https://orcid.org/0000-0002-4611-4770
Figures
Abstract
Background
In Colombia, Lachesis snakes are not commonly implicated in snakebite incidents. However, the considerable number of severe envenomation cases and associated mortality rates represent a significant challenge that must be overcome. A deeper comprehension of the composition of their venom, along with its variability across their distribution range, including ontogenetic and sexual variations, will contribute to the enhancement of the scarce knowledge about them and the mitigation of the consequences of snake envenomation.
Methodology/Principal Findings
Through the application of snake venomics and in conjunction with biochemical characterization, here we analyze the venoms of fourteen specimens of Lachesis acrochorda (adults and juveniles) and L. muta from several geographical regions within Colombia. The venoms of juveniles and adults of L. acrochorda exhibited distinct chromatographic profiles and varying relative abundances of their toxins, indicating the presence of ontogenetic shifts, as well as enzymatic activities. Furthermore, a principal component analysis (PCA) revealed significant differences between the male and female samples in the chromatographic region associated with small peptides and nucleosides. In contrast, L. muta exhibited a relatively simple venom composition with low phospholipase activity and intense fibrinogen hydrolysis towards Aα and Bβ subunits. Finally, the Caldas sample exhibited the highest diversity of compounds and phospholipase activity.
Author summary
Snakebite envenomation represents a critical public health concern on a global scale, with tropical regions being particularly affected. However, the venoms of Lachesis snakes in Colombia remain a subject of limited scientific understanding. Although Lachesis bites are not commonly reported in the country, when they do occur, they frequently result in severe symptoms and high mortality rates. We applied detailed protein analysis methods to venom samples from fourteen juvenile and adult specimens collected across multiple regions of Colombia. Differences in the relative abundance of several venom components were found. Juveniles of Lachesis acrochorda exhibited higher levels of enzymes that break down blood proteins, while Lachesis muta venom was dominated by enzymes that promote clot formation. Furthermore, male and female venoms differed in the concentration of low molecular weight compounds. These variations could explain the range of clinical symptoms observed after envenomation, the potential impact of the effectiveness of existing antivenoms, and a deeper understanding of Lachesis venom diversity.
Citation: Franco-Vásquez AM, Lazcano-Pérez F, Carbajal-Saucedo A, Mejía-Sánchez MA, Meléndez-Martínez D, Corzo G, et al. (2026) Comparative venomics reveals intra, interspecific and ontogenetic changes in the venom composition of Lachesis snakes from Colombia. PLoS Negl Trop Dis 20(2): e0014021. https://doi.org/10.1371/journal.pntd.0014021
Editor: Marco Aurélio Sartim, Universidade Federal do Amazonas, BRAZIL
Received: April 24, 2025; Accepted: February 9, 2026; Published: February 23, 2026
Copyright: © 2026 Franco-Vásquez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. Mass spectrometry proteomics data have been deposited in the “Portal de Datos Abiertos from the Universidad Nacional Autónoma de México (UNAM)”: https://datosabiertos.unam.mx/CGEP:RESDATA-CGEP:DCBQ_AF00001 under the project identifier DCBQ_AF00001.
Funding: This work was partially funded by CONAHCyT PRONAII (303045 to G.C) and PAPIIT-UNAM (IN215823 to R.A-E). This work was supported by an PhD scholarship (1011003 to A.M.F-V) from the Secretaría de Ciencia, Humanidades, Tecnología e Inovación (SECIHTI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
The biochemical characterization of snake venom components is crucial for understanding their toxicological properties and therapeutic potential. Lachesis species, commonly known as bushmasters, are the biggest vipers in the world attaining almost 3 m in length and are able to deliver a great amount of venom [1], containing enzymes such as phospholipases A2 (PLA2), snake venom metalloproteinases (SVMP), and snake venom serine proteases (SVSP), which are responsible for their potent effects on blood coagulation, tissue damage, edema and hemorrhage shown in patients envenomed by bushmasters [2,3]. The variability in venom composition observed across different populations and ontogenic stages of Lachesis species represents an additional layer of venom complexity, generating implications for the snakebite treatment correlated to the antivenom efficacy [4,5]. Studies related to the cross-reactivity of poly- and monospecific viperid antivenoms underscore the need for a deeper understanding of venom composition to improve clinical treatment [4,6].
Taxonomical and ecological studies have been pivotal explaining the evolutionary significance of venom variation within Lachesis and other viperid species. These variations are often driven by environmental factors such as prey availability, habitat, and genetic differences among populations [7,8]. Moreover, ontogenetic shifts in venom composition have been observed, which may reflect adaptative strategies for feeding and defense at different life stages [8,9].
Nowadays, the identification of venom components is possible through toxico-venomic studies, establishing the biological and biochemical functions of venom molecules, enhancing the development of promissory bioactive compounds, and allowing a significant advance in the understanding of the pathophysiological conditions observed in snakebite victims.
Several proteomics strategies have been developed in order to identify bioactive compounds. Liquid chromatography-based proteomics consists in the chromatographic separation of peptides resulting from a proteolytic digestion of a complex extract capable of elucidating the large number of proteins present in the sample. Features such as “simple” sample preparation, high-speed, high-throughput automated processing of LC-MS/MS runs, along with in-depth detection of protein components make chromatography-based strategies an attractive option for the study of snake venoms [10].
In the American continent, there are some proteomic studies focused on knowing the characteristics of the venoms of different viperids [11], which involve some of the species that annually cause the largest number of accidents on the continent [12–14] including the species of interest for this research [1,15,16]. Nevertheless, a comprehensive understanding of the venom compounds, their enzymatic and biological activities, and the efficacy of the different antivenoms that can be utilized in the event of a snakebite incident remain unclear. In this work, we aim to biochemically characterize and determine the proteomic profiles of the venoms of Lachesis acrochorda and Lachesis muta collected from different regions of Colombia. By explicitly examining intra and interspecific variation, together with ontogenetic and sexual differences, it is possible to assess how compositional variability contributes to observed differences in venom toxicity and to the recognition and efficacy of existing antivenoms, thereby improving understanding of the pathophysiological effects produced by these species.
2. Materials and methods
2.1. Venoms
Twelve individual samples of L. acrochorda (Ten adults, SVL > 100 cm and two juveniles, SVL < 80 cm) as well as two individual samples of L. muta (Adults, SVL > 100 cm), were included in this study. The venoms were obtained by direct milking of the organisms in situ, which were subsequently released. Venoms were frozen, lyophilized and stored at -50 °C until use. The samples are representative of the biogeographic regions where they are distributed (Fig 1). The organisms were handled and the venoms extracted in accordance with the Colombia’s National Authority for Environmental Licenses (in its Spanish acronym ANLA) regulations, authorization number 2457.
L. acrochorda Santander is represented by green star, L. acrochorda Pacifico by a red star, L. muta in a yellow star, L. acrochorda juveniles in purple, and L. acrochorda Caldas with a blue star. https://www.naturalearthdata.com/downloads/50m-physical-vectors/50m-rivers-lake-centerlines/. https://www.naturalearthdata.com/downloads/50m-physical-vectors/50m-ocean/. https://www.naturalearthdata.com/downloads/50m-cross-blend-hypso/50m-cross-blended-hypso-with-shaded-relief-and-water/.
The venoms samples were geographically characterized and grouped. Group 1 comprises L. acrochorda adults collected in the Santander zone (samples #1 to #5); group 2, a single L. acrochorda adult from Caldas area (#6); group 3, L. acrochorda adults collected in the Pacifico region (#7 to #10); group 4, two L. muta adults (#11 and #12) from Putumayo and Meta; and group 5 included two L. acrochorda juveniles (#13 and #14) from Santander (Table 1).
For some experiments, individual samples (5 mg dry venom/1 mL Tris-HCl 10 mM, pH 8.0) were quantified for protein content and then mixed based on this protein concentration in equal proportions to generate representative pooled samples of each zone/species.
2.2. Biochemical characterization
2.2.1. Electrophoresis.
The samples were loaded onto a 12.5% polyacrylamide gel using the discontinuous system described by [17]. Samples of each venom (15 μg protein) were dissolved in loading buffer (15.6 mM Tris-HCl pH 6.8, 37.5% [v/v] glycerol, 0.125% [m/v] bromophenol blue, 5% [m/v] SDS) in a 5:1 ratio (sample/loading buffer) under reducing and non-reducing conditions. As a molecular weight standard, 4.0 µL of the Page Ruler PLUS Standard marker (Thermo Fisher), with a range of molecular masses ranging from 10 kDa to 250 kDa was used. The gels were run at 100 V for 90 min and stained with Coomassie R-250.
2.2.2. Reverse phase liquid chromatography.
The venom chromatographic profiles were determined using a Shimadzu SPD-10A system (UV/Vis detector SPD 10) equipped with a Phenomenex Jupiter C18 column (4.6 x 250 mm, 5 μm), following the method proposed by [10]. The elution was performed with 0.1% [v/v] TFA in H2O (solvent A) and 0.1% [v/v] TFA in acetonitrile (ACN, solvent B) using the following gradient: 0–5 min 0% B, 5–15 min 15% B, 15–75 min 45% B, 75–85 min 70% B and 85–94 min, maintaining 70% of B at a flow rate of 1 (mL/min). The elution was monitored at 215 and 280 nm simultaneously.
2.2.3. Enzymatic activities.
Phospholipase A2 and protease activities were determined using the EnzChek PLA2 and EnzChek Protease kits (Invitrogen, USA), respectively. In a black 96-well microplate, 3 µg of each Lachesis venom and controls dissolved in 50 µL of buffer and 50 µL of the substrate (fluorophore-labeled lipids and BODIPY mixture) were added. Plates were incubated for 10 min at room temperature. Finally, fluorescence was measured with microplate reader equipment (Synergy HT, BioTek instrument, Winooski, USA) using a 450–490 nm wavelength range for excitation and 515–575 nm range for emission. To monitor kinetics, readings were taken every 2 min for 1 h. The assay was performed by triplicate and graphs were obtained using Prism Graph Pad 9 software.
2.2.4. Zymography.
A modification of the Laemmli, 1970 traditional electrophoresis technique was performed in order to determine protease activity in the gel. A polyacrylamide solution was co-polymerized with gelatin (1.5 mg/mL). Once co-polymerized, 10 µg of venom was loaded under non-reducing conditions, and the corresponding separation was performed at 150 V for 1 h. To monitor the effect of inhibitors, samples were preincubated with 150 mM ethylenediaminotetraacetic acid (EDTA) or 1 mM phenylmethyl sulfonyl fluoride (PMSF) for 30 min at 37°C. Following the incubation period, the venoms were loaded to the wells and subjected to electrophoresis following the previously described methodology. Subsequently, the gel was subjected to a renaturation process, comprising three continuous washes using a Tris-HCl 0.1 M, pH 8.0 with 5% [v/v], 0.05% and 0% of Triton X-100, respectively, for 30 min. Finally, the gel was stained with Coomassie R-250 for an hour. Protease bands were visualized as clear bands on blue background. The assay was performed at least 3 times with consistent results, and a representative gel is shown.
2.2.5. Fibrinogen degradation activity.
The activity of venoms on bovine fibrinogen was determined according to the method described by [18]. Briefly, 50 µL of a 4 mg/mL solution of bovine fibrinogen (MP Biomedicals) dissolved in isotonic saline solution was mixed with 4 µL of venom (1 mg/mL) in a 50:1 ratio (fibrinogen:venom). The mixtures were then incubated at 37 °C for 60 min. A 20 µL aliquot was taken and mixed with 5 µL of 5X loading buffer (15.6 mM Tris-HCl pH 6.8, 37.5% [v/v] glycerol, 0.125% [m/v] bromophenol blue, 5% [m/v] SDS), heated at 95 °C for 5 min, centrifuged at 13,000 rpm, and loaded onto a 10% SDS-Page gel. For the inhibition assays, samples were preincubated with 150 mM EDTA or 1 mM PMSF at 37 °C for 30 min before the incubation with the bovine fibrinogen solution for 60 min. The assay was performed at least 3 times with consistent results, and a representative gel is shown.
2.3. Proteomic characterization
2.3.1. Protein digestion with trypsin.
Venoms were diluted in a 6 M urea buffer. The proteins were reduced by adding 2.5 μL of the reduction buffer (45 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 37 °C and then alkylated by adding 2.5 μL of the alkylation buffer (100 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min at 24 °C in dark conditions. Before trypsin digestion, samples were diluted with water to reduce the urea concentration to 2 M. A protein/trypsin ratio of 20 was used for the tryptic digestion (trypsin sequencing grade from Promega, 50 mM ammonium bicarbonate). Protein digestion was performed at 37 °C for 18 h and stopped with 5 μL of 5% [v/v] formic acid. Protein digests were dried in a vacuum centrifuge and stored at -20 °C until LC-MS/MS analysis.
2.3.2. LC-MS/MS analysis.
Prior to LC-MS/MS analysis, protein digests were re-solubilized under agitation for 15 min in 10 μL of 0.2% [v/v] formic acid. Desalting/cleanup of the tryptic digests was performed by using C18 Zip Tip pipette tips (Millipore, Billerica, MA). Eluates were dried with a vacuum concentrator Savant SPD1010 system (Thermo Scientific, Waltham, MA, USA), reconstituted under agitation for 15 min in 12 μL of 2% ACN/1% formic acid (FA), and loaded into a 75 μm × 150 mm, self-pack C18 column, installed in the Easy-nLC II system (Proxeon Biosystems). Peptides were loaded on-column and eluted with a three-slope gradient at a flow rate of 270 nL/min. Solvent B first increased from 1 to 33% in 110 min, followed by an increase from 33 to 65% B in 10 min, and finally, from 65 to 84% B in 2 min. The HPLC system was coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific) through a Nanospray Flex Ion Source. Nanospray and S-lens voltages were set to 1.3-1.8 kV and 50 V, respectively. The capillary temperature was set to 250 °C. Full scan MS survey spectra (m/z 300–1,500) in profile mode were acquired in the Orbitrap with a resolution of 120,000 with a target value at 3e5. The 18 most intense peptide ions were fragmented by HCD (Higher Energy Collisional Dissociation, charges 2–4) and/or ETD (Electron Transfer dissociation, charges 4–7) and analyzed in the Orbitrap at a resolution of 15,000 and an Automatic Gain Control (AGC) target value set to 5e4. The peptide ion fragmentation parameters were as follow: a reaction time of 120 ms, a reagent target of 2e5 and a maximum reagent injection time of 200 ms for ETD, a normalized collision energy of 30% for HCD. Target ions selected for fragmentation were dynamically excluded for 45 sec after 3 MS/MS scan events.
Mass spectrometry proteomics data have been deposited in the “Portal de Datos Abiertos from the Universidad Nacional Autónoma de México (UNAM)”, (https://datosabiertos.unam.mx/CGEP:RESDATA-CGEP:DCBQ_AF00001) under the project identifier DCBQ_AF00001.
2.3.3. Protein/peptide identification.
The peak list files were generated with Proteome Discoverer (version 2.4) using the following parameters: minimum mass set to 500 Da, maximum mass set to 6,000 Da, no grouping of MS/MS spectra, precursor charge set to auto, and the minimum number of fragment ions set to 5. Protein database searching was performed with Mascot 2.6 (Matrix Science) against the UniProt Squamata database. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.6 Da, respectively. Trypsin was used as the enzyme allowing for up to 1 missed cleavage. Methionine oxidation and carboxylation of glutamic acid were specified as variable modifications.
Scaffold (version Scaffold_4.10.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if > 95% probability was established by the Peptide Prophet algorithm [19] with Scaffold delta-mass correction. Protein identifications were set at >95% probability. Protein probabilities were assigned by the Protein Prophet algorithm [20]. A minimum number of peptides was set to 2.
2.3.4. Protein quantification.
The Normalized Spectral Abundance Factor (NSAF) was used in order obtain protein abundance estimates for each protein based on spectral counts (SpC), which can serve as a proxy of protein abundance [21,22]. The NSAF was calculated, as presented below:
The NSAF for a protein is calculated by normalizing its SpC per its length (L), against the sum of all SpC/L for N proteins [23,24].
2.4. Statistical analysis
All experiments were performed by at least three independent replicates. One-way ANOVA with 95% confidence, was used to compare means between treatments in enzymatic activity. Post hoc analyses were performed using the Tukey multiple comparisons test. To evaluate the effect of the presence and abundance of the toxins (RP-HPLC data) on the venom enzymatic activities, we performed the multivariate Principal Component Analysis (PCA) and K-mean clustering. These analyses were made using Minitab 21 (PA, USA). Statistical analysis and all graphs were done with Prism Graph Pad 9 (Graph Pad Inc., San Diego, CA, USA). The results were expressed as mean ± 95% Confidence Intervals (CI).
3. Results and discussion
3.1. Venom profiles
All samples were subjected to electrophoretic analysis to compare the protein profiles [25]. Venom samples from adult specimens of L. acrochorda showed homogenous protein banding patterns under reducing conditions, especially in the components situated at apparent ∼30, ∼24 and ∼14 kDa molecular weight. The protein bands were also shared by the juveniles L. acrochorda and adult samples of L. muta, except for sample #6 (single sample from Caldas), where an abundant component around ∼17 kDa was evident. Despite these similarities, two regions that show high variability in the banding pattern among all fourteen samples, can be observed: one between 35–70 kDa and another between 15–20 kDa (Fig 2A and 2C).
Lanes #1 to #5 comprise L. acrochorda adults collected in the Santander zone (group 1); Lane #6 is a single L. acrochorda adult from Caldas area (group 2); Lanes #7 to #10 are L. acrochorda adults collected in the Pacifico region (group 3); Lanes #11 and #12 represent two L. muta adults (group 4) from Putumayo and Meta, respectively; and Lanes #13 and #14 included two L. acrochorda juveniles (group 5) from Santander. MWM = Molecular Weight Markers (kDa). A total of 15 µg of venom was loaded by lane.
Individual variability was also observed when the samples were resolved under non-reducing conditions, especially in the zones between 10–15 kDa and 35–70 kDa. A decrease in the abundance and complexity of the bands in the latter region can be observed in contrast to an increase of these same patterns in the former region (Fig 2). In addition, the darker stained bands, between 20–25 kDa, and the presence of a 125 kDa component in the samples under non-reducing conditions and their absence under reducing conditions, suggest the presence of oligomeric proteins or the possible presence of protein precursors [26].
When we compare a pool made from each region samples under reducing conditions, L. acrochorda profiles showed marked differences between them, such as a double band pattern around 60 kDa, only present in the organisms from the Pacifico group (likely related to SVMP-II or III), or the almost complete absence of a component ∼25 kDa (possibly SVMP-I or SVSP) observed in juveniles. When pooled, a high variation among all venoms were evident in the medium molecular weight proteins between 35–50 kDa (Fig 3), that was observed in the chromatographic profiles (Fig 4). In contrast L. muta electrophoretic profile shows a similar pattern compared with L. acrochorda from Caldas region under reducing conditions, and a similar pattern with all L. acrochorda samples, except Santander, under non-reducing conditions. The electrophoretic profiles did not show relevant differences when compared to those reported by [1], easily observable when comparing the more abundant components around 15, 25, 34 and 60 kDa under non-reducing conditions.
MWM = Molecular Weight Markers (kDa). Lanes Lacro_S, Lacro_C, Lacro_P, and Lacro_J are L. acrochorda venoms from Santander, Caldas, Pacifico region and Juveniles, respectively; Lane Lmuta are L. muta venoms. A total of 15 µg of venom were loaded by lane.
Venoms were resolved in a Phenomenex Jupiter C18 column (4.6 x 250 mm, 5 μm). Colors represent the different groups, for L. acrochorda: green – Santander (#1 - #5), blue – Caldas (#6), red – Pacifico (#7 - #10), yellow and purple for L. muta (#11, #12) and juveniles (#13, #14), respectively.
RP-HPLC was carried out for each sample, as a complementary approach to understand the nature of venom complexity. All chromatograms obtained were overlapped and compared in order to identify the most representative compounds, based upon their retention times and area under the curve (Fig 4). To make a better comparison, all chromatographic separations were carried out using equal amount of protein (200 µg).
The differences in venom compositions were observed after a comparison of the peak pattern of the individual chromatograms. The presence of a conserved peak around 40 min in all 14 samples and the existence of an abundant peak around 75 min in L. acrochorda adults from Santander (samples #1 - #5), with minor representation in the Caldas (#6) venom and absent in Pacifico (#7 - #10) samples as well as in juvenile (samples #13 and #14) and adult L. muta venoms were observed. A remarkable level of variation, in terms of peak diversity and proportion, was observed in the zone between 20–30 min, where two different peak patterns appear in L. acrochorda venoms (Fig 4), unrelated to sex, age, or locality. Small peptides and small organic components are commonly resolved in this time window in different viperid species [10] but these variation could be related to other patterns as mentioned elsewhere [27–29].
3.2. Venomics
Based on the individual variability observed after the biochemical characterization, individual samples were pooled according to regions and ages and used for proteomic analysis. A total of 151 proteins were identified and grouped in eleven toxin families of which SVSP, SVMP, and PLA2 represent more than 70% of the protein composition of venoms (Fig 5 and Table 2). These three toxin families comprise the predominant components of viperid venoms [30] and they are responsible for the vascular, myotoxic, coagulopathic, and hemorrhagic effects after viper envenomations [31].
A. L. acrochorda Santander; B. L. acrochorda Pacifico; C. L. muta; D. L. acrochorda juveniles; E. Representation of shared and unique total proteins amongst venoms of L. acrochorda Santander (green), L. acrochorda Pacifico (red), L. muta (yellow), and L. acrochorda juveniles (purple). Serine proteases (SVSP), metalloproteases (SVMP), phospholipases A2 (PLA2), C-type lectins (CTL), cysteine-rich secretory proteins (CRISP), phosphodiesterase (PDE), phospholipase B (PLB), hyaluronidase (HYL), nerve-growth factor (NGF), L-amino acid oxidase (LAAO) and vespryns (VESP). More detailed information in S1 Table.
SVSP represents 23–42% of toxin proteins, closely followed by SVMP that account for 18–35% of the identified proteins (Fig 5). Considerable differences between the proportions of these protein families were observed across samples, where L. acrochorda juveniles show a higher representation of SVSP (41.7%) (Fig 5D and Table 2). In contrast, L. muta and L. acrochorda (Santander) adults show a higher amount of SVMP (33.7% and 34.8%, respectively) (Fig 5C and Table 2), whereas L. acrochorda (Pacifico) showed the highest abundance of PLA2 (15.5%) and C-type Lectins (CTL, 7.7%) (Fig 5B and Table 2). Nevertheless, these findings align with previous proteomic reports for the species, where the venoms are rich in SVSP and SVMP [1,15,16]. No big differences between the relative abundance of secondary and minor protein families such as vespryns (VESP), hyaluronidase (HYL), nerve-growth factor (NGF), phosphodiesterase (PDE), and phospholipase B (PLB) were observed. Nevertheless, none of these proteins except NGF were identified by [15], and were identified, but not quantified by [1]. However, cysteine-rich secretory proteins (CRISP) were more abundant in L. acrochorda from Santander region (8.4%) (Fig 5A and Table 2) and L. muta (8.4%) venoms, whereas L-amino acid oxidase (LAAO), are more represented in L. acrochorda juveniles (7.3%).
Following a comprehensive analysis of the shared and non-shared components, the Venn diagram (Fig 5E) identified 49 toxins representing 12 protein families, serine proteases and metalloproteases were the most widely shared (13 and 14 components, respectively). While L. muta and Pacific L. acrochorda exhibited the most unique components (15 and 28, respectively). Juvenile L. acrochorda venoms more closely look like those of L. muta (sharing 24 components versus only 2 with adult L. acrochorda), and together with chromatographic differences indicate an ontogenetic shift in L. acrochorda venom as previously reported for L. stenophrys and L. m. rhombeata [5,15].
Previous reports [15,16,32] had provided a general, but limited overview of variability in the toxic arsenal of Lachesis snakes in countries such as Colombia. These observations have demonstrated notable parallels in the composition of the venoms of L. melanocephala and L. acrochorda, as well as L. muta and L. stenophrys. It is evident that both groups are composed of species with approximately 11–4 million years of phylogenetic divergence [33]. However, the similarities with those that have a closer evolutionary history, as in the case of L. acrochorda and L. muta, are less pronounced. In contrast, ontogenetic changes in the composition of venoms have been documented in species such as L. stenophrys as mentioned previously. These changes range from a decrease in the abundance of SVSPs from birth to adulthood to an increase in the concentration of SVMPs and vasoactive peptides throughout their developmental stages [15]. Additionally, Galizio et al. found differences in the biochemical and toxicological profiles of L. m. rhombeata between mother and her offspring [5]. This consistency across the genus underscores ontogenetic plasticity as a driver of venom diversity in Lachesis. Furthermore, they argue for integrative studies to be conducted, with the aim of linking proteomic change to ecological function.
Predation behavior is closely related to the presence of high amounts of SVMP, SVSP and PLA2 in viperid venoms. Although these enzymes are used in the digestion process, it is believed that they are more important in the killing [34]. The observed variability in the venom composition is also a consequence of the evolutionary history divergences of the lineages and direct selection on the ecological deployment of toxins [29], in addition to recent findings where the venom variation concept has been extended within species, thanks to the advances in proteomics or transcriptomics technologies [16,35–38]. Moreover, proteases and phospholipases are related to the most destructive and painful symptoms after a snake bite due to their enzymatic and biological activities that include local tissue damage, myonecrosis, several hemodynamics and coagulopathies problems [39]. These protein families are abundant in all venoms tested, which reinforces the importance of characterizing their differences, to better understand their chemical composition, mechanism of actions, and potential targets.
3.3. Venom enzymatic profile
Regarding the quantitative analysis of enzymatic activities, L. acro #6 exhibited the highest phospholipase activity when compared to all venoms. Statistically, there were no significant differences between L. acrochorda adults (except L. acro #6) when regions were compared. On the other hand, juvenile specimens of L. acrochorda and adults of L. muta did not show significant differences. However, when compared to adult specimens of L. acrochorda, the enzymatic activity was found to be lower (Fig 6A). In contrast, the protease activity of L. acro #6 was found to be significantly lower than that of all L. acrochorda adult venoms. Samples from the Pacifico region exhibited a notable reduction in activity when compared with those from Santander but exhibited a similar level of activity when compared to L. muta. Finally, juveniles showed the lowest enzymatic activity (Fig 6B).
A. PLA2 activity, B. Protease activity. The specific enzymatic activity was determined with the commercial EnzCheck PLA2 and EnzCheck Protease assay kit using the protocols provided by the manufacturer. Three micrograms of protein of each venom were used for the experiment performed in triplicate. C(+): Positive control, PLA2 isolated from bee venom for phospholipase assay and Trypsin for protease.
The observed differences in enzymatic activity can be attributed to fluctuations in the abundance of these enzymes; for instance, L. acro #6 exhibits a higher diversity of peaks (although smaller peaks) in the region between minutes 50 and 75, which could be related to its higher PLA2 activity (blue bar, Fig 4). Additionally, as previously indicated, samples from the Santander region showed a substantial component around 70 min, which could be related to either SVSP or SVMP (Fig 4), as proposed by [10].
All venoms exhibited proteolytic activity when analyzed by zymography. Most of the venoms showed the highest degradation bands between 35 and 50 kDa, where two bands could be appreciated at ∼ 31 and 37 kDa, being the former the one with the highest activity in all samples (Fig 7). In contrast, only L. acrochorda venoms exhibited a substrate hydrolysis at ∼ 40 kDa (Fig 7A and 7D). Except for L. acro J #14, all samples exhibited a complete inhibition by PMSF (Fig 7E), indicating a proteolytic activity against gelatin that is mediated mainly by SVSPs.
A. proteolytic activity of L. acrochorda adult’s venoms. B. proteolytic activity of L. acrochorda adult’s venoms and its inhibition by EDTA. C. proteolytic activity of L. acrochorda adult’s venoms and its inhibition by PMSF. D. proteolytic activity of L. muta venoms and its inhibition by EDTA and PMSF. E. proteolytic activity of L. acrochorda juvenile’s venoms and its inhibition by EDTA and PMSF. 10 µg of venom was loaded per lane. Hydrolysis is observed as clear halos on the blue background, 1.5 mg/mL gelatin was added to 10% SDS-PAGE. Gels were stained with Coomassie blue R-250. Molecular weight markers are indicated in the first lane from left to right (MWM) in kDa and trypsin was used as a positive control (Trp).
The fibrinogen degradation assays revealed distinct activity profiles among the venoms (Fig 8). The degradation pattern observed for L. acrochorda samples from Santander, particularly the hydrolysis of the α-chain in the presence of PMSF, is consistent with a prominent role of metalloproteinases (SVMPs), potentially of the P-I and P-III classes. In contrast, samples from the Pacifico region exhibited a comparatively weaker degradation profile. Inhibition with EDTA markedly reduced activity across most samples, supporting the significant contribution of metal-dependent enzymes. Notably, visible clot formation in test tubes for samples #6, #7, and #9, even in inhibitor-free conditions, provides functional evidence correlating with the proteomic identification of thrombin-like serine proteases (SVSPs) in the venoms. Together, these functional patterns complement quantitative proteomic data, highlighting how regional and compositional differences may lead to different pathophysiological effects. This activity was confirmed by the clot’s disintegration when PMSF was added, as reported for Crotalus spp. [40–42]. In contrast, L. muta samples were capable of hydrolyzing strongly α and β chains when PMSF was added, with L. muta #12 exhibiting almost complete hydrolysis of all fibrinogen subunits. Finally, the fibrinogenolytic hydrolysis observed in juveniles was comparable to that described for adults, indicating that ontogenetic differences do not influence this activity.
A. Lachesis acrochorda #1 - #10 samples in the presence and absence of inhibitors (EDTA and PSMF). B. L. muta (#11, #12) and L. acrochorda juveniles (#13, #14) in the presence and absence of inhibitors (EDTA and PSMF). Fibrinogen (4 mg/ml) was preincubated with the respective inhibitors or in their absence and subsequently exposed to a variety of samples, including controls. ISS was used as negative control sample (C-); 4 µg of Bothrops asper venom was used as positive control C (+). MWM = Molecular Weight Markers (kDa).
3.4. Lachesis venom variation
To evaluate the venom variation among Lachesis venom samples, the cumulative effect of the presence and abundance of the RP-HPLC (Fig 4) peaks in the enzymatic activity was analyzed through PCA. This analysis explained a 47.9% variance observed in the RP-HLPC profiles in Lachesis venoms (Fig 9). Moreover, the analysis demonstrated the presence of five venom clusters: L. muta and L. acrochorda juveniles, Santander 1, Santander 2, Caldas-Valle de Cauca, and Pacifico. The L. muta and L. acrochorda juvenile cluster showed the presence and abundance of the peaks contained at 20–40 and 65–70 min. Santander 1 and Santander 2 share similarities at the 20–40, 57–65, and 75–85 min but they differ at the 20–30 min region. Caldas-Valle de Cauca was the only conglomerate with peaks at 45–55 and with the highest signal at 85–95 min. Finally, Pacifico differs from L. muta and L. acrochorda juvenile and the two Santander clusters mostly at the peaks from 60-85 min. Furthermore, when we compared the PCA with the enzymatic activity, the Santander and Pacifico clusters demonstrated a higher protease activity, whereas phospholipase activity did not show any pattern among regions but demonstrated a lower activity in L. muta and L. acrochorda juvenile’s cluster.
Principal component analysis of the cumulative effect of the presence and abundance of the RP-HPLC peaks of the Lachesis venom samples. The PCA explained the 47.9% of the variance observed in the sample. The resulting clusters made by K-means conglomerates analysis are highlighted in yellow for L. muta and L. acrochorda juvenile, green for Santander 1, blue for Santander 2, red for Caldas-Valle de Cauca, and purple for Pacifico. The mean of the enzymatic activities was plotted in the spots of the samples, color gradient for protease activity and dot size for phospholipase A2 activity.
In a second PCA, we analyzed the presence and abundance of the RP-HPLC peaks at 15–40 min interval which are related to peptides as described by [10]. This analysis explained the 63.7% variance observed in Lachesis venom peptides (Fig 10). The samples were distributed into five clusters: Santander 1, Santander 2, Lacro#6, Lacro#7, and a complex cluster comprised by Pacifico, L. acrochorda, L. muta, and L. acrochorda juveniles. Santander 1 showed the higher signal peaks at 25.1, 28.4, and 29 min. Santander 2 showed the higher signal peaks at 20.2 and 38.9 min, but lack of signal at 15.1, 25.1, 28.4, and 29.0 min. Lacro6 and Lacro7 are one sample clusters, Lacro6 showed abundant peaks at 21.9 and 26.6 min, whereas Lacro7 showed the higher signals at 17.8, 34.6, and 40.1 min. Finally, the complex cluster was represented for all the samples that have mild concentration of all peaks.
Principal component analysis of the cumulative effect of the presence and abundance of the RP-HPLC peaks resolved at the expected peptide region proposed by Lomonte and Calvete (2017). The PCA explained the 63.7% of the variance observed in the sample. The resulting clusters made by K-means conglomerates analysis are highlighted in red for Santander I, blue for Santander II, purple for Lacro6, yellow for Lacro7 and green for the complex cluster comprised by Pacifico, L. acrochorda, L. muta, and L. acrochorda juveniles’ specimens.
When Lachesis peptides were analyzed through PCA, results were compared against sex of the donor specimen, females were grouped in two close groups (green and red), while males were presented in three different and separate groups (yellow, blue and purple), which suggest two kind of venom compositions based on the diversity and abundance of compounds related to nucleosides, disintegrins, and small peptides/proteins according to [10].
In comparison to the numerous studies of the most common medically important snakes in the American continent such Bothrops and Crotalus species [37,43–48], the knowledge about the biological role and envenoming mechanism of proteins and peptides from Lachesis venoms remains limited. In addition, the venom variation of the species among different biogeographical regions has not been adequately studied. These results demonstrated that L. acrochorda adults from all biogeographical regions exhibited slight differences in chromatographic and electrophoretic profiles. These differences could be associated with variations in enzymatic activities and the abundance of proteins, including SVSP, SVMP, and PLA2. Venoms from L. acrochorda juveniles exhibited biochemical similarities to L. acrochorda adults from Santander, with notable differences in the abundance of proteins like SVSP, and less pronounced variations in enzymatic activities. The same behavior was observed in L. muta samples when compared to all venoms, demonstrating less diversity of bands and peaks in electrophoretic and chromatographic analysis, respectively, and a lower enzymatic activity (in vitro and in gel).
Our findings on ontogenetic changes in L. acrochorda complement prior reports for L. stenophrys [15,49], aligning with a broader pattern of venom plasticity seen in pit vipers, such as rattlesnakes where both age and geographic location influence composition [50,51]. The substantial compositional differences between L. acrochorda and L. muta can be contextualized within their deep phylogenetic divergence (~11–4 million years) [33]. This extended period of independent evolution likely drove adaptations to distinct ecological niches. For instance, the simpler, fibrinogenolytic-rich profile of L. muta versus the more complex and geographically variable venom of L. acrochorda. Therefore, while historical biogeography explains major interspecific variation, environmental factors, geographic isolation, and ontogeny are key drivers of venom composition [7,8]. Our results confirm that these factors also shape the venom landscapes of neotropical bushmasters, suggesting common evolutionary mechanisms underlie venom diversity across widely separated viperid lineages.
4. Conclusion
Through a combination of proteomic and biochemical analyses, this work reveals significant sources of venom variation in Colombian Lachesis species. The results of this study demonstrate that the composition of the samples shifts with ontogeny, as evidenced by the higher serine protease (SVSP) content in L. acrochorda juveniles versus the metalloprotease (SVMP)-rich profile of adults L. acrochorda from Santander and L. muta. Furthermore, geographic origin and sex contribute to variability, with principal component analysis highlighting differences in low molecular weight components between males and females. These documented variations in toxin families and enzymatic activities provide a biochemical basis for understanding the range of clinical symptoms that can result from envenomation by these snakes.
Beyond composition, this variability has direct implications for the pathology of envenomation and the challenges of treatment. The observed differences in enzymatic activity and toxin abundance suggest that envenomations could present with varied symptom severity and may respond differently to antivenom, which poses a substantial challenge for effective snakebite management in Colombia. Future research integrating transcriptomics, expanded sampling, and in vivo neutralization studies is essential to move from correlative patterns to a mechanistic understanding of how ecology, evolution, and individual biology collectively shape the venom landscape in this medically relevant genus.
Supporting information
S1 Table. Detailed list of the proteins identified from Lachesis by MS/MS after tryptic digestion of venoms of each group showed on Fig 5.
https://doi.org/10.1371/journal.pntd.0014021.s001
(XLSX)
Acknowledgments
The authors wish to thank M.Sc. Eréndida García Rios, M.Sc. Lucero Mayra Ríos Ruiz, M.Sc. Lucía del Carmen Márquez Alonso for their expert and technical help during the research. The authors want to acknowledge the skillful field work of M. Sc. Giovanny Blandon Marin for the obtention of samples.
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