- Email: [email protected]
284. The Bio-Logic of Venom Complexity
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
ranges from southern Sonora northward into south-central Arizona. Methods: With the aid of temperature-sensitive radio transmitters we tracked tiger rattlesnakes in the Rincon (two sites) and Tortolita Mountains (one site) of Pima County, Arizona from 1997-2010. Results: Based on data pooled across years and sites, we determined that timing of ingress coincides for males and females. However our results revealed that timing of egress varied, with females leaving dens significantly earlier than males. Specifically, the average date of emergence for females was almost a full month (22-23 March) before males (20-21 April). Due to small sample sizes from our Rincon Mountain sites, we were only able to compare interannual variation in the timing of egress at our Tortolita Mountain site from 2004-2006. In each year, females emerged (3/19/2004, 3/16/2005, 3/28/2006) earlier than males (4/12/2004, 3/31/2005, 5/1/2006). Discussion: Our data provides support for the hypothesis that male rattlesnake species which do not mate in spring should not emerge before females. Unlike females, there is no need to come out to bask in early spring to facilitate the process of gametogenesis. We are also investigating the possibility of sexual variation in overwinter body temperature in C. tigris to further our understanding of this species winter ecology. Keywords: Rattlesnake, ecology, ingress, egress 10.1016/j.toxicon.2012.04.283
283. Relocator Proteins: Identification of the Chemical Component of Venoms Allowing Prey Recovery During Strike-induced Chemosensory Searching Anthony J. Saviola 1, Stephen P. Mackessy 1, David Chiszar 2, Chardell Busch 2 1 School of Biological Sciences, University of Northern Colorado, Greeley, CO, USA 2 Department of Psychology, University of Colorado, Boulder, CO, USA E-mail address: [email protected] (A.J. Saviola).
Background: Among advanced snakes, a chemical mode of dispatching prey is commonly utilized to obtain prey rapidly and with minimal contact. Venoms contain a variety of protein, peptide and small organic compounds, and a persistent issue in the study of venom evolution has been to explain the compositional complexity of venoms. Lethal toxicity toward particular prey has demonstrated the adaptive significance of several taxon-specific venom components, but for most venom proteins, particularly the low toxicity, non-enzymatic fractions, a well-defined role in envenomation and predation has not been established. During predatory episodes, rattlesnakes and other pitvipers commonly strike, envenomate and release prey, minimizing retaliation. They are then confronted with the additional task of relocating the carcass via chemosensory searching once venom has taken its course, which they do with a high degree of precision. However, the specific component of venom involved in altering the chemical cues of prey, allowing for relocation, has yet to be identified.
241
Methods: Two-hundred and fifty milligrams of crude Crotalus atrox (Western Diamondback) venom was fractionated using a BioGel P-100 size exclusion column. All fractions were tested for enzymatic activity of enzymes common to rattlesnake venoms. Peaks were dialyzed, lyophilized, and frozen at -20 C until behavioral testing. Eight C. atrox were tested for vomeronasal chemosensory responses to fractionated peaks. The significant peak (peak III) was further fractionated using RP-HPLC, and protein identification was confirmed by MALDI-TOF mass spectrometry and N-terminal sequencing. Results: Enzymatic and other major protein components of size exclusion-fractionated C. atrox venom had no effect on discrimination of envenomated vs. non-envenomated prey by snakes. Conversely, peak III elicited a statistically significant response to treated prey. Further purification by RP-HPLC and analysis by MALDI-TOF mass spectrometry and N-terminal sequencing confirmed that this peak contained only two monomeric disintegrins, crotatroxins 1 and 2. Discussion: In the field, this chemical tag on prey will help minimize foraging time and greatly expedite discrimination of a trail left by envenomated prey from the many trails of non-envenomated conspecifics. Our results demonstrate unequivocally that venom components, such as disintegrins, can have important biological roles which extend beyond those that are apparent from their biochemically functional roles. Conclusion: This is the first study to identify the component of venom allowing for relocation of envenomated prey, and we suggest that a major biological role of venom disintegrins for rattlesnakes is to allow these strikeand-release predators to relocate envenomated prey effectively. Keywords: Chemosensory searching, disintegrins, prey relocation 10.1016/j.toxicon.2012.04.284
284. The Bio-Logic of Venom Complexity David Morgenstern 1, 4, Brett Hamilton 2, Daniel Sher 3, 4, Alun Jones 1, Gideon Mattius 4, Eli Zlotkin 4, Deon Venter 2, Glenn F. King 1 1 Institute for Molecular Bioscience, University of Queensland, St. Lucia 4072, Australia 2 Department of Pathology, Mater Health Services, Raymond Terrace, South Brisbane, 4101, Australia 3 Department of Marine Biology, School of Marine Sciences, University of Haifa, Haifa, Israel 4 Department of Cellular and Animal Biology, Silberman Life Science Institute, Hebrew University, Jerusalem, Israel E-mail address: [email protected] (D. Morgenstern).
Background: The dependency of venomous species on their venom for prey capture has resulted in the evolution of very complex mixtures. Although this strategy is effective, the use of venom has an associated metabolic price tag, and consequently venomous animals tend to use their protein-rich venom sparingly. While complexity appears to stand in contrast to the need for metabolic economy, we still lack knowledge as to the significance of this complexity
242
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
for venom use. The aim of the present study was to characterize the molecular complexity of natural envenomations and to examine the contribution of individual venom components to overall venom toxicity. Methods: We used the Australian funnel-web spider Hadnyche infensa and the scorpion Hottentotta judaicus as models of two independently recruited venom systems in order to characterize venom use under natural conditions. In both cases, the animals were provoked to repetitively deliver venom, simulating a defensive secretion, leading to the gradual release of multiple venom droplets. The individual venom droplets were then assayed for their toxicity against flesh fly larvae (Chrysomya megacephala). The relative abundance of the various components between secretions of each series was assayed using LC-ESI-MS. Additionally, we have examined the distribution of the various components identified previously in the venom, in the venom gland using MALDI imaging. Results and Discussion: an analysis of the individual secretions, found them to be extremely heterogeneous with regard to their volume, protein content, protein profile, and toxicity. Nevertheless, a pattern was observed whereby the toxicity of the stings peaks with time. This increase correlated with a discernable change of venom composition, whereby the most potent toxins are secreted only in later stings. The correlation between toxicity and peptide abundance reveals that only a fraction of the toxins contribute significantly to venom toxicity. Remarkably, we demonstrate that this manipulation of the venom composition is achieved by a spatial organization of toxin production within the venom gland. Imaging mass spectrometry revealed that the most potent toxins, which are abundantly secreted only late in the secretion series, are stored deeper within the gland than the toxins seen in the initial venom secretions. Conclusion: We suggest that this arrangement allows venomous arachnids to reduce the selection pressure on their most potent toxins through a reduction in the occasion of their use. This strategy appears to be successful enough to have evolved at least twice, though preliminary evidence exists to suggest this modulation may also occur in spitting cobras, implying an evolutionary favorable strategy Keywords: Venom complexity, venom optimization, proteomics 10.1016/j.toxicon.2012.04.285
R. Venomous Animal Collections 285. Venomous Workshop: Evolution of a Professional Training Course Douglas L. Hotle Albuquerque BioPark, Department of Herpetology, Albuquerque, NM, USA E-mail address: [email protected].
Background: Venomous animals pose a myriad of unique challenges for those who work with them. From propagation to politics, few other animals are surrounded with such mystique and misinformation.
Methods: The Albuquerque BioPark in conjunction with specialists from around the world have funneled their expertise into this week long workshop. Covering topics including handling, exhibit design, fieldwork, envenomations, antivenom, venomics and more, this workshop is a first of its kind. All types of venomous animals will be covered including marine life and terrestrial invertebrates. The workshop is open to zoo staff, field biologists, academic researchers, Federal and State authorities and even the serious private enthusiast. Results: There has been an appreciable amount of interest in this workshop with attendees expected from countries outside of the United States as well. Conclusions: A workshop covering all aspects of venomous animal handling has drawn widespread interest. Results will be reported in future presentations. Keywords: Venomous animals, professional training 10.1016/j.toxicon.2012.04.286
286. Legal Conundrums Impeding Patient Safety Initiative to Prevent Exotic Envenomation in the United States Joshua Z. Silverberg 1, 2, Michael Touger 1, Donal M. Boyer 3 1 Jacobi Medical Center, Department of Emergency Medicine, Albert Einstein College of Medicine, Bronx, NY, USA 2 Montefiore Medical Center, Department of Emergency Medicine, Albert Einstein College of Medicine, Bronx, NY, USA 3 Department of Herpetology, Bronx Zoo/Wildlife Conservation Society, Bronx, NY, USA E-mail address: [email protected] (J.Z. Silverberg).
Background: During 2011 In New York state there were fifty-seven venomous snakes confiscated comprising fortynine species of world-wide origin after the possible suicide of their owner. The owner was found with puncture wounds consistent with a snakebite and her black mamba's (Dendroaspis polylepis) enclosure was unlocked. The large collection was brought to the Bronx Zoo and placed in quarantine. This presentation will discuss how current federal state and local law affects the availability of venomous reptiles and the clinical relevance of that availability. Discussion: There is currently limited federal law regarding the importation, possession and interstate transport of these animals. A great variability between states in this area exists. An example pertinent to this case is the relatively strict regulations of New York State as compared to nearby states–especially Pennsylvania. To possess a venomous reptile in New York State, a permit from the New York Department of Environmental Conservation is required. Outside of a few local municipalities there is no regulation in Pennsylvania. There are large snake expos in Western Pennsylvania that have been responsible for multiple envenomations in the New York Metropolitan area based on feedback from snakebite patients. This inconsistent regulatory situation makes primary preventative measures against snake envenomation difficult. Recently, federal regulations banning the interstate transport of large constrictor snakes have been adopted. A proposal for similar
ranges from southern Sonora northward into south-central Arizona. Methods: With the aid of temperature-sensitive radio transmitters we tracked tiger rattlesnakes in the Rincon (two sites) and Tortolita Mountains (one site) of Pima County, Arizona from 1997-2010. Results: Based on data pooled across years and sites, we determined that timing of ingress coincides for males and females. However our results revealed that timing of egress varied, with females leaving dens significantly earlier than males. Specifically, the average date of emergence for females was almost a full month (22-23 March) before males (20-21 April). Due to small sample sizes from our Rincon Mountain sites, we were only able to compare interannual variation in the timing of egress at our Tortolita Mountain site from 2004-2006. In each year, females emerged (3/19/2004, 3/16/2005, 3/28/2006) earlier than males (4/12/2004, 3/31/2005, 5/1/2006). Discussion: Our data provides support for the hypothesis that male rattlesnake species which do not mate in spring should not emerge before females. Unlike females, there is no need to come out to bask in early spring to facilitate the process of gametogenesis. We are also investigating the possibility of sexual variation in overwinter body temperature in C. tigris to further our understanding of this species winter ecology. Keywords: Rattlesnake, ecology, ingress, egress 10.1016/j.toxicon.2012.04.283
283. Relocator Proteins: Identification of the Chemical Component of Venoms Allowing Prey Recovery During Strike-induced Chemosensory Searching Anthony J. Saviola 1, Stephen P. Mackessy 1, David Chiszar 2, Chardell Busch 2 1 School of Biological Sciences, University of Northern Colorado, Greeley, CO, USA 2 Department of Psychology, University of Colorado, Boulder, CO, USA E-mail address: [email protected] (A.J. Saviola).
Background: Among advanced snakes, a chemical mode of dispatching prey is commonly utilized to obtain prey rapidly and with minimal contact. Venoms contain a variety of protein, peptide and small organic compounds, and a persistent issue in the study of venom evolution has been to explain the compositional complexity of venoms. Lethal toxicity toward particular prey has demonstrated the adaptive significance of several taxon-specific venom components, but for most venom proteins, particularly the low toxicity, non-enzymatic fractions, a well-defined role in envenomation and predation has not been established. During predatory episodes, rattlesnakes and other pitvipers commonly strike, envenomate and release prey, minimizing retaliation. They are then confronted with the additional task of relocating the carcass via chemosensory searching once venom has taken its course, which they do with a high degree of precision. However, the specific component of venom involved in altering the chemical cues of prey, allowing for relocation, has yet to be identified.
241
Methods: Two-hundred and fifty milligrams of crude Crotalus atrox (Western Diamondback) venom was fractionated using a BioGel P-100 size exclusion column. All fractions were tested for enzymatic activity of enzymes common to rattlesnake venoms. Peaks were dialyzed, lyophilized, and frozen at -20 C until behavioral testing. Eight C. atrox were tested for vomeronasal chemosensory responses to fractionated peaks. The significant peak (peak III) was further fractionated using RP-HPLC, and protein identification was confirmed by MALDI-TOF mass spectrometry and N-terminal sequencing. Results: Enzymatic and other major protein components of size exclusion-fractionated C. atrox venom had no effect on discrimination of envenomated vs. non-envenomated prey by snakes. Conversely, peak III elicited a statistically significant response to treated prey. Further purification by RP-HPLC and analysis by MALDI-TOF mass spectrometry and N-terminal sequencing confirmed that this peak contained only two monomeric disintegrins, crotatroxins 1 and 2. Discussion: In the field, this chemical tag on prey will help minimize foraging time and greatly expedite discrimination of a trail left by envenomated prey from the many trails of non-envenomated conspecifics. Our results demonstrate unequivocally that venom components, such as disintegrins, can have important biological roles which extend beyond those that are apparent from their biochemically functional roles. Conclusion: This is the first study to identify the component of venom allowing for relocation of envenomated prey, and we suggest that a major biological role of venom disintegrins for rattlesnakes is to allow these strikeand-release predators to relocate envenomated prey effectively. Keywords: Chemosensory searching, disintegrins, prey relocation 10.1016/j.toxicon.2012.04.284
284. The Bio-Logic of Venom Complexity David Morgenstern 1, 4, Brett Hamilton 2, Daniel Sher 3, 4, Alun Jones 1, Gideon Mattius 4, Eli Zlotkin 4, Deon Venter 2, Glenn F. King 1 1 Institute for Molecular Bioscience, University of Queensland, St. Lucia 4072, Australia 2 Department of Pathology, Mater Health Services, Raymond Terrace, South Brisbane, 4101, Australia 3 Department of Marine Biology, School of Marine Sciences, University of Haifa, Haifa, Israel 4 Department of Cellular and Animal Biology, Silberman Life Science Institute, Hebrew University, Jerusalem, Israel E-mail address: [email protected] (D. Morgenstern).
Background: The dependency of venomous species on their venom for prey capture has resulted in the evolution of very complex mixtures. Although this strategy is effective, the use of venom has an associated metabolic price tag, and consequently venomous animals tend to use their protein-rich venom sparingly. While complexity appears to stand in contrast to the need for metabolic economy, we still lack knowledge as to the significance of this complexity
242
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
for venom use. The aim of the present study was to characterize the molecular complexity of natural envenomations and to examine the contribution of individual venom components to overall venom toxicity. Methods: We used the Australian funnel-web spider Hadnyche infensa and the scorpion Hottentotta judaicus as models of two independently recruited venom systems in order to characterize venom use under natural conditions. In both cases, the animals were provoked to repetitively deliver venom, simulating a defensive secretion, leading to the gradual release of multiple venom droplets. The individual venom droplets were then assayed for their toxicity against flesh fly larvae (Chrysomya megacephala). The relative abundance of the various components between secretions of each series was assayed using LC-ESI-MS. Additionally, we have examined the distribution of the various components identified previously in the venom, in the venom gland using MALDI imaging. Results and Discussion: an analysis of the individual secretions, found them to be extremely heterogeneous with regard to their volume, protein content, protein profile, and toxicity. Nevertheless, a pattern was observed whereby the toxicity of the stings peaks with time. This increase correlated with a discernable change of venom composition, whereby the most potent toxins are secreted only in later stings. The correlation between toxicity and peptide abundance reveals that only a fraction of the toxins contribute significantly to venom toxicity. Remarkably, we demonstrate that this manipulation of the venom composition is achieved by a spatial organization of toxin production within the venom gland. Imaging mass spectrometry revealed that the most potent toxins, which are abundantly secreted only late in the secretion series, are stored deeper within the gland than the toxins seen in the initial venom secretions. Conclusion: We suggest that this arrangement allows venomous arachnids to reduce the selection pressure on their most potent toxins through a reduction in the occasion of their use. This strategy appears to be successful enough to have evolved at least twice, though preliminary evidence exists to suggest this modulation may also occur in spitting cobras, implying an evolutionary favorable strategy Keywords: Venom complexity, venom optimization, proteomics 10.1016/j.toxicon.2012.04.285
R. Venomous Animal Collections 285. Venomous Workshop: Evolution of a Professional Training Course Douglas L. Hotle Albuquerque BioPark, Department of Herpetology, Albuquerque, NM, USA E-mail address: [email protected].
Background: Venomous animals pose a myriad of unique challenges for those who work with them. From propagation to politics, few other animals are surrounded with such mystique and misinformation.
Methods: The Albuquerque BioPark in conjunction with specialists from around the world have funneled their expertise into this week long workshop. Covering topics including handling, exhibit design, fieldwork, envenomations, antivenom, venomics and more, this workshop is a first of its kind. All types of venomous animals will be covered including marine life and terrestrial invertebrates. The workshop is open to zoo staff, field biologists, academic researchers, Federal and State authorities and even the serious private enthusiast. Results: There has been an appreciable amount of interest in this workshop with attendees expected from countries outside of the United States as well. Conclusions: A workshop covering all aspects of venomous animal handling has drawn widespread interest. Results will be reported in future presentations. Keywords: Venomous animals, professional training 10.1016/j.toxicon.2012.04.286
286. Legal Conundrums Impeding Patient Safety Initiative to Prevent Exotic Envenomation in the United States Joshua Z. Silverberg 1, 2, Michael Touger 1, Donal M. Boyer 3 1 Jacobi Medical Center, Department of Emergency Medicine, Albert Einstein College of Medicine, Bronx, NY, USA 2 Montefiore Medical Center, Department of Emergency Medicine, Albert Einstein College of Medicine, Bronx, NY, USA 3 Department of Herpetology, Bronx Zoo/Wildlife Conservation Society, Bronx, NY, USA E-mail address: [email protected] (J.Z. Silverberg).
Background: During 2011 In New York state there were fifty-seven venomous snakes confiscated comprising fortynine species of world-wide origin after the possible suicide of their owner. The owner was found with puncture wounds consistent with a snakebite and her black mamba's (Dendroaspis polylepis) enclosure was unlocked. The large collection was brought to the Bronx Zoo and placed in quarantine. This presentation will discuss how current federal state and local law affects the availability of venomous reptiles and the clinical relevance of that availability. Discussion: There is currently limited federal law regarding the importation, possession and interstate transport of these animals. A great variability between states in this area exists. An example pertinent to this case is the relatively strict regulations of New York State as compared to nearby states–especially Pennsylvania. To possess a venomous reptile in New York State, a permit from the New York Department of Environmental Conservation is required. Outside of a few local municipalities there is no regulation in Pennsylvania. There are large snake expos in Western Pennsylvania that have been responsible for multiple envenomations in the New York Metropolitan area based on feedback from snakebite patients. This inconsistent regulatory situation makes primary preventative measures against snake envenomation difficult. Recently, federal regulations banning the interstate transport of large constrictor snakes have been adopted. A proposal for similar