John A. Burns School of Medicine

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jelly®sh (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and puri®cation procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by d-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20 mM EDTA buffer; it was optimal in the presence of 10 mM Ca 21 or Mg 21 . Two chromatographic puri®cation methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42 kDa by SDS±PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG. q

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hingelike fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.

Abstract: Just over a century ago, animal responses to injections of jellyfish extracts unveiled the phenomenon of
anaphylaxis. Yet, until very recently, understanding of jellyfish sting toxicity has remained limited. Upon contact,
jellyfish stinging cells discharge complex venoms, through thousands of barbed tubules, into the skin resulting in painful
and, potentially, lethal envenomations. This review examines the immunological and toxinological responses to stings by
prominent species of jellyfish including Physalia sp. (Portuguese Man-o-War, Blue-bottle), Cubozoan jellyfish including
Chironex fleckeri, several Carybdeids including Carybdea arborifera and Alatina moseri, Linuche unguiculta (Thimble
jellyfish), a jellyfish responsible for Irukandji syndrome (Carukia barnesi) and Pelagia noctiluca. Jellyfish venoms are
composed of potent proteinaceous porins (cellular membrane pore-forming toxins), neurotoxic peptides, bioactive lipids
and other small molecules whilst the tubules contain ancient collagens and chitins. We postulate that immunologically,
both tubular structural and functional biopolymers as well as venom components can initiate innate, adaptive, as well as
immediate and delayed hypersensitivity reactions that may be amenable to topical anti-inflammatory-immunomodifier
therapy. The current challenge for immunotoxinologists

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.

Abstract: Cnidarians are the oldest extant lineage of venomous animals. Despite their
simple anatomy, they are capable of subduing or repelling prey and predator species that are
far more complex and recently evolved. Utilizing specialized penetrating nematocysts,
cnidarians inject the nematocyst content or “venom” that initiates toxic and immunological
reactions in the envenomated organism. These venoms contain enzymes, potent pore
forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that
catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust
membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion
channel specific activities. In addition, certain cnidarian venoms contain or induce the
release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and
caissarone accelerating the pathogenic effects of other venom enzymes and porins. The
cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation
of humans may result in death, in some cases within a few minutes to an hour after sting.
The complexity of venom components represents a unique therapeutic challenge and probably
reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are
invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.