Fig 1.
Percentage expression of toxin families found in the venom gland transcriptomes of Bitis arietans from three different geographic locales.
a) Percentage abundance, calculated from relative toxin abundance data, of toxin families that constituting 90% of Nigerian, Tanzanian and South African B. arietans venom gland transcriptomes shown as pie-charts (produced using Canva). b) Side-by-side relative abundances (percentage expression of the toxinome) of detected toxins comprising ≥97% of the venom gland transcriptome and Nigerian. Tanzanian and South African B. arietans, displayed as bar charts. Snake venom metalloproteinases (SVMP), snake venom serine proteinases (SVSP), Phospholipase A2s (PLA2), C-type Lectin-like proteins (CLP), disintegrins (DIS), Kunitz-like proteins (KUN), Bradykinin potentiating peptide (BPP), Vascular endothelial growth factor (VEGF), and cystatin (CYS).
Fig 2.
RP-HPLC profiles of Bitis arietans venoms from three different geographical locales.
Whole venom was dissolved in PBS and a 30 μg sample was applied to a BioBasic C4 RP-HPLC column. The proteins were separated in a gradient of acetonitrile in SDS-PAGE sample buffer. The CLPs were identified by their characteristic electrophoretic patterns: paired bands of alpha and beta subunits at 14–18 kDa under reducing conditions, but a single band at 30–32 kDa under non-reducing conditions (S1 Fig). The position of the SVMPs was determined by comparison with that of B. arietans SVMPs purified and identified in a separate study [52]. Relative toxin percentages for SVMPs and CLPs are shown as a percentage of whole venom and were calculated from integrated peak areas.
Fig 3.
Enzymatic activity assays show significant intraspecific differences in Bitis arietans venom.
Three enzymatic assays were used to quantify snake venom metalloproteinase (SVMP), phospholipase A2 (PLA2), and snake venom serine proteases (SVSP) activity in venoms. a) For SVMP activity, venom activity was assayed with the fluorogenic substrate ES010. Venoms were used at 1 μg/well, in triplicate. Fluorescence was read at an excitation wavelength of 320nm and an emission wavelength of 405 nm for 1 hour. At 1 hour, the fluorescence values were taken as end-read values for statistical comparison, and plotted ± SD. b) For PLA2 activity, venoms were tested in triplicate, at 10 ng/well using a commercially available Abcam secretory PLA2 kit. Absorbance was read at 405 nm and specific PLA2 activity was calculated as per manufacturer’s instructions and plotted ± SD c) SVSP activity was measured using the commercially available chromogenic S-2288 substrate. Venoms were used in triplicate at 0.066 mg/mL. Absorbance was read at 405 nm. Data was then used to calculate activity in U/I, plotted ± SD. Quantified activity was then statistically compared between different locales using a Two-way ANOVA with Tukey’s multiple comparison tests. Significance is denoted by *, with * P = 0.05, ** P = 0.005, *** P = 0.0005.
Fig 4.
Venoms from Bitis arietans show different cytotoxic potencies to human keratinocytes.
Cell metabolic (MTT) assays were used to measure cell viability 24 hours after exposure to B. arietans venom sourced from three different locales. HaCaT cells were seeded at 20,000 cells/well and exposed to serial dilutions of B. arietans venom for 24 hours before Thiazole Blue tetrazolium bromide was used to quantify cell viability using a BMG Clariostar plate reader. Data was used to generate dose-response curves for IC50 calculations. IC50 values were then statistically compared using Two-way ANOVA with Tukey’s multiple comparison test. Each experiment was performed on three independent occasions with triplicate readings collected for each condition on the plate. The data shown represent means of triplicate experiments and error bars represent standard deviations. Significance is denoted by *, with * P = 0.05, ** P = 0.005, *** P = 0.0005.
Fig 5.
Intraspecific venom variation has minimal impact on Bitis arietans venom recognition by polyvalent antivenoms measured by end-point titration ELISA.
B. arietans venom was adsorbed to ELISA plates at 100 ng/well and before being incubated with serial dilutions of three medically relevant antivenoms (SAIMR Polyvalent, Fav-Afrique, and EchiTAb-Plus; dilution range: 1:100–1:39,062,500). Absorbance was measured at 405 nm and plotted ± SD to visualise dilution curves. Dotted line represents comparison point selected, at 1 in 62,500. Graphs prepared using GraphPad Prism 9.
Fig 6.
Immunoblotting of three polyvalent antivenoms against three different geographical variants of B. arietans venom.
B. arietans venoms were run on pre-cast SDS-PAGE gels under reduced conditions at 1 μg/lane before being transferred to nitrocellulose membranes for immunoblotting. Membranes were incubated overnight with antivenom before being visualised for quantification via a LiCor Odyssey System at 800 nm.
Fig 7.
Antivenoms broadly inhibit Bitis arietans venom in vitro despite intra-specific venom variation.
The inhibitory capability of three polyvalent antivenoms, SAIMR, Fav-Afrique, and EchiTAb-Plus, against the enzymatic activity of (a) SVMP (b) PLA2 and (c) SVSP was assessed against the three geographically distinct B. arietans venoms. The data shown are percentage inhibition calculated relative to a venom only control and represents the mean of triplicate measurements captured over three replicate experiments. Error bars represent ± SD. Inhibition data was statistically compared using a Two-Way ANOVA with Tukey’s multiple comparison tests to determine statistical significance in the inhibitory activity of each antivenom between the three locales. Significance is denoted by *, with * P = 0.05, ** P = 0.005, *** P = 0.0005.