Nematocyst ratio and prey in two Australian cubomedusans, Chironex fleckeri and Chiropsalmus sp.

This study examined differences in the nematocyst ratios between two species of Australian cubozoans. In Chiropsalmus sp., a species that feeds exclusively on shrimp, no changes in the ratio of the three groups of nematocyst present in the cnidome were detected with size of the individual animals. In Chironex fleckeri, the ratio of different types of nematocysts in the cnidome for small animals (less than 40 mm) was similar to that of Chiropsalmus sp. However, with an increase in body size in C. fleckeri, the nematocyst ratio changed, with mastigophores (nematocysts believed to hold the lethal venom component for prey) increasing in proportion. The change in cnidome ratio is correlated with a change in the prey of C. fleckeri with increased size. Small C. fleckeri appeared to feed exclusively on prawns, medium sized animals fed on fish and prawns and large animals fed predominantly on fish. An increase in the proportion of mastigophores (and presumably the lethal venom component) in the cnidome of C. fleckeri may also be responsible for why this species has caused numerous human fatalities, while the Australian Chiropsalmus sp. has not.     

The in vitro effects of two chirodropid (Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom.

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 micro g/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 micro g/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 micro g/ml). Antivenom (5U/ml) added 1h after the addition of C. fleckeri venom (50 micro g/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 micro g/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin.Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically.     

A functional comparison of the venom of three Australian jellyfish--Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana--on cytosolic Ca2+, haemolysis and Artemia sp. lethality.

Cnidarian venoms produce a wide spectrum of envenoming syndromes in humans ranging from minor local irritation to death. Here, the effects of Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana venoms on ventricular myocyte cytosolic Ca2+, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca2+ in myocytes as measured using the Ca2+ sensitive fluorescent probe Indo-1. The L-type Ca2+ channel antagonist verapamil had no effect on Ca2+ influx whilst La3+, a non-specific channel and pore blocker, inhibited the effect. Haemolytic activity was observed for all venoms, with C. xaymacana venom displaying the greatest activity. These activities are consistent with the presence of a pore-forming toxin existing in the venoms which has been demonstrated by transmission electron microscopy in the case of C. fleckeri. The venom of C. fleckeri was found to be more lethal against Artemia sp. than the venom of the other species, consistent with the order of known human toxicities. This suggests that the observed lytic effects may not underlie the lethal effects of the venom, and raises the question of how such potent activities are dealt with by envenomed humans.     

The in vivo cardiovascular effects of an Australasian box jellyfish (Chiropsalmus sp.) venom in rats.

Using a new technique to extract venom from the nematocysts of jellyfish, the in vivo cardiovascular effects of Chiropsalmus sp. venom were investigated in anaesthetized rats. Chiropsalmus sp. venom (150 microg/kg, i.v.) produced a transient hypertensive response (44+/-4 mmHg; n=6) followed by hypotension and cardiovascular collapse. Concurrent artificial respiration or pretreatment with Chironex fleckeri antivenom (AV, 3000 U/kg, i.v.) did not have any effect on the venom-induced hypertensive response nor the subsequent cardiovascular collapse. The cardiovascular response of animals receiving venom after the infusion of MgSO4 (50-70 mM @ 0.25 ml/min, i.v.; n=5) alone, or in combination with AV (n=5), was not significantly different from rats receiving venom alone. Prior administration of prazosin (50 microg/kg, i.v.; n=4) or ketanserin (1 mg/kg, i.v.; n=4) did not significantly attenuate the hypertensive response nor prevent the cardiovascular collapse induced by venom (50 microg/kg, i.v.). In contrast to previous work examining C. fleckeri venom, administration of AV alone, or in combination with MgSO4, was not effective in preventing cardiovascular collapse following the administration of Chiropsalmus sp. venom. This indicates that the venom of the two related box jellyfish contain different lethal components and highlights the importance of species identification prior to initiating treatment regimes following jellyfish envenoming.     

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.     

Australian venomous jellyfish, envenomation syndromes, toxins and therapy.

The seas and oceans around Australia harbour numerous venomous jellyfish. Chironex fleckeri, the box jellyfish, is the most lethal causing rapid cardiorespiratory depression and although its venom has been characterised, its toxins remain to be identified. A moderately effective antivenom exists which is also partially effective against another chirodropid, Chiropsalmus sp. Numerous carybdeids, some unidentified, cause less severe illness, including Carybdea rastoni whose toxins CrTX-A and CrTX-B are large proteins. Carukia barnesi, another small carybdeid is one cause of the 'Irukandji' syndrome which includes delayed pain from severe muscle cramping, vomiting, anxiety, restlessness, sweating and prostration, and occasionally severe hypertension and acute cardiac failure. The syndrome is in part caused by release of catecholamines but the cause of heart failure is undefined. The venom contains a sodium channel modulator. Two species of Physalia are present and although one is potentially lethal, has not caused death in Australian waters. Other significant genera of jellyfish include Tamoya, Pelagia, Cyanea, Aurelia and Chyrosaora.     

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri.

Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal delta-endotoxins Cry1Aa, Cry3Bb and Cry3A.     

The in vitro vascular effects of two chirodropid (Chironex fleckeri and Chiropsella bronzie) venoms

Clinical observations suggest a primary cardiotoxic role in fatal Chironex fleckeri stings. The limited research available indicates that Chiropsella bronzie venom acts in a similar manner although appears to be less potent. The aim of the present study was to elucidate the vascular effects of C. fleckeri and C. bronzie venoms using rat isolated aorta. Both venoms produced a sustained contraction of endothelium-denuded aorta which was not significantly affected by prazosin or box jellyfish antivenom. Felodipine significantly reduced the contractile response to C. fleckeri venom but not C. bronzie venom. Both venoms produced an initial relaxation (Phase 1), followed by a sustained contraction (Phase 2), in pre-contracted endothelium-intact aorta. Removal of the endothelium significantly inhibited both phases of the response. NOLA significantly inhibited Phase 1, but not Phase 2, of the response to both venoms. Atropine, HOE 140 or BQ 123 did not have any significant inhibitory effect on either phase. In conclusion, neither C. fleckeri nor C. bronzie venoms appear to contain components with activity at alpha(1)-adrenoceptors. Antivenom was ineffective in reversing the effects of the venom suggesting it is incapable of completely neutralising nematocyst-derived venom. Determining the mechanism of action of these venoms will allow for the development of better treatment strategies.     

The Jellyfish hunter--Jack Barnes: a pioneer medical toxinologist in Australia.

Dr Jack Handyside Barnes (1922-1985) was one of the small and elite group of Pacific marine toxinologists whose work was characterised by an uncompromising rugged persona, a focussed resolve to solve challenging problems of human clinical envenomation, and who conducted curiosity-driven research under conditions of scientific isolation. He was a pioneering advocate for the preservation of marine heritage, particularly that of the Great Barrier Reef. A former military commando who later became a general medical practitioner with extensive surgical and obstetric skills, in 1947 he was appointed the Medical Superintendent of Thursday Island in the Torres Strait. Later (from January 1953), he worked indefatigably as a general medical practitioner in Cairns, in tropical North Australia. For four decades (1945-1985), Jack Barnes undertook detailed laboratory, clinical and field research into invertebrate envenomation, particularly medusan toxinology. In 1960 he discovered the species responsible for the Irukandji syndrome, a small carybdeid named Carukia barnesi in his honour. He invented the research technique for the extraction of venom from the world's most venomous creature, the Pacific box Jellyfish, Chironex fleckeri. By 1960, he had published taxonomic details of nematocyst structure and clinical envenomation details consequent upon stings by Physalia, Cyanea, Chironex, Chiropsalmus and Carukia. This paper is a précis of the chronology of his life, contributions and influence.     

An in vivo examination of the stability of venom from the Australian box jellyfish Chironex fleckeri.

We have previously characterised the pharmacological activity of a number of jellyfish venoms with a particular emphasis on the profound cardiovascular effects. It has been suggested that jellyfish venoms are difficult to work with and are sensitive to pH, temperature and chemical changes. The current study aimed to examine the working parameters of the venom of the Australian box jellyfish Chironex fleckeri to enable fractionation and isolation of the toxins with cardiovascular activity. C. fleckeri venom was made up fresh each day and subjected to a number of different environments (i.e. a pH range of 5-9 and a temperature range of 4-30 degrees C). In addition, the effect of freeze drying and reconstituting the venom was investigated. Venom (50 microg/kg, i.v.) produced a transient hypertensive response followed by cardiovascular collapse in anaesthetised rats. This biphasic response was not significantly effected by preparation of the venom at a pH of 5, 7 or 9. Similarly, venom (50 microg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30 degrees C for 1.5h. However, the cardiovascular activity was abolished by boiling the venom. Freeze drying, and then reconstituting, the venom did not significantly affect its cardiovascular activity. However, repeated freeze drying and reconstituting of extracted venom resulted in a significantly loss of activity. This study provides a more detailed knowledge of the parameters in which C. fleckeri venom can be used and, while supporting some previous studies, contradicts some of the perceived problems of working with the venom.     

The in vivo cardiovascular effects of box jellyfish Chironex fleckeri venom in rats: efficacy of pre-treatment with antivenom, verapamil and magnesium sulphate.

Using a new technique to extract venom from the nematocysts, the efficacy of CSL box jellyfish antivenom (AV) and adjunct therapies, verapamil and magnesium sulfate (MgSO(4)), were investigated against the in vivo cardiovascular effects of Chironex fleckeri venom in anaesthetised rats. C. fleckeri venom (30 microg/kg; i.v.) produced a transient hypertensive response followed by hypotension and cardiovascular collapse within 4 min of administration. Prophylactic treatment of anaesthetised rats with CSL box jellyfish AV (3000 U/kg; i.v.) did not have any effect on the venom-induced pressor response, but prevented cardiovascular collapse in four out of 10 animals. Administration of verapamil (20mM@0.25 ml/min; i.v.) either alone or in combination with AV, did not have any effect on the C. fleckeri venom-induced pressor response nor the consequent hypotension or cardiovascular collapse of animals. However, the administration of verapamil negated the partially protective effects of AV. Concurrent artificial respiration of animals with the above treatments did not attenuate the C. fleckeri venom-induced cardiovascular effects. MgSO(4) (0.05-0.07M@0.25 ml/min; i.v.) alone did not have any effect on the venom-induced pressor response nor the consequent cardiovascular collapse of animals. However, although combined AV and MgSO(4) administration could not inhibit the transient pressor effect following the administration of C. fleckeri venom, it prevented cardiovascular collapse in all animals. We show for the first time, the cardiovascular effects of a C. fleckeri venom sample free of tentacular contamination and the potential of MgSO(4) as an adjunct therapy for the treatment of potentially fatal C. fleckeri envenomings.     

Venom lethality and diet: Differential responses of natural prey and model organisms to the venom of the saw-scaled vipers (Echis)

The composition of snake venoms shows a high degree of variation at all taxonomic levels, and natural selection for diet has been implicated as a potential cause. Saw-scaled vipers (Echis) provide a good model for studying this phenomenon. The venoms of arthropod feeding species of Echis are significantly more toxic to natural scorpion prey than those of species which feed predominantly upon vertebrate prey. Although testing venom activity on natural prey is important for our understanding of the evolution of venom, natural prey species are often difficult to obtain in sufficient numbers for toxinological work. In order to test the viability of using cheaper and more easily available model organisms for toxicity assessments in evolutionary research, and the extent to which toxicity of arthropod-eating Echis venoms is increased to arthropods in general or targeted to certain groups, we conducted median lethal dosage (LD₅₀) and time to death trials using the desert locust (Schistocerca gregaria) as a model arthropod, rarely consumed by wild Echis. The venoms of arthropod specialist Echis were found to be significantly more toxic to locusts than the venom of a vertebrate feeding outgroup (Bitis arietans), and one arthropod specialist venom was found to be more toxic than those species which feed upon arthropods infrequently or not at all. The venoms of arthropod specialists were also found to cause death and incapacitation faster than the vertebrate feeding outgroup. Despite some similarity of trends, there are considerable differences between the response of natural prey (scorpions) and a model arthropod (locust) to the venoms of Echis species. This suggests that when possible, natural prey rather than convenient model organisms should be used to gain an understanding of the functional significance of variation in venom composition in snakes.     

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats.

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2-5 microg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 microg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET(A) receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 microg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 microg/kg venom and 6 of 6 rats at 10 microg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 microg/kg) exerted only modest effects and the rats survived. Heating venom (50 degrees C for 10 min) before injection practically abolished the haemodynamic effects of 10 microg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.     

The effectiveness of antivenom in countering the actions of box-jellyfish (Chironex fleckeri) nematocyst toxins in mice

The neutralizing ability of commercially available antivenom prepared against 'milked' box-jellyfish (Chironex fleckeri) venom was tested intravenously in mice against crude nematocyst venom obtained by crushing isolated nematocysts and against each of two lethal toxins (T1 and T2) present in this venom. The in vitro neutralizing ability of the antivenom against crude venom was reduced markedly compared with its reported neutralizing ability against 'milked' venom whilst the in vivo neutralizing ability of the antivenom tested in both prophylactic and rescue experiments involving crude nematocyst venom was reduced approximately threefold. When tested in vitro and prophylactically in vivo the neutralizing ability of the antivenom was much more pronounced against T2 than against T1. This finding was in accord with the view that T1 was absent from the 'milked' venom against which the antivenom was prepared. Doses of crude venom in excess of twice the lethal dose killed mice within 2-3 min emphasizing the need for speed in the administration of antivenom.     

Cyanea capillata tentacle-only extract as a potential alternative of nematocyst venom: Its cardiovascular toxicity and tolerance to isolation and purification procedures

As the proteins with cardiovascular toxicity in jellyfish nematocyst venom and tentacle-only extract (TOE) are probably encoded by the same gene, TOE provides a potential alternative of nematocyst venom with much richer source for acquisition of such proteins. In this study, Cyanea capillata nematocyst venom and TOE (5 mg/kg) both exhibited cardiovascular toxicity in rats, and TOE caused blood pressure reduction in slightly greater amplitude than nematocyst venom 3 min after intravenous administration. SDS-PAGE suggested high likeliness that they both contained the same bioactive protein. The activity of TOE was dose-dependent within 1.25-5 mg/kg, but not at higher concentrations. The cardiovascular activity of TOE sustained a major loss after exposure to 60 °C, and was totally abolished after exposure to 80 °C. Within the pH range of 7-11, the activity of TOE was well preserved, and rapidly attenuated in pH below 5. At 4 °C, TOE lost cardiovascular toxicity after preservation for 7 days, which occurred only after an 8-h preservation at 20 °C. Repeated freeze-thawing and freeze-drying did not significantly affect the toxicity of TOE. Buffer solutions obviously affected the toxicity of TOE, and 0.02 mol/L HAc (pH 6.0) was optimal. These results provide experimental data for optimizing the conditions for isolating the proteins with cardiovascular toxicity from jellyfish TOE, which serves as a promising alternative source of nematocyst venom.     

Venomous pelagic coelenterates: chemistry, toxicology, immunology and treatment of their stings

Ten years have elapsed since our last review article on the toxicology of venomous pelagic coelenterates was published (Burnett and Calton, 1977). Investigation on important medusae and the chemistry of their nematocyst venoms have been expanding. The venomous jellyfish discussed here include the Portuguese man-o'war, (Physalia physalis), the sea nettle (Chrysaora quinquecirrha), the box jellyfish (Chironex fleckeri and/or Chiropsalmus quadrigatus), the cabbage head jellyfish (Stomolophus meleagris), the lion's mane jellyfish (Cyanea capillata), the Irukandji jellyfish (Carukia barnesi), the Moreton Bay Carybdeid medusa (Morbakka), and the mauve blubber (Pelagia noctiluca).     

The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats

Previous studies in our laboratory have shown that tentacle-only extract (TOE) has similar hypotensive effects with nematocyst venom from jellyfish Cyanea capillata, and the experimental studies on the in vivo cardiovascular effects of TOE were further performed to explore the leading cause of death and analyze the basic physiopathologic change in anaesthztized SD rats. Plots of TOE dose versus time to death showed dose-dependent curvilinear relationship. ECG changed in a dose- and time-dependent manner. Haemodynamic parameters, including the heart rate, mean femoral arterial pressure, left ventricular developed pressure and the first derivative of left ventricular pressures, decreased, but left ventricular end-diastolic pressure did not increase. Arterial partial pressure of oxygen and oxygen saturation did not change. Lactate dehydrogenase, creatine kinase and MB isoenzyme of creatine kinase increased significantly. Histopathological examination showed congestion, haemorrhage, edema and denaturation in the heart; congestion, haemorrhage in the lung and acute congestion in the liver. Transmission electron microscopy examination found that parts of sarcomeric filaments disrupted, dissolved and disappeared, and parts of mitochondria swelled in cardiocytes. Laser scanning confocal microscope examination found that ventricular myocytes from adult rat were deformed and ultimately died within 30 min after TOE treatment. Our results reveal that cardiodepressive effect of C. capillata TOE is the leading cause of death and acute total heart failure is the basic physiopathologic change in anaesthetized SD rats.     

Sphingomyelinase D in sicariid spider venom is a potent insecticidal toxin

Spider venoms have evolved over hundreds of millions of years with a primary role of immobilizing prey. Sphingomyelinase D (SMase D) and homologs in the SicTox gene family are the most abundantly expressed toxic protein in venoms of Loxosceles and Sicarius spiders (Sicariidae). While SMase D is well known to cause dermonecrotic lesions in mammals, little work has investigated the bioactivity of this enzyme in its presumed natural role of immobilizing insect prey. We expressed and purified recombinant SMase D from Loxosceles arizonica (Laz-SMase D) and compared its enzymatic and insecticidal activity to that of crude venom. SMase D enzymatic activities of purified protein and crude venom from the same species were indistinguishable. In addition, SMase D and crude venom have comparable and high potency in immobilization assays on crickets. These data indicate that SMase D is a potent insecticidal toxin, the role for which it presumably evolved.     

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye

Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID₅₀ was about 0.6 μg protein/g fish. Toxin samples were stable when kept at −80 ^°C, but after 48 h, an 80% decline in lethality occurred at −20 ^°C. Poor stability of the venom was observed within the range of 65-80 ^°C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60 kDa and 50 kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.     

Physiological resistance of grasshopper mice (Onychomys spp.) to Arizona bark scorpion (Centruroides exilicauda) venom

Predators feeding on toxic prey may evolve physiological resistance to the preys' toxins. Grasshopper mice (Onychomys spp.) are voracious predators of scorpions in North American deserts. Two species of grasshopper mice (Onychomys torridus and Onychomys arenicola) are broadly sympatric with two species of potentially lethal bark scorpion (Centruroides exilicauda and Centruroides vittatus) in the Sonoran and Chihuahuan deserts, respectively. Bark scorpions produce toxins that selectively bind sodium (Na(+)) and potassium (K(+)) ion channels in vertebrate nerve and muscle tissue. We previously reported that grasshopper mice showed no effects of bark scorpion envenomation following natural stings. Here we conducted a series of toxicity tests to determine whether grasshopper mice have evolved resistance to bark scorpion neurotoxins. Five populations of grasshopper mice, either sympatric with or allopatric to bark scorpions, were injected with bark scorpion venom; LD50s were estimated for each population. All five populations of grasshopper mice demonstrated levels of venom resistance greater than that reported for non-resistant Mus musculus. Moreover, venom resistance in the mice showed intra- and interspecific variability that covaried with bark scorpion sympatry and allopatry, patterns consistent with the hypothesis that venom resistance in grasshopper mice is an adaptive response to feeding on their neurotoxic prey.