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]. Envenomation syndromes induced by cnidarian animals represent a therapeutic challenge especially to bathers, swimmers and surfers. Envenomation symptoms are painful hemorrhagic skin lesions, systemic reactions (e.g., direct effects on muscle and nerve tissue), long-term immunological responses, and occasionally fatalities due to Chironex fleckeri cardiovascular and pore-forming toxins [6,7,8,9].
In recent years, cnidarian venoms have begun to be investigated as a potential source of novel bioactive therapeutic compounds [10,11,12,13,14]. However, in comparison with the vast number of studies conducted on the venoms of other venomous animals such as snakes, cone snails, spiders and scorpions, cnidarian venoms have received scant attention from toxinologists. The principal technical impediment in cnidarian research is the fact that the venom does not exist in a large discrete gland as an aqueous mixture in milligram quantities but instead is distributed in microscopic individual nematocysts, each containing picogram of protein . 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]. As a consequence, a modern proteomics approach based on high-throughput mass spectrometry analysis is ideal .
Previous venom preparation techniques have been based upon electrical discharge of nematocysts through human amnion , or homogenization  and pulverization or maceration  of whole frozen tentacles and saline or phosphate buffer wash. However, the “venom” recovered utilizing these methods is in fact total tentacular extracts comprised of the contents of both nematocysts and other tentacle cell types. Current venom preparation techniques are based on mechanical rupture of the isolated nematocysts with mortar and pestle grinding , glass beads  and sonication [9,21] in the presence of extraction solutions such as distilled water or saline. However, difficulties relating to equipment availability and contamination of the venom by structural components (e.g., nematocyst capsule-walls) are the major disadvantages of a mechanical disruption and solvent based extraction approach. Another approach has been developed in which density purified intact cnidae are disrupted using pressure followed by rapid centrifugation to harvest the contents without contaminating then with tentacular material or structural components . This technique results in venom with very high specific activity and complexity but is a laborious process.
Since certain chemicals such as ethanol, or 5% acetic acid in distilled water, cause massive cnidae discharge in some cnidarian species [22,23,24] (Hydrozoa and Cubozoa, respectively), in this study we utilized ethanol to obtain venom proteins and peptides from box jellyfish, C. fleckeri, because of its significant envenomation consequences and need of opportune therapeutic tools [7,8]. This study highlights the advantages of this new technique, which results in pure venom, free of contaminants from the tentacles or structural components of nematocysts.
2. Results and Discussion
The venom composition of C. fleckeri has previously been studied, although the methodological approaches used to obtain venom varied between authors [16,17,18,19,20,21]. Moreover, due to the lack of a transcriptomic database underpinning the annotation of the isolated proteins, proteomic approaches were unlikely to discover novel toxins unique to this species. Notably, when pulverization based approaches are used on purified nematocysts in combination with a solvent extraction, the recovered proteins include structural components of the nematocyst capsule rather than just intra-capsular material. Even more concerning is that many “venom” preparations are in fact solvent extracts of the whole tentacles, and thus contain all tentacular biomolecules soluble in the chosen solvent. In addition, many current venom obtaining techniques based upon mechanical disruption of nematocysts are time-consuming and expensive. Here we report a new venom recovery technique based on chemically induced discharge of nematocysts that maximizes and accelerates the identification of the toxic molecules comprised in jellyfish venom. Also in order to rule out that the identified proteins are produced by tentacular epithelial cells, (e.g., toxin Nv1 localized to ectodermal gland cells in the tentacles rather than nematocysts ), we have isolated nematocysts from C. fleckeri, disrupted them in vitro and analyzed and compared the released protein mixture with identified proteins in our method.
2.1. Microscopy Examination of Undischarged and Chemically Discharged Nematocysts
Thus far, there have been differences in the reported nematocyst types and morphology of C. fleckeri cnidome. Despite this diversity in the results of studies conducted by various research groups [19,26,27,28], the consensus is that the cnidome includes four types of nematocyst: (i) those that contain the lethal venom component (microbasic p-mastigophores); (ii) those that penetrate the prey’s skin or cuticle and ensnare it with hook-like structures in order to secure close contact with the tentacles (small and large tri-rhopaloids); (iii) adherent cnidae which adhere to the prey via a coiled shaft upon discharge (holotrichous isorhizas); and (iv) enigmatic spineless adhesive cnidae that secrete sticky fluid (atrichous isorhizas) [19,26,27,28].
In this study the effectiveness of ethanol in inducing discharge discharge C. fleckeri nematocysts was proved by both light microscopy and scanning electron microscopy (SEM). Prior to immersion in ethanol, SEM examination of tentacles revealed undischarged nematocysts, which were categorized as rod-shaped atrichous isorhizas (Figure 1A and Figure 2B), banana-form microbasic p-mastigophores (Figure 1B and Figure 2C), large oval p-rhopaloids (Figure 1C,E, and Figure 2B) and small sub-spherical p-rhopaloids (Figure 1D and Figure 2C). In order to achieve a better understanding of nematocyst orientation within the tissue and the morphological characteristics of the discharged and undischarged nematocysts, the histological samples of tentacles were examined using light microscopy (Figure 2). The transverse section of the tentacle clearly showed three groups of nematocyst batteries: top, intermediate and lower (Figure 2A,E); with nematocysts located at the tips of the batteries. Before chemical discharge, tubules were observed to be coiled and twisted inside the intact nematocyst capsule (Figure 2A–C). After discharge, the capsule remained intact, although the capsular components including shaft, tubule and venom were expelled (Figure 2D–F and Figure 3A–C). Moreover, after immersion in ethanol, the tentacle surface was found to be densely packed with discharged nematocysts (Figure 3D), with a few nematocysts, mostly those placed in lower nematocyst batteries remain undischarged (Figure 2E). As previously suggested , the “roofed-over” effect of the top nematocyst batteries likely prevents the less-prominent intermediate and inferior batteries from being exposed to ethanol. It should be mention that this new ethanol recovery based method demonstrated higher yield in term of the number of proteins identified. Therefore, it is reasonable to propose that this method is more effective in obtaining a good yield of venom, as compared to a previously reported high activity and recovery protocol. The effect of ethanol on nematocyst discharge in
Believe in the chocolate diet? I have a box jelly antivenom to sell you.
By Christie Wilcox | May 31, 2015 8:00 am
On Wednesday, journalist John Bohannon revealed to the world how he “fooled millions into thinking chocolate helps weight loss.” In a boastful piece for i09, he details how he and German television reporter Peter Onneken performed a faulty clinical trial and used flawed statistics to make it seem like chocolate was a weight loss wonder. The team then wrote a bad paper and managed to publish it in a (non-peer-reviewed) journal. They intentionally concocted an enticing press release to tell the world about their not-so-reliable results, and managed to get a few large sites to bite the hook they carefully baited. “For far too long, the people who cover this beat have treated it like gossip, echoing whatever they find in press releases,” Bohannon wrote to explain why he agreed to the elaborate sting. He hopes that the shame of being called out for bad journalism will be enough to get reporters and the public to be a bit more skeptical of science news.
Of course, some were quick to point out that Bohannon mostly fooled the most well-known churnalistic sites, and that overall, science journalists didn’t fall for the ruse. I’m inclined to agree with their criticisms both of ethics of how the sting was conducted and the bold conclusions about the lazy nature of science journalists drawn from it. But it’s hard to stand on my soapbox, fist in the air, when it seems like every week there’s another example of just how shoddy science journalism often is, even when the studies reported on are actually quite wonderful.
Diane Brinkman is first author on a new paper that tells us more about the venom of the deadly box jelly Chironex fleckeri than ever before — too bad the news media has done such a shoddy job of reporting about it. Image from Wikipedia.
You see, I’m in a particularly sour mood because I didn’t want to bring up John Bohannon or the failings of science journalists today. Instead, I had planned to write this awesome post about a fascinating new paper published in BMC Genomics. I wanted to talk about how this research (which details the venom transcriptome and proteome of the largest of the deadliest class of invertebrates in the world, the box jellyfish Chironex fleckeri) is an incredible, fresh look at an evolutionarily old venom. I wanted to expound extravagantly on the novel toxin types Diane Brinkman and her colleagues from Queensland found in the terrifying tentacles of a species that has killed more than 60 people and caused serious injury in multitudes more. Most importantly, I would have loved to dive deeply into the study’s methods and results, discuss what this new information tells us about some of my favorite venomous animals, and how it builds the foundation for future studies.
But instead, I was so nauseated by the coverage of this study that I feel obligated to take the time to correct the lazy reporting of others. Bohannon’s chocolate fake-out may not have been right, but it’s hard to say he’s wrong about science news coverage.
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MORE ABOUT: Box Jellyfish, Chironex fleckeri, Cnidaria, Science Journalism