Abstract
Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or “venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.
Highlights
Stinging cells are distinctive of venomous marine animals of Cnidaria phylum.They contain microscopic organelles that discharge explosively, injecting a mixture of compounds into prey or potential predators [1,2]
Pressure disrupted nematocysts (PDN); Chemically discharged nematocysts (CDN); a: comparative protein-level results across multiple searches using ProteinPilot; b: The percentage of matching amino acids from identified peptides having confidence greater than or equal to 95%; c: The number of distinct peptides having at least 95% confidence
The venom pellets generated by chemically discharged nematocysts (CDN) and pressure disrupted nematocysts (PDN) were dissolved in 8 M urea, 50 mM ammonium bicarbonate buffer
Summary
Stinging cells (cnidocytes) are distinctive of venomous marine animals of Cnidaria phylum. They contain microscopic organelles (cnidae) that discharge explosively, injecting a mixture of compounds into prey or potential predators [1,2]. Upon contact with human skin or other surface (e.g., prey and predator), penetrant cnidae or nematocysts evert harpoon-like tubules laden with spines that act like hypodermic devices to inject venom (proteinaceous porins, neurotoxic peptides and bioactive lipids) [3,4,5]. Venom analysis at the picogram scale presents challenges as cnidarian venom is a complex mixture of bioactive molecules, some of which are aqueous while others are lipidic [7,8]. A modern proteomics approach based on high-throughput mass spectrometry analysis is ideal [15]
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