Fig 1.
Geographic distribution of Micrurus camilae in Colombia and proteomic composition of its venom.
(A) Map showing the known distribution of M. camilae based on published records. The inset highlights the region where the species has been reported. The type locality is indicated by a star, the origin of the specimen analyzed in this study is marked with a red dot, and other reported records from the literature are shown with blue dots [34–36,59]. Photograph by Jose Vieira, ExSitu Project. (B) Proteomic composition of M. camilae venom (full details of identifications are provided in S1 Table). The pie chart shows the relative abundance (%) of venom components classified into protein families, including phospholipase A2 (PLA2), L-amino acid oxidase (LAO), three-finger toxins (3FTx), metalloproteinases (MP), peptides/non-proteinaceous components (P/NP), nucleotidases (ND), platelet-derived growth factors (PDGF), glutathione peroxidase (GPOX), C-type lectins (CTL), Kunitz-type inhibitors (KUN), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hyaluronidase (HYA), phospholipase B (PLB), cysteine-rich secretory proteins (CRISP), Kazal-type inhibitors (KAZ), cysteine protease inhibitors (CySP), and nucleases (NUC).
Fig 2.
Phylogenetic relationships of Micrurus species inferred from mitochondrial ND4 sequences.
Phylogenetic tree inferred using the Maximum Likelihood (ML) method in IQ-TREE, based on a 627 bp alignment of the mitochondrial ND4 gene. The GTR + G + I substitution model was selected as the best-fit model by the software. Node values represent ultrafast bootstrap support (UFBoot) based on 10,000 replicates. Calliophis bivirgatus and Micruroides euryxanthus were included as outgroups. The triadal clade is highlighted in pink, and the monadal clade in green. Nodes with bootstrap support >80% and Bayesian posterior probability >0.95 are marked with red circles. Although ML and Bayesian Inference (BI) yielded congruent topologies, only the ML tree is presented. Photograph represent the usual behavior of this species of curling its tail, in defense situations. Photo: Jose Vieira, ExSitu Project.
Fig 3.
Separation and analysis of M. camilae venom proteins by RP-HPLC and SDS-PAGE.
(A) Reverse-phase high-performance liquid chromatography (RP-HPLC) profile of M. camilae venom. Two mg of crude venom were applied to a C18 column (4.6 × 250 mm) and eluted with an acetonitrile gradient (dotted line). The asterisk indicates the only lethal fraction of venom, by i.p. injection of mice at a dose of 50 μg. (B) SDS-PAGE (15%) of the collected HPLC fractions, under reducing conditions. Molecular mass markers are indicated at the left, in kDa.
Fig 4.
Biochemical and toxic activities of the whole venom of Micrurus camilae.
(A) Phospholipase A2 (PLA2) activity on 4-nitro-3-octanoyloxy-benzoic acid, using 5 μg of venom. (B) Myotoxic activity induced by intramuscular injection of venom (5 μg) in groups of three mice; phosphate-buffered saline (PBS) was used as a control. (C) L-amino acid oxidase (LAO) activity, using 20 μg of venom. (D) Edematogenic activity induced by subcutaneous injection of venom (5 μg) into the right footpad of mice (n = 3); PBS was injected as control in left footpad. The venoms of M. mipartitus and M. dumerilii were included for comparative purposes in the in vitro assays. Asterisks indicate statistically significant differences (*** p < 0.001 ** p < 0.01) between venoms. Bars represent mean ± standard deviation (SD) of three replicates.
Fig 5.
Biochemical and toxic activities of the whole venom of M. camilae.
(A) Proteolytic activity, on azocasein, using 20 µg of venom (B) Coagulant activity on citrated human plasma using 50 µg of venom. (C) Hemorrhagic activity in mice using 50 µg of venom. The in vitro activities were compared with M. mipartitus, M. dumerilii, and B. asper venom (the latter included as a positive control). PBS was used as negative control. Statistically significant differences from controls are indicated by asterisks (*** p < 0.001 ** p < 0.01, * p < 0.05). Bars represent mean ± SD of three replicates.
Fig 6.
Characterization of the lethal fraction in M. camilae venom peak 10.
Intact mass of Camilaetoxin-I. (A) Multicharge series; (B) Deconvolution, showing a monoisotopic mass of 6744 Da (Mo), and additional peaks interpreted as an oxidized (+16, Moox) form and a potassium (+38, MoK+) adduct; (C) Complete sequence of Camilaetoxin-I; (D) Three-dimensional structure modeled by AlphaFold3 (E) Alignment of Camilaetoxin-I with the 3FTx sequences with the highest identity; The colors in the sequence logo (consensus sequence) indicate the different amino acids.
Table 1.
Neutralization of the lethal effect of Micrurus camilae venom in mice by INS-Anticoral and an experimental antivenom.
Fig 7.
Immunorecognition of Micrurus camilae venom by commercial coral snake antivenoms.
(A) Comparison of the immunobinding of the equine antivenom INS-Anticoral against the venoms of M. camilae, M. mipartitus, and M. dumerilii. (B) Comparative recognition of M. camilae venom by two antivenoms: INS-Anticoral and ICP-Anticoral. (C) Immunorecognition of the main RP-HPLC fractions (numbering as in the Fig 3) of M. camilae venom by the INS-Anticoral. Non-immune equine serum was used as a negative control. Asterisks indicate statistically significant differences (p < 0.05) between venoms (A) or respect to controls (B and C). Each point represents mean ± standard deviation (SD) of triplicates.
Fig 8.
Immunorecognition of Micrurus camilae venom by experimental coral snake antivenom.
(A) Comparison of the immunobinding of the experimental rabbit antivenom against the venoms of M. camilae, M. mipartitus, and M. dumerilii. (B) Immunorecognition of the main RP-HPLC fractions (numbering as in Fig 3) of M. camilae venom by an experimental antivenom. Non-immune rabbit serum was used as a negative control. Asterisks indicate statistically significant differences (p < 0.05) between venoms (A) or respect to controls (B). Each point represents mean ± standard deviation (SD) of triplicates.