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, 11, 20

Molecular Basis for Prey Relocation in Viperid Snakes


Molecular Basis for Prey Relocation in Viperid Snakes

Anthony J Saviola et al. BMC Biol.


Background: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation.

Results: During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with size-fractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A(2), showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions.

Conclusions: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.


Figure 1
Figure 1
Discrimination of envenomated prey is not dependent on enzymatic toxins. (A) Size exclusion fractionation of 250 mg crude C. atrox venom on a 90 × 2.8 cm BioGel P-100 column equilibrated with HEPES/NaCl/CaCl2 buffer. Fractionation occurred at a flow rate of 6.3 mL per hour at 4°C, and eluting proteins/peptides were followed by absorbance at 280 nm. Enzyme activities common to rattlesnake venoms were assayed and are limited to the first two peaks. Arrow indicates the peak containing crotatroxins 1 and 2 (Peak III). (B) MALDI-TOF-MS analysis of peptides in BioGel size exclusion Peak III. Approximately 0.5 μg protein was spotted onto sinapinic acid matrix and analyzed using a mass window of 3 to 25 kD. Several peptides with masses typical of monomeric disintegrins (7,245 to 7,655 Da) were present, but no larger proteins were observed.
Figure 2
Figure 2
Peak III consist only of 7 kDa peptides. (A) Reversed-phase chromatography of Peak III from the gel filtration step (BioGel P-100). Two hundred microliters was injected onto a Vydac C18 (4.6 × 250 mm) column, and disintegrin peaks were eluted at 23% buffer B (13 to 14 minutes). (B) MALDI-TOF-MS analysis of crotatroxin 1 from the reverse-phase chromatography purification step (fraction 13). Mass of 7,440.35 was observed for crotatroxin 1. (C) MALDI-TOF-MS analysis of crotatroxin 2 from the reverse-phase chromatography purification step (fraction 14). Mass of 7,383.29 was observed for crotatroxin 2.
Figure 3
Figure 3
N-terminal sequence of Peak III peptides (Relocator) confirms identity with crotatroxins (CT) 1 and 2. Note that CTs 1 and 2 are identical in sequence except for the additional N-terminal alanine residue in CT1. Protein sequencing of the relocator peak showed lower yield (approximately 3 pmol, compared to approximately 6.5 pmol for residues 2 to 6) and presence of an N-terminal alanine at residue 1, indicating that both CTs were present. No secondary sequence (indicative of potential contaminant proteins) was observed.

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