aPPT assay (Figure 6, F), assessing for coagulation induction via the intrinsic pathway, bot h venoms again show similar coagulation times with stagnating dose -dependency and very similar effects (10 µg/ml: male 31 s, female 32 s; 1 µg/ml: male 52 s, female 54 s; 0.1 µg/ml: male 62 s, female 62 s; 0.01 µg/ml: male 63 s, female 63 s). At the highes t concentration evaluated, a strong decrease in aPPT could be observed. However, no variation between sexes was determined. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint Figure 6 – Effects on coagulation exerted by adult male and female Vipera berus venom: Displayed is the enzymatic activity in the coagulation cascade with A) Factor Xa (FXa)-like activity, B) Thrombin-like activity, and C) Plasmin- like activity. Further is presented the impact on coagulation times in human platelet-poor plasma with the D) Venom- induced coagulation time; E) Prothrombin time; F) Activated partial thromboplastin time. The maximum coagulation at each concentration, compared to the respective positive control, is defined as the highest change in fibrin deposition over time, measured as absorbance a t λ = 405. Data are means ± standard deviations of technical triplicates (n = 3, A-C) and octuplicates (n = 8, D-F). 10 1 0.1 0.01 Venom-free 0 10 20 30 40 50 60 70 80 90 100Activated partial thromboplastin time (s) Concentration (µg/ml) 10 1 0.1 0.01 Venom-free 0 100 200 300 400 500 600 700Venom-induced coagulation time (s) Concentration (µg/ml) 10 1 0.1 0.01 Venom-free 0 10 20 30 40 50 60 70 80 90 100Prothrombin time (s) Concentration (µg/ml) 50 25 12.5 6.25 3.125 0 20 40 60 80 100 120 200 220 240 260 280 Male Female Thrombin activity (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 0 20 40 60 80 100 120 200 220 240 260 280Plasmin activity (%) Concentration (µg/ml) 50 25 12.5 6.25 3.125 0 20 40 60 80 100 120 200 220 240 260 280Factor Xa activity (%) Concentration (µg/ml) A) B) D)C) F)E) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint 5 Discussion 5.1 Low extent of sex-based venom variation in Vipera berus As other functional traits relevant to an animal’s fitness, snake venoms are shaped by selective pressures and environmental cues (Casewell et al., 2013; Schendel et al., 2019). Venoms generally serve the three primary functions of intraspecific interaction, defense, and predation (Schendel et al., 2019). However, the trophic role of venom appears to be the most critical function in snakes, while defense ap pears to be pivotal only in few lineages (Ward-Smith et al., 2020; Kazandjian et al., 2021) and intraspecific interaction has not yet been shown to play a role (Daltry et al., 1996b; Casewell et al., 2013). That said, identifying the factors determining the occurrence of intraspecific venom variation in snakes and contextualizing them within an ecological framework is crucial for understanding venom evolution as well as improv ing snakebite treatment. Particularly, intersexual size differences have been proposed to drive venom variation in several taxa (e.g., Calloselasma rhodostoma (Daltry et al. , 1996a) , B. jararaca (Menezes et al., 2006; Zelanis et al., 2016), B. moojeni (Hatakeyama et al., 2021; Ferreira-Rodrigues et al., 2024), and B. leucurus (Machado Braga et al., 2020)). Differences in body size ultimately caus e differences in gape size, and hence influence the ability to swallow prey (Arnold, 1993). Such discrepancies between male and female conspecifics are often associated with dietary preferences between sexes (Nogueira et al., 2003; Sasa et al., 2009). Hence, intersexual venom variation has typically been explained with the need to target different prey spectra as a result from the physical constraints stemming from size differences (Menezes et al., 2006; Zelanis et al., 2016). However, to date a relatively limited number of studies has explored the frequency and extent of sex -related differences in snake venoms. Vipera berus, a snake that has been shown to exhibit sexually dimorphism and to display venom variation, has seldomly been studied for the role of sex in manifesting aberrant venom profiles. We determined the venom profiles of male and female V. berus from a German po pulation. The SDS-PAGE showed no differences in band patterning between sexes, although slightly increased intensity of certain bands could be detected in female venom s. Nonetheless , considering earlier reported discrepancies in SDS -PAGE venom profiles between individual Hungarian V. berus specimens (Malina et al., 2017), this variation seems unlikely to be strictly sex-based and likely falls within the individual venom variation already detected in this species. Similarly, our HPLC profiles present highly similar peak landscapes for both sexes, suggesting qualitatively and quantitatively similar venom profiles. Furthermore, we performed shotgun proteomics, revealing svSPs, PLA2s, CTLs, svMPs, and LAAOs as major diverse components of venoms from the analyzed specimens. We also detected considerable overlap of hits retrieved from publicly available databases between both samples, suggesting minor variability (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint between the male and the female venoms tested. These proteomic findings are in agreement with the low extent of venom variation already seen by our SDS-PAGE and RP-HPLC profiles. We also gathered additional evidence for supporting the similarity of male and female venoms in our functional screening. The results of PLA2 and protease activity assays, as well as those regarding the assessment of thrombin -, plasmin - and FXa -like activities, were generally comparable between sexes. The effects on coagulation times are comparable between male and female venoms. Likewise, no relevant differences were detected on mammalian cells. These findings are in line with the results of Schulte et al. (2024), comparing the cytotoxic effects of the venoms V. berus specimens exhibiting different color phenotypes. Our data suggests that venom composition and activity seem to be nearly identical between male and female venoms in V. berus . Especially when facing the variability of individual venoms (Malina et al., 2017), the extent of venom variation observed across our proteomic and functional comparison appears negligible. While we retrieved some smaller differences for some activities, we believe that these differences have little to no role in an ecological or clinical framework since they usually occurred only at the lowest concentrations. We come to this

based on the >10 mg dry venom that was gathered from the analyzed snakes in our study and by facing that V. berus can inject up to 10-18 mg (Al-Shekhadat et al., 2019). Therefore, it can be expected that the injected amounts in real -world encounters symptomatically overshadow such subtle differences at low dosage s. Our data agrees with natural history data , as no intersexual differences in foraging behavior and prey choice are reported for V. berus to this day (Forsman, 1991). Therefore, it is likely that both sexes of V. berus produce chemically and functionally highly comparable venoms in response to similar prey spectra targeted despite their sexually dimorphic nature. 5.2 Clinical considerations and the effect of sex on envenoming Parts of our work were set out to unveil intersexual differences in venom function and activity of V. berus venom. While these revealed no striking differences between the venoms of male and female common adders, this investigation helps to shed new light on the pharmacological effects caused by this species' venom. For instance, our work revealed that V. berus venom acts very potent ly on the coagula tion cascade and targets several of its elements. Considering the assessment of FXa-like, thrombin-like, and plasmin-like activity, both venoms showed similarly strong effects (Figures 6, B and C ). Considering that endogenic thrombin and plasmin are serine proteases, these findings possibly align with the high protease activity measured for V. berus venom (Figure 5, A). On the other hand , no FXa -like activity was detectable in either venom at the tested concentrations. The performed plasma coagulation time assays provided further evidence for the ability of V. berus venom to disrupt the normal coagulation process of human plasma. The (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint venom was able to induce coagulation in recalcified human plasma in a concentration - dependent manner, with male and female venom possessing comparable potency. At venom concentrations of 10 µg/ml and 1 µg/ml, both male and female venom induced an increase in the prothrombin time (PT) while the aPPT was strongly reduced at the highest conc entration tested (10 µg/ml). The increase in PT might be due to the disruption of membranes by Phospholipases in the venom , as the tissue factor used for the PT assay is enveloped in phospholipid vesicles. Considering the observed thrombin-like activity, it has been shown that some svSP s exhibit thrombin -like activity, often recalled as snake venom Thrombin -like enzymes (svTLEs), summarized in fibrino(geno)lytic activity . Such svTLEs have been identified in the proteomic data of both sexes (see Supplementary Table S4). The svTLEs vary in their mode of action; however, in general, they only partially mimic thrombin activity and do not activate further coagulation factors, resulting in incomplete or unstable formation of fibrin clots. Furthermore, some thrombin-like svSPs and svMPs have been shown to act fibrinolytic. This results in a plasmin-like activity as shown in our assessments, degrading the (unstable) fibrin clots (Castro et al., 2004; Lu et al., 2005; Sajevic et al., 2011). This presents the venom as an overall pro -coagulant, acting on the common pathway of the coagulation cascade , by promoting an unstable clot formation and the consumption of coagulation factors. An impaired coagulation at the bite site might cause the described local bleeding due to V. berus envenomation (Warrell, 2005; Paolino et al., 2020). However, systemic coagulopathy is rarely reported in clinical cases (Persson, 2014; Jollivet et al., 2015; Dyląg-Trojanowska et al., 2018; Hermansen et al., 2019), likely due to an insufficient venom dosage to induce a systemic effect in humans. Besides the effects on the coagulation cascade, our investigation of cell viability allows us to draw conclusions upon the cytotoxicity of V. berus venom. Our experiments showed that male and female venoms had similar effects on mammalian cells. Cell viability of MDCK II cells was not detectable at venom concentration ≥ 12.5 µg/ml, whereas viability of Calu-3 cells was barely detectable at 25 µg/ml venom. In line with our results, V. berus venom is known to cause local tissue damage, hemostatic imbalance, and organ damage (Al-Shekhadat et al., 2019; Siigur and Siigur, 2022; Shchypanskyi et al., 2024). The most common symptoms caused are local swelling and tissue damage and in more severe cases renal damage, which aligns with our results from cell viability assays (Warrell, 2005; Persson, 2014; Valenta et al., 2014; Dyląg- Trojanowska et al., 2018; Hermansen et al., 2019; Paolino et al., 2020). The high potency in our cell assays may, at least partially, also explain frequent reports of long-lasting local swelling (Warrell, 2005; Persson, 2014). Concerning the hemolytic activity assay, in line with previous reports (Schulte et al., 2024), we did not observe any activity at the tested concentrations. We therefore hypothesize that described symptoms in clinical reports indicating erythrocytopenia (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint (Persson, 2014; Hermansen et al., 2019) are secondary effects of V. berus venom, e.g. due to hemorrhage due to local tissue destruction and not stemming from direct hemolytic activity. Our analysis of V. berus venom demonstrates strong effects on the coagulation cascade . It causes disturbances in the common coagulation pathway, likely caused by its thrombin-like and plasmin -like activity . It also has noteworthy effects on cell viability, providing the mechanistic basis for the clinically observed tissue damage. The major enzymatic activities are protease and PLA 2 activity, both of which are conside rable in this species. Interestingly, while the venom profiles and activities between males and females were found to be highly similar, there might be a clinically relevant effect of sex. In virtually all conducted assays, we retrieved very clear dose -dependencies. When analyzing our venom yields, gathered by mimicking defensive bites, we found that females were capable of delivering much higher amounts of venom. Ergo, while venom biochemistry is conserved between both sexes, female V. berus may bear a higher potential in causing severe envenoming by delivering bites with a higher dosage of venom. 5.3 Novel proteomic perspectives on Vipera berus venom Vipera berus is Earth´s most widespread medically relevant snake, and is responsible for the highest fraction of snakebites in Europe (Paolino et al., 2020). Surprisingly little is known about its venom composition and activity from across its range. Our investigation of sex-based venom variation allows us to gather important new insights into the venom of this severely overlooked snake. Most importantly, our work provides the first venom proteomes for V. berus specimens from Germany, and Central Europe in general. Previous studies on the venom of V. berus were mostly conducted on specimens from Eastern and Southern Europe. They identified a similar set of major components, such as svSPs, PLA 2s, and svMPs. The families CTL and LAAO occur consistently but in varying proportions in the total venom proteomes (Latinović et al., 2016; Bocian et al., 2016; Al-Shekhadat et al., 2019; Damm et al., 2024). A recently published quantitative venom proteome of Norwegian V. berus (Nicolaysen et al. , 2024) presented LAAOs as the most abundant venom components, followed by svMP and svSP. Overall, V. berus venom appears to comprise the same major building blocks across the species´ range, all belonging to the classical major viperine venom components sensu Damm et al. (2021). However, the relative contribution of each compo und appears to differ between localities, probably in response to locally varying selective pressures, or based on differ ent methods. Nonetheless, many populations of Western and Central European countries , as well as of Central and East Asia have never been investigated , and the whole spectrum of V. berus venom components and properties is yet to be fully understood. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint 6 Conclusion Animal venom is a highly functional ecological trait, complex and dynamic between and within taxa. Especially snakes have been shown to produce intraspecifically diverse venom profiles, affecting the mode of action and symptoms of envenomation. Therefore, it is of great interest to unravel the factors shaping venom composition and bioactivity in the context of the evolution of the snake’s ecological niche and to improve the treatment of snake bite patients. Among others, the snake’s sex, in the context of sexual dimorphism, has been discussed. It emerged to be an important factor influencing venom composition in some snakes but overall received comparatively little scientific attention . Using V. berus , a promising model snake to study venom variation, o ur study provide s strong evidence for a low extent of sex-based venom variation in this spe cies. While shedding new light on its functional and compositional phenotype via proteomics, profiling, and bioassays, we did not retrieve striking differences between male and female venoms for having an ecologically or medically relevant impact . However, since our work is limited by low sample sizes and only considers a single population, further comparative studies on a larger scale and covering multiple localities are desirable. As obtaining relevant sample sizes is often a considerable hurdle, especial ly when working with rare, heavily protected, or dangerous species, we acknowledge that this metric cannot always be optimized to the desired extent, such as in the present study. Furthermore, it is worth noticing that the term ‘venom variation’ has so far been applied quite subjectively and in a non- standardized way to address both subtle variations in SDS-PAGE band patterns to dramatic differences in the relative abundances of major venom protein families . Additionally, while some works used rigorous methodological approaches, statistics, and large sample sizes, others were based on less stringent frameworks. As our study highlights, different approaches generating qualitative venom proteomes can have a marked impact on the extent of observed differences in protein diversity within the same sample. We believe that the field of snake venom research would tremendously benefit from a unified definition of best practices and methodological standards to address snake venom variation. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2025. ; https://doi.org/10.1101/2025.03.27.645687doi: bioRxiv preprint 7 Supplementary material Table S1 - Metrics Table S2 - RP-HPLC Table S3 - Proteomics settings Table S4 - Proteomics data Table S5 - Protease activity Table S6 - PLA2 activity Table S7 - Factor Xa activity Table S8 - Thrombin activity Table S9 - Plasmin activity Table S10 - Coagulation summary Table S11 - VICT Table S12 - PT Table S13 - aPPT Table S14 - Cell viability Table S15 - Hemolytic activity 8 Competing interest statement All authors declare that they have no conflicts of interest. 9 Acknowledgements We thank the members of the “Terrarienclub Bayreuth und Umgebung e.V.” for donating the venom samples used in this study. We highly appreciate the intellectual support given by various members of the Venture for Interconnection, Protection, Education and Research in Adders, VIPERA e.V.. 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