Pogona Research Articles

GASTRIC NEUROENDOCRINE CARCINOMA IN A BEARDED DRAGON (POGONA VITTICEPS)

There are multiple reports of gastric neuroendocrine carcinoma in bearded dragons (Pogona spp), but other types of alimentary neoplasia are rarely reported. In a retrospective study, we identified 51 cases of neoplasia, including gastric (n = 26), oral (n = 18), intestinal (n = 13), oesophageal (n = 3) and cloacal (n = 2) neoplasms. Round cell neoplasia was diagnosed in the alimentary tract in 14 cases, all of which had extra-alimentary involvement, most commonly in the liver, lung, spleen and kidney, and was considered to be the cause of death or euthanasia in all cases. Oral neoplasms included sarcoma, adenomatous polyp, round cell neoplasia, fibromatous epulis of periodontal ligament origin and myxoma. Apart from disseminated round cell neoplasia, all the oral neoplasms, including sarcomas, were not associated with death or euthanasia and had a good to excellent prognosis. Oesophageal neoplasms included round cell neoplasia. Gastric neoplasms included neuroendocrine carcinoma, round cell neoplasia, adenocarcinoma and sarcoma. Intestinal neoplasms included round cell neoplasia, sarcoma, adenocarcinoma and metastatic sarcoma. All oesophageal, gastric and intestinal neoplasms were associated with death or euthanasia and had a poor to grave prognosis. Cloacal neoplasms included round cell tumours and squamous cell carcinoma, and all were primary alimentary tumours except for the round cell tumours and one metastatic sarcoma. Round cell neoplasia and gastric neuroendocrine carcinoma were the most common diagnoses. Sarcomas and polyps were common in the oral cavity and rare to absent elsewhere. Adenocarcinoma was rare and only identified in the stomach and intestines.

Immunohistochemical staining patterns of alpha-keratins in normal tissues from two reptile species: implications for characterization of squamous cell carcinomas

BackgroundCytokeratins with epitopes in common with those of alpha (acidic and basic) mammalian keratins have been immunohistochemically demonstrated in the epidermis of reptiles. However, there are no reports of immunohistochemical staining patterns of alpha-keratins in other tissues from reptiles. Because the epithelial tumours usually retain the keratin patterns of their normal epithelial origin, it is necessary to know in advance these patterns in the major normal epithelia and organs. We used anti-alpha human keratin AE1 and AE3 monoclonal antibodies to study the staining patterns of alpha-keratins in the major normal epithelia and organs from two reptile species [the bearded dragon (Pogona vitticeps) and the loggerhead sea turtle (Caretta caretta)]. We also studied the immunolocalization of alpha-keratins in squamous cell carcinomas (SCCs) in a bearded dragon and two loggerhead turtles.ResultsAcidic alpha-keratin (AE1 positive) was detected in many of the epithelial tissues of the bearded dragons; however, the detection of basic alpha-keratin (AE3 positive) was much more limited. Alpha-keratins were detected in a greater number of tissues of loggerhead turtles compared with those observed in bearded dragons. In the bearded dragon SCC, all layers of the nests of neoplastic cells, including the cornified layer of the keratin pearls, were strongly reactive with the AE1 antibody. However, a weak reactivity using the AE3 antibody was detected in the basal and intermediate layers of these nests. In the cutaneous SCCs of both sea turtles, acidic alpha-keratin was detected in the basal and suprabasal layers, and in all of the invasive neoplastic cords, while basic alpha-keratin was mainly detected in the invasive neoplastic cords. The pattern observed in the metastases in both turtles consisted of immunohistological detection of acidic alpha-keratin in all metastatic foci, and limited or lack of detection of basic alpha-keratin.ConclusionsThis study provides, for the first time, information about the immunohistochemical staining patterns of alpha-keratins in normal tissues from bearded dragons and loggerhead sea turtles, and confirms the usefulness of AE1 and AE3 monoclonal antibodies in these reptile species. The use of these antibodies also contributed to a better characterization of SCCs in these species.

Relative position of the atrioventricular canal determines the electrical activation of developing reptile ventricles.

Squamate reptiles appear to lack the specialized His-Purkinje system that enables the cardiac ventricle to be activated from apex to base as in mammals and birds. Instead, activation may simply spread from where the atrioventricular canal connects to the base. Gja5, which encodes Cx40, which allows fast impulse propagation, was expressed throughout the ventricles of developing anole lizards. Activation was optically recorded in developing corn snake and central bearded dragon. Early embryonic ventricles were broad in shape, and activation propagated from the base to the right. Elongated ventricles of later stages were activated from base to apex. Before hatching of the snake, the ventricle developed a cranial extension on the left and activation propagated from the base to the caudal apex and the cranial extension. In squamate reptiles, the pattern of electrical activation of the cardiac ventricle is dependent on the position of the atrioventricular canal and the shape of the ventricle.

Temperature-induced colour change varies seasonally in bearded dragon lizards

The benefits of colour change are expected to vary seasonally because of changes in reproductive activity, temperature and, potentially, predation risk; yet temporal variation in colour change has seldom been examined. We measured colour change in spring and autumn using captive individuals from two differently coloured populations of the central bearded dragon lizard, Pogona vitticeps. We predicted that colour change should be greater in spring than autumn because of the added requirements of reproductive and territorial activity. To elicit colour change in a standardized way, we placed lizards inside temperature-controlled chambers and measured colour at 15, 25, 35 and 40 °C, repeating experiments in spring and autumn. Lizards from both populations changed from dark grey to light yellowish or orange-brown (increasing luminance and saturation) with increasing temperature in both seasons, and both populations changed colour to a similar extent. As predicted, the maximal extent of temperature-induced colour change (in particular, luminance change) was greater in spring than autumn. Our results confirm that temperature-induced colour change is greater in the peak activity season, probably an adaptation to the greater thermal and/or signalling needs of that time of year.

Intravenous Alfaxalone and Propofol Anesthesia in the Bearded Dragon (Pogona vitticeps)

Abstract Anesthesia is an essential part of veterinary care for reptiles; however, there is limited literature available for the anesthetic management of commonly kept species such as the bearded dragon (Pogona vitticeps). This study compares the intravenous use of 10 mg/kg propofol with 12 mg/kg alfaxalone. Both protocols resulted in smooth and rapid induction and subsequent intubation. A surgical plane of anesthesia, defined as a loss of reaction to noxious stimuli and absent muscle tone, was achieved in 5/8 (62.5%) alfaxalone animals and 0/8 of the propofol animals. Reflexes and muscle tone were regained from a cranial to caudal direction. There was no effect on the heart rate; however, both protocols resulted in respiratory depression, including 5 and 20 min durations of apnea in two of the alfaxalone animals. Differences in the characteristics of these two protocols are likely attributable to the different depths of anesthesia achieved. Further research is required to establish equipotent dosages to ...

Sex determination mode does not affect body or genital development of the central bearded dragon (Pogona vitticeps)

BackgroundThe development of male- or female-specific phenotypes in squamates is typically controlled by either temperature-dependent sex determination (TSD) or chromosome-based genetic sex determination (GSD). However, while sex determination is a major switch in individual phenotypic development, it is unknownhow evolutionary transitions between GSD and TSD might impact on the evolution of squamate phenotypes, particularly the fast-evolving and diverse genitalia. Here, we take the unique opportunity of studying the impact of both sex determination mechanisms on the embryological development of the central bearded dragon (Pogona vitticeps). This is possible because of the transitional sex determination system of this species, in which genetically male individuals reverse sex at high incubation temperatures. This can trigger the evolutionary transition of GSD to TSD in a single generation, making P. vitticeps an ideal model organism for comparing the effects of both sex determination processes in the same species.ResultsWe conducted four incubation experiments on 265 P. vitticeps eggs, covering two temperature regimes (“normal” at 28 °C and “sex reversing” at 36 °C) and the two maternal sexual genotypes (concordant ZW females or sex-reversed ZZ females). From this, we provide the first detailed staging system for the species, with a focus on genital and limb development. This was augmented by a new sex chromosome identification methodology for P. vitticeps that is non-destructive to the embryo. We found a strong correlation between embryo age and embryo stage. Aside from faster growth in 36 °C treatments, body and external genital development was entirely unperturbed by temperature, sex reversal or maternal sexual genotype. Unexpectedly, all females developed hemipenes (the genital phenotype of adult male P. vitticeps), which regress close to hatching.ConclusionsThe tight correlation between embryo age and embryo stage allows the precise targeting of specific developmental periods in the emerging field of molecular research on P. vitticeps. The stability of genital development in all treatments suggests that the two sex-determining mechanisms have little impact on genital evolution, despite their known role in triggering genital development. Hemipenis retention in developing female P. vitticeps, together with frequent occurrences of hemipenis-like structures during development in other squamate species, raises the possibility of a bias towards hemipenis formation in the ancestral developmental programme for squamate genitalia.

Incubation environment impacts the social cognition of adult lizards.

Recent work exploring the relationship between early environmental conditions and cognition has shown that incubation environment can influence both brain anatomy and performance in simple operant tasks in young lizards. It is currently unknown how it impacts other, potentially more sophisticated, cognitive processes. Social-cognitive abilities, such as gaze following and social learning, are thought to be highly adaptive as they provide a short-cut to acquiring new information. Here, we investigated whether egg incubation temperature influenced two aspects of social cognition, gaze following and social learning in adult reptiles (Pogona vitticeps). Incubation temperature did not influence the gaze following ability of the bearded dragons; however, lizards incubated at colder temperatures were quicker at learning a social task and faster at completing that task. These results are the first to show that egg incubation temperature influences the social cognitive abilities of an oviparous reptile species and that it does so differentially depending on the task. Further, the results show that the effect of incubation environment was not ephemeral but lasted long into adulthood. It could thus have potential long-term effects on fitness.

Characterization of Nannizziopsis guarroi with genomic and proteomic analysis in three lizard species

Fungal infections in captive as well as in free-living reptiles caused by emerging obligate pathogenic fungi appear with increasing frequency and give occasion to establish new and fast methods for routine diagnostics. The so-called yellow fungus disease is one of the most important and common fungal dermatomycoses in central bearded dragons (Pogona vitticeps) and green iguanas (Iguana iguana) and is caused by Nannizziopsis guarroi. The aim of this study was to prove reliability in identification of N. guarroi with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in comparison to molecular biological analysis of ribosomal DNA genes. In seven lizards from three different species, including central bearded dragons, green iguanas, and a European green lizard (Lacerta viridis), dermatomycoses caused by N. guarroi were diagnosed by isolation of the fungal pathogen as well as histopathological confirmation of the granulomatous inflammatory reaction in deep skin biopsies. With this survey, we proved that MALDI-TOF MS is a diagnostic tool for accurate identification of N. guarroi. Besides small subunit 18S rDNA (SSU) and internal transcribed spacer (ITS)1-5.8S rDNA, a large fragment of the large subunit of the 28S rDNA (LSU), including the domain (D)1 and D2 have been sequenced, for phylogenetical analysis. Large fragment of the LSU from N. guarroi has been sequenced for the first time. Yellow fungus disease in a European lizard species is described for the first time to our knowledge as well, which could be of importance for free-ranging populations of European lizards.

A survey of diseases in captive bearded dragons: a retrospective study of 529 patients

The aim of this clinical retrospective study was to analyse the prevalence of common disorders in 529 captive bearded dragons that were presented to three exotic animal clinics in Central Europe (Austria and Czech Republic) over a period of three years. A diagnosis was made on the basis of the presenting clinical signs and physical examination in 30.8% of the cases, whereas various additional diagnostic tests were performed in the other cases (69.2%). These included diagnostic imaging (radiography, ultrasound and computed tomography), examination of faecal samples for the presence of parasites, a blood-profile analysis, histological, as well as bacteriological and mycological examinations and necropsy. Gastrointestinal diseases (42.67%) like endoparasitism, constipation, sand ingestion, tympany and meteorism were the most common disorders. In 51.92% of the cases of constipation, endoparasites were present, whereas in 38.46% of the cases of constipation, metabolic bone diseases and imbalances in calcium and phosphorus levels were detected. Most of the analysed faecal samples (83.27%) were positive for the presence of endoparasites (48.7% pinworms, 25.39% coccidians and 16.06% flagellates). Dermatological problems (22.4%) contained skin tumours, which were observed in adult lizards (age range from three to eight years) and included spindle cell tumour on the eyelid, as well as spindle cell sarcoma, squamous cell carcinoma and melanoma on the trunk. Osteodystrophy, limb fractures and various types of necrosis on the limbs and tail were frequently observed musculoskeletal disorders (18.93%). Over half of all animals (57.14%) that underwent a blood test, showed a deviation in the calcium-phosphorus ratio, 63.98% showed hypocalcaemia and 26.71% hyperphosphataemia. Diseases of the urogenital system (9.47%) included renal diseases and dystocia. Neoplastic diseases besides skin tumours included two cases of leukaemia in 4-year-old bearded dragons, one of these with lymphoma in kidneys, lungs and liver and one ganglioneuroma in the body cavity of a bearded dragon of unknown age. Orthopaedic surgeries were most frequently performed to amputate the limbs or tail, whereas soft-tissue surgeries most often included the treatment of skin wounds and correction of cloacal prolapse, in addition to ovariectomy, salpingotomy or salpingectomy in female bearded dragons. Similar to the situation in Australia and the USA, infectious as well as non-infectious diseases are common in captive bearded dragons in Central Europe. Due to the high occurrence of endoparasitism, skin diseases and metabolic bone diseases in this present study, regular veterinary controls in bearded dragons including parasitological faecal examinations and optimisation of feeding and housing are necessary to improve the standard of health of bearded dragons kept as pet lizards in Europe.

Microsporidial Keratoconjunctivitis in a Pet Bearded Dragon (Pogona vitticeps)

A 4-month-old female bearded dragon (Pogona vitticeps) was presented with unilateral conjunctivitis. The conjunctivitis was not responsive to topical antibiotic or anti-inflammatory therapy and continued to progress in severity. Conjunctival tissue was sampled and microsporidial conjunctivitis was diagnosed on histopathological evaluation of submitted samples. Medical therapy was initiated and included topical itraconazole, systemic itraconazole, and systemic fenbendazole. Serial serum chemistry panels and complete blood counts were monitored for potential side effects associated with the medications. Despite treatment, the conjunctivitis progressed and enucleation was performed. Histopathology on the enucleated globe confirmed the diagnosis of microsporidial keratoconjunctivitis. Systemic itraconazole was administered at 5mg/kg orally for approximately 3 months. No abnormalities were detected on hematologic diagnostic testing during the treatment period. A computed tomographic scan was performed to monitor for systemic lesions, but no abnormalities were detected. About 12 months after initial presentation, the bearded dragon is healthy and there are no overt signs of disease.

Thermoregulatory behavior and orientation preference in bearded dragons

The regulation of body temperature is a critical function for animals. Although reliant on ambient temperature as a heat source, reptiles, and especially lizards, make use of multiple voluntary and involuntary behaviors to thermoregulate, including postural changes in body orientation, either toward or away from solar sources of heat. This thermal orientation may also result from a thermoregulatory drive to maintain precise control over cranial temperatures or a rostrally-driven sensory bias. The purpose of this work was to examine thermal orientation behavior in adult and neonatal bearded dragons (Pogona vitticeps), to ascertain its prevalence across different life stages within a laboratory situation and its interaction with behavioral thermoregulation. Both adult and neonatal bearded dragons were placed in a thermal gradient and allowed to voluntarily select temperatures for up to 8h to observe the presence and development of a thermoregulatory orientation preference. Both adult and neonatal dragons displayed a non-random orientation, preferring to face toward a heat source while achieving mean thermal preferences of ~ 33–34°C. Specifically, adult dragons were more likely to face a heat source when at cooler ambient temperatures and less likely at warmer temperatures, suggesting that orientation behavior counter-balances local selected temperatures but contributes to their thermoregulatory response. Neonates were also more likely to select cooler temperatures when facing a heat source, but required more experience before this orientation behavior emerged. Combined, these results demonstrate the importance of orientation to behavioral thermoregulation in multiple life stages of bearded dragons.

Fungal dermatitis, glossitis and disseminated visceral mycosis caused by different Metarhizium granulomatis genotypes in veiled chameleons (Chamaeleo calyptratus) and first isolation in healthy lizards

Metarhizium (M.) granulomatis (formerly Chamaeleomyces granulomatis) invariably causes fatal fungal glossitis and systemic mycosis in veiled chameleons (Chamaeleo calyptratus). Isolation of M. granulomatis in other lizards thus far has not been described. The aim of this study therefore was to obtain information on the presence of M. granulomatis in reptiles kept as pets, and to examine whether there was an association between specific genotypes and clinical/pathological outcomes. Besides 18S ribosomal (r) DNA (SSU) and internal transcribed spacer1-5.8S (ITS1-5.8S) rDNA, a fragment of the large subunit of the 28S rDNA (LSU), including the domains 1 (D1) and D2, were sequenced for identification of the fungus and phylogenetic analysis. Metarhizium granulomatis was isolated from 23 veiled chameleons, two panther chameleons (Furcifer pardalis) and one central bearded dragon (Pogona vitticeps). Only the veiled chameleons revealed corresponding pathological findings in the form of glossal hemorrhage, granulomatous glossitis, pharyngitis, dermatitis and/or visceral mycosis. The infection site correlated to survival times of infected veiled chameleons. Combined long-term treatment with terbinafine and nystatin based on susceptibility testing may be helpful for prevention of disease and visceral spreading of the fungus, but elimination of the fungal pathogen or successful treatment of diseased veiled chameleons have not been achieved yet. Sequencing of the ribosomal genes yielded five different genotypes, with genotype A being strongly correlated with dermatitis, and remaining genotypes with pharyngitis and glossitis. However, disseminated visceral mycosis developed irrespective of the genotypes.

Cholelithiasis in adult bearded dragons: retrospective study of nine adult bearded dragons (Pogona vitticeps) with cholelithiasis between 2013 and 2015 in southern Germany.

With an increased number of pet reptiles, many diseases occur due to nutritional disorders. Between 2013 and 2015, irregular gallbladder contents (sludge/choleliths) in adult bearded dragons (Pogona vitticeps) were recorded in many of the routinely conducted necropsies at the reptile rescue station in Munich (Auffangstation für Reptilien, München e.V., Munich, Germany). Nine animals, six from the rescue station and three from an associated veterinary practice (Tierärztliche Praxis für Exoten, Augsburg, Germany), were studied. Gallbladder contents from all animals were analysed at the Institute for Clinical Chemistry in Zurich, Switzerland. In three of nine animals, one cholelith composed of 100% calcium carbonate (CaCO3 ) was detected and it precipitated either as pure calcite or as a calcite:vaterite combination. In the remaining six animals, analyses suggested a protein-based material. The detection of choleliths/sludge was not anticipated at necropsy or surgery in eight of nine animals. The diet of the six animals from the rescue station was retrospectively described as mainly insects, whereas the diet of the three animals from the veterinary practice also contained little plant matter. Fed insect species were mealworm larva (Tenebrio molitor), house cricket (Acheta domestica), migratory locust (Locusta migratoria) and zophobas larva (Zophobas morio), all high in protein and fat. In other species, a nidus must be present for CaCO3 to precipitate. As a protein-based sludge was detected in six gallbladders, it is possible that a high-protein diet could lead to such a nidus and subsequently to cholelith formation. Cholelithiasis seems to be a rising problem in adult bearded dragons and is likely underdiagnosed, as many choleliths were found at necropsy. This rise in cholelithiasis may correlate with an unnatural high-protein, high-fat insect-based diet instead of a balanced plant-based diet.

A comparison of UVb compact lamps in enabling cutaneous vitamin D synthesis in growing bearded dragons.

The effect of exposure to different UVb compact lamps on the vitamin D status of growing bearded dragons (Pogona vitticeps) was studied. Forty-two newly hatched bearded dragons (<24h old) were allocated to six treatment groups (n=7 per group). Five groups were exposed to different UVb compact lamps for two hours per day, with a control group not exposed to UVb radiation. At 120days of age, blood samples were obtained and concentrations of 25(OH)D3 , Ca, P and uric acid were determined. In addition, plasma 25(OH)D3 concentration was determined in free-living adult bearded dragons to provide a reference level. Only one treatment resulted in elevated levels of 25(OH)D3 compared to the control group (41.0±12.85 vs. 2.0±0.0nmol/L). All UVb-exposed groups had low 25(OH)D3 plasma levels compared to earlier studies on captive bearded dragons as well as in comparison with the free-living adult bearded dragons (409±56nmol/L). Spectral analysis indicated that all treatment lamps emitted UVb wavelengths effective for some cutaneous vitamin D synthesis. None of these lamps, under this regime, appeared to have provided a sufficient UVb dose to enable synthesis of plasma 25(OH)D3 levels similar to those of free-living bearded dragons in their native habitat.

Response to novelty as an indicator of reptile welfare

Whilst a great deal of research has been focused on identifying ways to assess the welfare of captive mammals and birds, there is comparatively little knowledge on how reptilian species are affected by captivity, and the ways in which their welfare can be accurately assessed. The present study investigated response to novelty – a commonly used approach to assess anxiety-like behaviour and hence welfare in non-human animals – in two species of reptile with the aim of determining whether this approach could be successfully translated from use in mammalian and avian species for use in reptiles, and whether we could also identify reptile-specific and/or species-specific behaviours. Eight red-footed tortoises (Chelonoidis carbonaria) and seventeen bearded dragons (Pogona vitticeps) were observed individually in both familiar and novel environments for 10min time periods, and their behaviour recorded. Tortoises were found to begin locomotion sooner when placed in a familiar environment than when placed in a novel environment, they extended their necks further in a familiar environment and their neck length increased over time in both familiar and novel environments, suggesting an overall anxiety-like response to novelty as seen in non-reptilian species. In contrast, whilst bearded dragons exhibited significantly more tongue-touches in a novel, compared to a familiar, environment, they showed no difference between familiar and novel environments in their latency to move. This result suggests that, whilst the dragons appeared to discriminate between the two environments, this discrimination was not necessarily accompanied by an anxiety-like response. This study has confirmed the translatability of response to novelty as an approach to assess anxiety-like behaviour in one species of reptile, as well as identifying species-specific behaviours that have the potential to be used in future studies when assessing the welfare of reptiles in response to captive environments, but our results also highlight the need to be aware of species differences within a class as diverse as reptilia.

Geographic divergence and colour change in response to visual backgrounds and illumination intensity in bearded dragons.

Animals may improve camouflage by both dynamic colour change and local evolutionary adaptation of colour but we have little understanding of their relative importance in colour-changing species. We tested for differences in colour change in response to background colour and light intensity in two populations of central bearded dragon lizards (Pogona vitticeps) representing the extremes in body coloration and geographical range. We found that bearded dragons change colour in response to various backgrounds and that colour change is affected by illumination intensity. Within-individual colour change was similar in magnitude in the two populations but varied between backgrounds. However, at the endpoints of colour change, each population showed greater similarity to backgrounds that were representative of the local habitat compared with the other population, indicating local adaptation to visual backgrounds. Our results suggest that even in species that change colour, both phenotypic plasticity and geographic divergence of coloration may contribute to improved camouflage.

Induction to inhalation anaesthesia in agamid lizards with alfaxalone

The aim of this study was to evaluate intravenous anaesthesia with alfaxalone for tracheal tube insertion in three species of agamid lizards. Alfaxalone, at a dose rate of 5 mg/kg, was administered intravenously to 80 inland bearded dragons (Pogona vitticeps), 10 Rankin’s dragons (Pogona henrylawsoni) and 10 Chinese water dragons (Physignathus cocincinus) following 24 h of fasting. The righting reflex in inland bearded dragons, Rankin’s dragons and Chinese water dragons was lost within 12–45 s (17.52 ± 8.80 s), 15–40 s (24.60 ± 9.61 s) and 12–40 s (21.70 ± 9.53 s), respectively. The mean tracheal tube insertion time for inland bearded dragons, Rankin’s dragons and Chinese water dragons was 23.61 ± 10.91 s, 35.30 ± 14.71 s and 32.00 ± 14.32 s, respectively. Intravenous use of alfaxalone proved to be a suitable method for safe tracheal tube insertion and induction to inhalation anaesthesia in agamid lizards.

Validation of a thermal threshold nociceptive model in bearded dragons (Pogona vitticeps)

ObjectivesTo validate a thermal threshold (TT) nociceptive model in bearded dragons (Pogona vitticeps) and to document TT changes after administration of morphine. Study designA two-part randomized, blinded, controlled, experimental study. AnimalsFive adult bearded dragons (242–396 g). MethodsA TT device delivered a ramped nociceptive stimulus (0.6 °C second−1) to the medial thigh until a response (leg kick/escape behavior) was observed or maximum (cut-off) temperature of 62 °C was reached. In phase I, period 1, six TT readings were determined at 20 minute intervals for evaluation of repeatability. Two of these readings were randomly assigned to be sham to assess specificity of the behavioral response. The same experiment was repeated 2 weeks later (period 2) to test reproducibility. In phase II, animals were administered either intramuscular morphine (10 mg kg−1) or saline 0.9%. TTs (maximum 68 °C) were determined before and 2, 4, 8, 12 and 24 hours after treatment administration. Data were analyzed using one-way anova (temporal changes and repeatability) and paired t tests (reproducibility and treatment comparisons) using Bonferroni correction (p < 0.05). ResultsMean TT values were 57.4 ± 3.8 °C and 57.3 ± 4.3 °C for periods 1 and 2, respectively. Data were repeatable within each period (p = 0.83 and p = 0.07, respectively). Reproducibility between periods was remarkable (p = 0.86). False-positive responses during sham testing were 10%. TTs were significantly increased after morphine administration at 2, 4 and 8 hours compared with baseline, and at 2 and 4 hours compared with saline 0.9%. The highest TT was 67.7 ± 0.7 °C at 4 hours after morphine administration. Conclusions and clinical relevanceTesting was repeatable, reproducible and well tolerated in bearded dragons. TT nociceptive testing detected morphine administration and may be suitable for studying opioid regimens in bearded dragons.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps)

Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons (Pogona vitticeps), six veiled chameleons (Chamaeleo calyptratus), and six panther chameleons (Furcifer pardalis) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity.

Expression of a hindlimb-determining factorPitx1in the forelimb of the lizardPogona vitticepsduring morphogenesis

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the central bearded dragon (Pogona vitticeps) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (GHR, Pitx1 and Shh) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimbs and hindlimbs of P. vitticeps. While the highest level of GHR expression occurred at the hatchling stage, Pitx1 and Shh expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of Pitx1 expression—a hindlimb-determining gene—in the forelimbs of P. vitticeps to that in a closely related Australian agamid lizard, Ctenophorus pictus, where we found Pitx1 expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis—a result consistent with that found across other tetrapods. Expression of Pitx1 in forelimbs has only rarely been documented, including via in situ hybridization in a chicken and a frog. Our findings from both RT-qPCR and IHC indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology.