DOI: 10.5061/dryad.v6wwpzh3x
title: Data from: Comparative venom analysis between melanistic and normally-colored phenotypes of the common adder (Vipera berus)

description: Snake venom is an ecologically relevant functional trait directly linked
 with a snake’s fitness and survival, facilitating predation and defence.
 Snake venom variation occurs at all taxonomic levels, but the study at the
 intraspecific level is still in its early stages. The common adder (Vipera
 berus) exhibits considerable variation in colour phenotypes across its
 distribution range. Melanistic (fully black) individuals are the subject
 of myths and fairytales, and in German folklore such ‘hell adders’ are
 considered more toxic than their normally coloured conspecifics despite
 any formal investigation. Here, we provide the first comparative analysis
 of venoms from melanistic and normally coloured common adders.
 Specifically, we compared the venom profiles by sodium dodecylsulfate
 polyacrylamide gel electrophoresis and reversed-phase high-performance
 liquid chromatography and tested the venoms’ protease, phospholipase
 A2 and cytotoxic activities. Phospholipase A2 activity
 was similar in both phenotypes, whereas general protease activity was
 higher in the melanistic venom, which was also more cytotoxic at two
 concentrations (6.25 and 12.5 µg ml−1). These minor differences between
 the venoms of melanistic and normally coloured adders are unlikely to be
 of clinical relevance in the context of human envenomation. In light of
 our results, the claim that melanistic adders produce more toxic venom
 than their normally coloured conspecifics appears rooted entirely in
 folklore.
description: <strong>Compositional venom profiling
 (SDS-PAGE)</strong> Compositional venom
 profiling was carried out as previously described (Schulte et al. 2023) by
 combining reducing and non-reducing sodium dodecylsulfate polyacrylamide
 gel electrophoresis (SDS-PAGE) with 5 µg of each sample and reversed-phase
 high-performance liquid chromatography (RP-HPLC) with 125 µg of each
 sample. Chromatograms (220 nm) were subtracted by an initial equilibration
 run to reduce noise. Raw gel image is provided in electronic supplementary
 material, figure S1.  
 <strong>Phospholipase A2 assay</strong>
 Phospholipase activity was measured using the EnzChek
 Phospholipase A<sub>2</sub> Assay Kit (Invitrogen, cat. no.
 E10217) for 96-well plates. Venoms were redissolved in 1 × Reaction Buffer
 and added in triplicates at final concentrations of 3.125, 6.25, 12.5, 25
 and 50 µg ml<sup>−1</sup>. Autolytic activity was minimized by
 handling all samples on ice and reducing the pre-incubation time. After
 incubation for 30 min at room temperature, the plates were transferred to
 a Synergy H4 Hybrid Microplate Reader (BioTek) and analysed using Gen 5 v.
 2.09 software (BioTek). The signal was detected at 515 nm following
 excitation at 470 nm. Values were averaged and normalized against the
 positive control (5 U ml<sup>−1</sup> purified bee venom
 phospholipase A<sub>2</sub>, 100%) and negative control (1×
 Reaction Buffer, 0%). Normalized raw data from the phospholipase
 A<sub>2</sub> activity assays are provided in electronic
 supplementary material, table S1.  
 <strong>Protease activity
 assay</strong> We used a non-specific
 Protease Activity Assay Kit (Calbiochem, cat. no. 539125) for 96-well
 plates as previously described (Schulte et al. 2023). Venoms were
 redissolved in double-distilled water and added in triplicates at final
 concentrations of 25, 50, 100, 200 and 400 μg ml<sup>−1</sup>,
 as described above. The reactions were incubated for 2 h at 37°C, shaking
 at 120 rpm on a Multitron device (Infors HT) and the
 OD<sub>492</sub> was measured in an Eon microplate reader
 (BioTek). The signals were averaged and normalized to the positive control
 (166 µg ml<sup>−1</sup> trypsin, 100%) and negative control
 (double-distilled water, 0%). Normalized raw data from the protease
 activity assay are provided in electronic supplementary material, table
 S2.   <strong>Cell
 viability assay</strong> The cytotoxicity of
 venoms was assessed in Madin-Darby canine kidney II (MDCKII) cells using
 the CellTiter-Glo Luminescent Cell Viability Assay (Promega, cat. no.
 G7570) as previously reported (Hurka et al. 2022). The venoms were
 redissolved in cultivation medium and added to final concentrations of
 1.56, 3.125, 6.25, 12.5 and 25 µg ml<sup>−1</sup>.
 Luminescence was measured using a Synergy H4 Hybrid Microplate Reader, and
 mean values for each treatment were normalized against the positive
 control (100 µM ionomycin, 0% growth) and negative control (cultivation
 medium, 100% growth). Normalized raw data from the cell viability assay
 are provided in electronic supplementary material, table S3.
   <strong>Hemolytic
 assay</strong> The haemolytic activity was
 based on a previously described method (Sæbø et al. 2023) that was
 adjusted to a 96-well format. To purify horse erythrocytes, whole blood
 was washed in Alsever buffer until the supernatant prepared by
 centrifugation (804<em>g</em>, 5 min, 4°C) was clear. The
 pellet was resuspended using a cut 1000 µl tip to reduce shear forces.
 Purified erythrocytes were diluted in Alsever buffer to a final
 concentration of 1% (w/v). Venom was redissolved to final assay
 concentrations of 5, 10, 20, 40 and 80 µg ml<sup>−1</sup> and
 added in triplicate to the erythrocyte suspension at a 1 : 1 ratio in
 V-bottom 96-well plates. The signal was detected at
 OD<sub>405</sub> and mean values were normalized to the
 positive control (1% Triton X-100, 100% lysis) and negative control
 (Alsever buffer, 0% lysis). Normalized raw data from the haemolytic
 activity assay are provided in electronic supplementary material, table
 S4.  
 <strong>References</strong>
 Schulte L et al. 2023 Venomics of the milos viper
 (<em>Macrovipera schweizeri</em>) unveils patterns of venom
 composition and exochemistry across blunt-nosed viper venoms. Front. Mol.
 Biosci. 10<strong> </strong>, 1254058.
 (doi:10.3389/fmolb.2023.1254058) Hurka S, et al.
 2022 Bioactivity profiling of in silico predicted linear toxins from the
 ants <em>Myrmica rubra</em> and <em>Myrmica
 ruginodis</em>. Toxins 14 , 846.
 (doi:10.3390/toxins14120846) Sæbø IP et al. 2023
 Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity.
 International Journal of Molecular Sciences 24, 2914.
 (doi:10.3390/ijms24032914)
description: # Data from: Comparative venom analysis between melanistic and
 normally-colored phenotypes of the common adder (*Vipera berus*) Here we
 provide the raw images of the reducing and non-reducing SDS-PAGE (Figure
 S1) as well as the normalized technical triplicates (1, 2, 3) of the
 protease activity assay (Table S1), phospholipase A2 assay (Table S2),
 cell viability assay (Table S3) and hemolytic activity assay (Table S4) by
 pooled venom of melanistic (MEL) and cryptic (CRY) *Vipera berus* in a
 five-fold dilution series (see below). ## Description of the data and file
 structure Abbrevations: Tech. rep. = Technical replicates (1, 2, 3) MEL =
 pooled venom of melanistic *Vipera berus* CRY = pooled venom of cryptic
 *Vipera berus* Pos. control = positive control Neg. control = negative
 control mean = Averaged normalized triplicates (%) SD = Standard deviation
 of normalized, averaged triplicates (%) Supplementary Figure: Figure S1
 SDS-PAGE raw images of pooled Vipera berus venom samples from melanistic
 (MEL) and cryptic (CRY) phenotypes under A) reducing and B) non-reducing
 conditions. Non-relevant lines, left and right of the section of interest,
 are cropped out. Supplementary Tables: Table S1 Phospholipase A2 activity
 of crude venom pools (concentrations: 3.125, 6.25, 12.5, 25 and 50 µg/ml)
 from melanistic (MEL) and cryptic (CRY) *Vipera berus*. The assay was
 performed in technical triplicates (1, 2, 3), subtracted by the buffer
 treatment (negative control, 0% activity) and normalized against 5 U/ml
 purified bee PLA2 treatment (positive control, 100% activity). Normalized
 activity was averaged (mean) and the standard deviation (SD) was
 calculated. Table S2 Protease activity of crude venom pools
 (concentrations: 25, 50, 100, 200 and 400 µg/ml) from melanistic (MEL) and
 cryptic (CRY) *Vipera berus*. The assay was performed in technical
 triplicates (1, 2, 3), subtracted by the double-distilled water treatment
 (negative control, 0% activity) and normalized against 1.66 µg/ml Trypsin
 treatment (positive control, 100% activity). Normalized activity was
 averaged (mean) and the standard deviation (SD) was calculated. Table S3
 Cytotoxicity of crude venom pools (concentrations: 1.56, 3.125, 6.25, 12.5
 and 25 µg/ml) from melanistic (MEL) and cryptic (CRY) *Vipera berus*
 against canine MDCKII cells. The assay was performed in technical
 triplicates (1, 2, 3), subtracted by the Media treatment (negative
 control, 100% growth) and normalized against 100 µM Ionomycin treatment
 (positive control, 0% growth). Normalized activity was averaged (mean) and
 the standard deviation (SD) was calculated. Table S4 Hemolytic activity of
 crude venom pools (concentrations: 5, 10, 20, 40 and 80 µg/ml) from
 melanistic (MEL) and cryptic (CRY) *Vipera berus* against purified equine
 erythrocytes. The assay was performed in technical triplicates (1, 2, 3),
 subtracted by Buffer treatment (negative control, 0% lysis)and normalized
 against 1% Triton X-100 treatment (positive control, 100% lysis).
 Normalized activity was averaged (mean) and the standard deviation (SD)
 was calculated.