A functional analysis of varanid vemon proteins and dirty ecology .
This research information has been provided to me directly from the author Alexandra Matossian on behalf of starting the website off right . Big thanks to her you can finder on Instagram @neversun! Check out a short video presentation also by A. Matossian in the media section under Venomus research.
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NSF Grant Proposal
Matossian -
IOS Preliminary Proposal: A functional analysis of varanid venom proteins and dietary ecology.
Conceptual Framework and Specific Objectives
Komodo dragons, the largest of the monitor lizards, were previously thought to possess bacteria-infested mouths, causing blood poisoning and mortality in their prey. However, researchers have examined Varanus komodoensis extensively, and discovered advanced venom glands in the bottom jaw (Fry et al., 2009). Other varanids have also been shown to possess venom glands; V. acanthurus, V. mitchelli, V. panoptes rubidus, and V. varius. V. griseus and V. scalaris bites have also been shown to be consistent with symptoms of envenomation (Sopiev et al., 1987, Ballard & Antonio, 2001). Despite the presence of venom glands, varanids lack the specialized dentition delivery system that is present in other members of the proposed toxicofera clade, Serpentes and Heloderma (Fry et al., 2006). Varanids also possess several means of predation and defense, such as whipping the tail at attackers, or using the mouth and claws to subdue prey (Fry et al., 2009).
It has been shown that diet plays a major role in the evolution of venom variability (Barlow et al., 2009). Even ontogenetic shifts in diet can cause changes in venom composition, as seen in B. jacara, which feeds primarily on ectotherms as a juvenile and endotherms as an adult; this is not seen in B. alternatus which preys almost entirely on endotherms throughout its life (Andrade and Abe, 1999). Varanids vary greatly in diet: the vast majority of the genus is carnivorous, but there are several frugivores (V. olivaceus, V. bitatawa) and other specialists such as V. dumerilli, which predominantly eat crustaceans (Brandenburg, 1983). Therefore, it is reasonable to suggest the possibility of varanid venom composition varying by diet as well.
This proposed research will focus primarily on the ecological function of varanid venom. Is the primary function of venom to aid in predation? I will focus on exploring the venom proteome of three species of varanid available in the United States: V. acanthurus, V. panoptes horni, the New Guinea subspecies of V. panoptes, and V. salvadorii, the closest relative of V. komodoensis. This will be done by gathering data from HPLC, N-terminal sequencing, mass spectrometry, and by using BLAST analysis to determine known protein families present in varanid venom. This research also seeks to understand the role that diet plays in determination of venom characteristics- does varanid venom vary by species with respect to its favored prey species? Analysis of the diet of snakes which possess the same proteins in their venom will be compared to the diet of each varanid species.
Rationale
It has been shown that species which develop other means of predation (e.g, constriction) or develop an ovophagous diet, undergo rapid degeneration of the venom glands and venom delivery system due to the high energetic cost of venom production (Fry et al., 2012). Given the size of the glands and venom yield of V. komodoensis and V. varius, their biology argues that venom has an important function ((Barlow et al., 2009). Furthermore, the complexity of Komodo venom glands has been studied; the species possesses serous glands along the lower jaw, which have central lumina and ducts leading to the tooth bases. This study also documented the diversity of toxins present in V. komodoensis venom, including AVIT, CRISP, kallikrein, natriuretic, and PLA2, which primarily cause hypotension, anticoagulation, stimulation of inflammation, and smooth muscle paralysis (Fry et al., 2012).
Monitor lizards frequently shake their heads, ravage the prey item with their claws, and otherwise traumatize it in order to subdue the animal; simply allowing the lizard to bite a prey item will provide no useful data with regard to venom efficacy. For example, V. komodensis possesses large, serrated teeth, and employs a grip and rip strategy that inflicts deep parallel wounds on large prey animals in addition to envenomation. It is important to note that this is a major component of monitor lizard predation behavior, to the degree where the venom may only be effective when prey is already experiencing physical trauma and blood loss given its anticoagulant and hypotensive properties already mentioned (Fry et al., 2009).
Hypotheses
Question 1: Why do varanids possess advanced venom glands? Q1 Hypothesis 1: The primary function of varanid venom is to aid in predation through anticoagulation protein factors and shock induction. This analysis predicts that venom possesses anticoagulation factors that should functionally increase prey blood flow through injuries inflicted by the monitor, and may also decrease blood pressure. We also predict that frugivorous varanids possess degenerated venom glands.
Question 2: How and why does varanid venom differ in composition between species? Q2 Hypothesis 1: Specific toxic proteins in varanid venom have selectively evolved to be most effective on their favored prey. Therefore, venom will vary between varanids that differ significantly in diet. It also follows that varanid venom may have varying effects on different species, being most effective on what it most commonly preys upon. Multiple in-situ observations of varanid diet will be analyzed and compared with other compilations of monitor lizard predation in order to assess which prey species each varanid species favors the most. Q2 Hypothesis 2: Geographic variation and speciation has allowed for the diversification of venom proteins. Crotalus scutulatus scutulatus populations produce different types of venom based upon their geographic variation, and where the ranges integrate, a third type of venom results (Glenn & Straight, 1989). This hypothesis predicts that the greater the geographic and evolutionary difference, the more differences in venom composition and concentrations will occur. More varanid species will be needed to conclude significant differences based upon geography, but for this research, V. acanthurus is sufficiently distant enough being from Northwestern Australia, away from V. panoptes horni and V. salvadorii which overlap in range in southern New Guinea. V. panoptes horni and V. salvadorii data will be analyzed for degree of relatedness in comparison to V. acanthurus.
Research Design
Venom Extraction
There is no standard protocol for venom extraction in varanids, so I will be following guidelines for Heloderma venom extraction, with some modifications to compensate for the varanids’ physiological differences.
Lizards are safely handled with minimal risk of injury to keeper and animal by firmly grasping the animal at the base of the neck and shoulders. This is done for smaller animals by putting the neck between the middle and ring finger while wrapping the thumb around, and for larger animals, by wrapping one hand around the neck. Then the animal is grasped by the tailbase and lifted into the air, supported by the hand on its neck and chest, while the other hand holds underneath the hips and back legs. The lizard is then drawn close to the body, with the tail pressed between the arm and abdomen, or between the legs. If the tail is not secured, the lizard risks injuring it in defensive whips to free itself.
The lizards are enticed to bite repeatedly on a soft rubber tube (a sterilized nipple used for milking calves has been reported as very effective by the Kentucky Reptile Zoo) towards the back of the mouth, and the venom glands are then gently massaged to facilitate the release of venom that subsequently will be pipetted directly from the mouth. Varanids only have venom glands in their bottom jaw, so the pipette will draw from that region of the mouth. Some venom may also collect in the tube, so the process is performed over a traditional venom collecting mechanism for snakes to ensure that all venom is captured in the process.
It has been shown that rattlesnake venom is largely unaffected by storage conditions varying up to 117C. Therefore, the temperature the venom is stored at is not something we are concerned highly about, and thus we are storing the venom at room temperature.
Captive Husbandry
Venom requires energy to make, so varanids that are milked for their venom must be fed well to ensure that there is no drop in body condition as a result of stress or heightened metabolism from venom production. Each individual is extracted from at a maximum of every 14 days. They are fed a varied frozen-thawed whole-prey diet that mimics what they would prey on in nature, with food offered after extraction, and otherwise every other day.
Lizards will be maintained in enclosures that have temperatures, furnishings, humidity, precipitation, and light cycles (including UVA/UVB) which mimic their natural environment. Each species will have its needs specially met based on geographic variation and requirements, and kept constant across all individuals of the same species. Individuals are housed in separate enclosures that measure at minimum one body length wide, and two body lengths tall and long. Terrestrial species provided a minimum of 18” of dirt to allow for proper cycling of females and to aid in the upkeep of humidity, as well as to enrich and support natural behaviors. Fresh water is available at all times. Each animal is given a tub large enough to fully submerge in, and this is changed daily. All animals are sexually mature. Handling will be kept to a minimum in order to reduce stress on the animal, but cage maintenance will be performed daily.
Testing
To test the hypothesis that venom aids in incapacitating prey, venom will be collected from three species of varanids. V. acanthurus, V. panoptes horni, and V. salvadorii were selected due to availability within the United States, especially as captive-bred specimens, and because of the distinct variation between species despite all being Indo-Australian monitor lizards (Fitch et al, 2006). V. acanthurus is a dwarf, terrestrial monitor species from Australia that primarily eats insects; V. panoptes horni is a mid-sized terrestrial monitor from New Guinea and a generalist predator on both vertebrates and invertebrates equally; V. salvadorii is a large arboreal lizard also from New Guinea that feeds almost exclusively on birds, eggs, and mammals (Arbuckle, 2009).
Toxicological analysis will be performed on crude venom collected from these species. Toxins will be isolated to determine which ones are shared between varanid species and other venomous reptiles. Upon extraction, measurements will be taken with regard to quantity of venom, body weight, and SVL (snout-to-vent length). Comparative analysis through the use of ANOVA testing will be performed. This is to determine if there is any difference in venom within a species, particularly based on size, as monitor lizards prey on different items throughout their lifespan. However, all monitor lizards used in this study are sexually mature adults.
Venom proteome testing will be performed with these three species known to possess venom glands, by collecting venom and performing venomonic analysis. This will start with crude venom analysis using reverse-phase high-performance liquid chromatography (HPLC). Each protein fraction will be characterized by N-terminal sequencing and mass spectrometric determination of molecular masses and cysteine content. Proteins with a single N-terminal sequence, molecular mass, and a single electrophoretic band will be characterized using BLAST analysis. From the BLAST analysis, a known protein family should be discernible, unless varanids possess novel toxins, which is beyond the scope of this intended research. This approach has allowed for the advanced sequencing and analysis of snake venom (Bazaa et al., 2005, Calvete et al., 2007). ANOVAs will be performed to determine if there is significant variation between the toxins present in each of the three species.
Further analysis will be performed to isolate the prey item most affected by each species' venom, and seeing if that matches observations of that species' natural diet. This will be extrapolated by comparison of known snake venom proteins and their effects on various species to the proteins found via BLAST analysis in varanid venom.
Overall, I seek to analyze and sequence the proteins present within varanid venom. I also will attempt to analyze possible connections between species-specific toxins, their relative abundance, and the varanid’s favored prey species.
Broader Impacts of the Proposed Work
If there is no significant effect of varanid venom on any of its prey types, then further testing is needed to determine what the ecological function of venom in monitor lizards is, if not to aid in predation. Some researchers have argued that due to the aggressive physical nature of varanid predation, there is possibly no ecological purpose for varanids possessing venom (Sweet, 2016). This is still significant, as we may then be able to observe the degeneration of energetically expensive venom glands. Testing it for antimicrobial/antiparasitic properties, as well as for any digestive enzymes and properties, is further research that needs to be done regardless of the results of this study. In viperids, elapids, and colubrids, venom has multiple functions, so it is likely that venom serves multiple functions for Varanus as well.
If there is a significant effect of varanid venom on any of its prey types, it is significant in multiple ways. First, it lends additional support to the proposed Toxicofera clade, which suggests that all reptiles descend from a common venom-producing squamate ancestor. Reptile phylogenetics are constantly evolving, so this research provides further evidence to support or refute the theory. Secondly, it shows that the biochemical oral secretions of squamates require further study, since only recently has it been proposed that members of Varanidae possess venom as opposed to just bacteria-infested mouths. Third, it has implications on the pet trade, as all states have regulations on the private keeping of venomous snakes, but varanids are less regulated and more commonly kept. Finally, this research may help to open up the possibilities of using varanid venom as medical technology. Captopril and Tirofiban are ACE inhibitors and anti-platelet drugs respectively, that were isolated from venom from Bothrops jararaca and Echis carinatus. I believe that varanid venom may also possess toxins that can be utilized to create therapeutic drugs and pharmaceuticals that enhance human life.
Reference List
Andrade, D. & Abe, A. (1999). Relationship of Venom Ontogeny and Diet in Bothrops. Herpetologica
55(2), 200-204.
Arbuckle, Kevin. (2009). Ecological Function of Venom in Varanus, with a Compilation of
Dietary Records from the Literature. Biawak, Journal of Varanid Biology and Husbandry, 3(2), 46-56.
Ballard, V. and F.B. Antonio. (2001) Varanus griseus (desert monitor): toxicity. Herpetological Review 32: 261.
Barlow, A., Pook, C. E., Harrison, R. A., & Wuster, W. (2009). Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proceedings of the Royal Society B: Biological Sciences, 276(1666), 2443-2449. doi:10.1098/rspb.2009.0048
Bazaa, A., Marrakchi, N., Ayeb, M. E., Sanz, L., & Calvete, J. J. (2005). Snake venomics: Comparative analysis of the venom proteomes of the Tunisian snakes; Cerastes cerastes, Cerastes vipera and Macrovipera lebetina. Proteomics, 5(16), 4223-4235. doi:10.1002/pmic.200402024
Brandenburg T. (1983). Monitors of the Indo-Australian Archipelago. M.Sc. Thesis, University of Leyden, The Netherlands.
Calvete, J. J., Juárez, P., & Sanz, L. (2007). Snake venomics. Strategy and applications. Journal of Mass Spectrometry, 42(11), 1405-1414. doi:10.1002/jms.1242
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Fry, B.G., Wroe, S., Teeuwisse, W., Van Osch, M., Moreno, K., Ingle, J., . . . Wake, D. (2009). A Central Role for Venom in Predation by Varanus komodoensis (Komodo Dragon) and the Extinct Giant Varanus (Megalania) priscus. Proceedings of the National Academy of Sciences of the United States of America, 106(22), 8969-8974.
Glenn, J. L., & Straight, R. C. (1989). Intergradation of venom A and venom B populations of the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona. Toxicon, 27(1), 47-54. doi:10.1016/0041-0101(89)90288-2
Sopiev, O., B.M. Makeev, S.B. Kudryavtsev and A.N. Makarov. 1987. A case of intoxication by a bite of the gray monitor (Varanus griseus). Izvestiva Akademii Nauk Turkmenskoi SSR, Seriya Biologitsheskikh Nauk 87: 78.
Sweet, S. (2016). Chasing Flamingos: Toxicofera and the Misinterpretation of Venom in Varanid Lizards. Institute for Research and Development, Suan Sunandha Rajabhat University. 123-149.