Hedgecock-Rowe, Ashlee and Fletcher, Paul

College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC and Brody School of Medicine, East Carolina University, Greenville, NC, USA

Because scorpions employ venom for immobilization of prey as well as defense against predators, the development of complex venoms containing multiple toxins may be adaptations to specific ecological conditions in terms of the dual functions of predation and defense. At the present time, few studies have either examined venom from an ultimate perspective or addressed the ecological and evolutionary significance of venom complexity. The goal of this study is to use the bark scorpion (Centruroides exilicauda) and the grasshopper mouse (Onychomys spp.) as a model representing a coevolved predator-prey system, to determine if venom complexity is, in part, the product of natural selection.

The relationship between C. exilicauda and Onychomys provides an excellent opportunity to test hypotheses dealing with the effects of selection on the evolution of venom neurotoxins, not only because grasshopper mice are voracious predators on scorpions, but also because there are areas where the mouse is both sympatric and allopatric with the scorpion. Preliminary data from my pilot study, conducted July-August 1999, are reported here. In feeding trials, Onychomys from areas both with and without C. exilicauda unhesitatingly attacked and voraciously consumed the scorpion. During these attacks, the mice were frequently stung; individuals from areas sympatric with the bark scorpion showed no systemic symptoms resulting from the stings, while mice from areas allopatric with the bark scorpion exhibited classic symptoms of neurotoxin envenomation. These observations suggest that Onychomys may have evolved resistance to the venom of C. exilicauda in those regions where the mice feed heavily on the scorpion. Thus, the evolution of one or more vertebrate-specific neurotoxins as components of C. exilicauda's venom may be explained, in part, by natural selection due to Onychomys, with counter selection on the grasshopper mouse to neutralize these toxins.

The basis for neutralization of the toxicity of this venom in Onychomys is not known. It is highly likely that resistance begins in the young grasshopper mouse via transfer of maternal immunity. The young may then gain active immunity upon exposure to scorpion venom during early feeding excursions. The mechanisms of resistance could implicate non-specific molecular binding interactions followed by degradation of the bioactive toxins apart from immunity. Plans are made for collection of geographically disperse grasshopper mice as well as associated C. exilicauda to carry out in vitro analysis of Onychomys serum. Experiments are planned to reveal the identification of the inactivation mechanisms, whether components of immunity or related detoxification. Additionally, we expect to identify interacting venom toxins and specific neutralizing molecules. If immune mechanisms are implicated, these experiments will be followed by transfer to native individuals for passive immunity.

Collections: Both scorpions as well as Onychomys sp. mice will be collected in both sympatric and allopatric areas of the US Southwest and hopefully Mexico. Records will be maintained regarding specific collection locations and topography in order that the presence or absence of scorpion venom resistance can be related to sympatric and allopatric associations in the wild. Animals will be shipped according to approved procedures and maintained in USDA and AAALAC approved facilities at East Carolina University, Greenville, NC and North Carolina State University, Raleigh, NC. Quarantine and potential infection control procedures will be followed until risks of hantavirus infection have been ruled out (wild mice only).

Determinations of Resistance: Assessment of resistance to scorpion venom toxins will be carried out initially with serum samples using in vitro ELISA. If serum immunoglobulins are found to specifically neutralize the scorpion venom toxins their classification, quantification, and specificity will be determined.

Eventually, both humoral and cell-mediated immunity factors will be examined. Furthermore, we expect to determine whether the resistance is innate or acquired. Since resistance has been found in Onychomys young it is of considerable interest to examine for possible modes of maternal transfer of immunity. Characterization of both passive and active mechanisms of resistance will provide insight into the potential for transfer to use in humans.

Antibody Production: Active immunity to scorpion venom toxins is not known in humans, even following multiple accidental envenomations. The resistance recorded in Onychomys sp. mice observed in the early phases of these studies is very likely to be based upon classical immune mechanisms of maternal immunity transferred passively to neonates followed by development of active immunity acquired by young animals exposed to envenomations during subsequent natural feeding on available scorpions of the genus Centruroides. Earlier studies in our laboratory (not reported) as well as reports from others indicate that antisera produced for human use against multiple Centruroides venoms appears to produce neutralization in all species of this genus. Likewise, antisera produced against multiple Tityus species neutralize venom toxins found in all species of Tityus. Additionally, antisera produced against Centruroides will neutralize Tityus sp. venom toxins and vice versa. This information infers the presence of common epitopes shared by these two geographically distinct genera of scorpions. Since there are similarities found in the protein structure and function of toxins thus far characterized, the common features indicate relative homology. Similarities are so far best describes in terms of secondary and tertiary, rather than primary structure. The common denominator would appear to he a set of shared venom protein toxin epitopic structures that produce shared immunity. This observation would support a proposal for preparation of a monoclonal antibody derived from these mice that could be effective for human use, even if a limited mixture of specific monoclonal antibodies were required to achieve desirable clinical effects. For these as well as many other reasons, the production of monoclonal antibodies from Onychomys sp. mice is planned.

Collections: Travel, lodging, food, and expendable supplies for trapping and recording, and express shipment of live wild animal species. Collections are planned beginning in early July through mid-August by Dr. Matthew Rowe assisting Mrs. Ashlee Rowe in the extensive field collections.

Animal Care: Care, feeding, routine maintenance, disease surveillance (mainly hantavirus), specimen collection, and storage in quarantined and later isolated animal quarters (AAALAC approved). The university veterinarians (Drs. William Pryor and John Bradfield) will collect blood samples for hantavirus screening and house sentinel mice on used bedding until assurance of freedom from infectious agents is gained.

Samples and Analysis: Both Onychomys sp. wild nice and sympatric scorpions will be collected and returned to North Carolina for these studies. Serum samples will be obtained as soon as possible from retro-orbital sinus punctures under anesthesia. These samples will provide initial information regarding viral (hantavirus) infection and titers of anti-Centruroides scorpion venom immunoglobulins. Circulating scorpion venom antigen levels will also be assessed. ELISA kits in addition to our own protocols for these determinations will be obtained from Silanes Bioclon Institute, Mexico City, Mexico. Individual animal data will be maintained as well as geographical information regarding collection sites, associated animals, and prevalent scorpion species. Collections will be repeated bi-monthly (or as recommended) to provide information regarding ongoing antibody titers. We anticipate having sufficient sample size to permit both regular and occasional feeding with Centruroides exilicauda scorpions to provide material for determining the requirements for resistance to scorpion venom exposure.

Additional information will be collected to indicate immunoglobulin classification, possibility for class shifts, presence of cell-mediated immunity factors, and identification of scorpion venom immunogens targeted by Onychomys sp. serum antibodies. Of additional interest is the ability to obtain passive transfer of immunity or resistance, as would be the case for neutralizing antibodies. A long-range goal for this study is the production of an Onychomys sp. monoclonal antibody with scorpion venom neutralizing capacity that might be humanized and produced in bulk quantity for treatment of sting accidents in humans.

Alternative Hypothesis: There is a small possibility that the resistance to Centruroides sp. scorpion stings is not based upon any form of immunity. Other mammals are known to be resistant to the venom of rattlesnake (Crotalus sp.). This resistance was found to be associated with enzymatic hydrolysis of the highly toxic phospholipase A2 contained in this venom. We have extensive experience with a variety of chromatographic and electrophoretic separations of scorpion ]]>