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FIG. 1. Medicine dropper (60 ml) with rubber bulb, over plastic tube.Note: that only the squeeze bulb with plastic tube is used with this technique.Reichert 1883; Russell and Bogert 1981). In addition, Stuart et al.(1998) demonstrated the toxic effects that anesthesia and parasympatheticstimulation can have on Heloderma while collectingvenom. These techniques were found to be impractical, or injuriousto the lizards, and/or ineffective. However, modifying the techniqueof offering a rubber-covered object to bite (Russell andBogert 1981) worked extremely well.Materials and Methods.—After the lizard was safely restrained(Poulin and Ivanyi 2003), had opened its mouth and the mouthcavity had been rinsed with water or saline, we presented it with aplastic-reinforced rubber squeeze bulb (14.5 mm diameter) froma Nalgene® 60 ml medicine dropper (Fig. 1). As soon as the bulbcontacted the lizard’s mouth, the lizard would voluntarily bite downon the bulb. Care was taken to ensure that the posterior teeth (towardthe angle of the jaw) engaged the bulb. The lizard was thenheld with its head at a 30–45° angle (below horizontal) and itsmouth was suspended above a glass or plastic vessel (Fig. 2).Results and Discussion.—After a lizard bites the squeeze bulb,released and compressed its jaws several times, drops of venomwould be produced and the venom would drop into the vessel fromalong the gums of the lower jaw (Fig. 2). Initially, 40 drops (~ 2ml) of venom were obtained from a several adult specimens. Subsequenttrials suggest that this volume of venom can frequently beobtained using this technique but the quantity of venom varieswith animal size and vigor (range of 1–4 ml), and it is important tonote that the venom may be mixed with saliva and blood. Thoughthe amount of venom per bite diminished after several jaw compressions,generally enough venom was collected in two minutes,allowing us to stop the trial. After each trial, the animal was placedback in its enclosure. In every case, as soon as the lizard was released,it would release the medicine dropper, making it easy toretrieve.This method was used on six each of H. horridum and H.suspectum during late morning to early afternoon hours in spring,summer, and autumn. The lizards ranged from subadults (2–3 yrs.old) to large adults (up to 21+ yrs. old), and in both species, malesand females were used for venom collection. All of the animalshad been in captivity for a minimum of 2 years and a maximum of21 years. Each lizard would bite and release (but not let go of) thebulb in a rhythmic pattern (i.e., bite and hold down for 4 seconds,bite and hold for 6 seconds, etc.). Though the defensive attitude ofanimals varied, only the oldest animal (an adult male Gila Monster),displayed less vigor in biting frequency (quantity of bitecompressions).Collected venoms (pooled by species/collection event) werestored at 4°C and immediately transported to a facility for lyophilizationand then kept at -80°C. After CITES permits were obtained,lyophilized venoms were shipped to the research laboratoryfor genomic and proteomic analysis. The lyophilized venomsfrom H. suspectum and H. horridum were separately analyzed bygel permeation chromatography, reverse-phase HPLC, and massspectrometry for the isolation and structural characterization ofbioactive peptides. The typical and novel helodermatid venom peptides,exendins and helokinestatin respectively, were both isolatedand characterized from the lyophilized venoms collected by thismethod (Chen et al. 2006; Kwok et al. 2008). The precursor cDNAsof exendin-4 and exendin-3 were also cloned where the lyophilizedvenoms used as the material for reverse transcriptase PCR (Chenet al. 2006).Data published by Chen et al. (2006) and Kwok et al. (2008)showed that this technique for helodermatid venom collectionworked extremely well. The advantages of this method are that 1)generally it is non-injurious to the lizard; 2) reduces animal stressand the amount of blood from breaking teeth that might be inadvertentlycollected using other methods (Mitchell and Reichert1883); and 3) maximizes the quantity of venom that can be safelycollected.FIG. 2. Restrained Gila monster biting down on rubber bulb. The animal’shead is held at a 30-45° angle (below horizontal). Note drop of venomhanging from lower jaw of lizard.180 Herpetological Review 39(2), 2008