Outer surface protein E (OspE) mediates Borrelia burgdorferi sensu stricto strain-specific complement evasion in the eastern fence lizard, Sceloporus undulatus.

In North America, Lyme disease is primarily caused by the spirochetal bacterium Borrelia burgdorferi sensu stricto (Bb), which is transmitted between multiple vertebrate hosts and ixodid ticks, and is a model commonly used to study host-pathogen interactions. While Bb is consistently observed in its mammalian and avian reservoirs, the bacterium is rarely isolated from North American reptiles. Two closely related lizard species, the eastern fence lizard (Sceloporus undulatus) and the western fence lizard (Sceloporus occidentalis), are examples of reptiles parasitized by Ixodes ticks. Vertebrates are known to generate complement as an innate defense mechanism, which can be activated before Bb disseminate to distal tissues. Complement from western fence lizards has proven lethal against one Bb strain, implying the role of complement in making those lizards unable to serve as hosts to Bb. However, Bb DNA is occasionally identified in distal tissues of field-collected eastern fence lizards, suggesting some Bb strains may overcome complement-mediated clearance in these lizards. These findings raise questions regarding the role of complement and its impact on Bb interactions with North American lizards. In this study, we found Bb seropositivity in a small population of wild-caught eastern fence lizards and observed Bb strain-specific survivability in lizard sera. We also found that a Bb outer surface protein, OspE, from Bb strains viable in sera, promotes lizard serum survivability and binds to a complement inhibitor, factor H, from eastern fence lizards. Our data thus identify bacterial and host determinants of eastern fence lizard complement evasion, providing insights into the role of complement influencing Bb interactions with North American lizards.

Functional remodeling of adrenal steroidogenic tissue by food deprivation in the lizard, Sceloporus undulatus.

The present study examined how food availability interacts with age to modulate lizard adrenal steroidogenic function at the cellular level. Adult male and juvenile male and female Eastern Fence Lizards (Sceloporus undulatus) underwent a period of food deprivation with or without a shorter re-feeding period. Lizards maintained on a full feeding regimen served as the controls. Across the feeding regimens, plasma corticosterone of adult lizards was unchanged whereas that of food-deprived juvenile lizards was increased nearly 7 times and this increase was normalized by a short re-feeding period. Freshly dispersed adrenocortical cells derived from these lizards were incubated with ACTH and the production of selected steroids was measured by highly specific radioimmunoassay. Net maximal steroid rates of juvenile cells were 161% greater than those of adult cells. Adult and juvenile progesterone rates were consistently suppressed by food deprivation (by nearly 48%) and were normalized by a re-feeding period, whereas divergent effects were seen with corticosterone and aldosterone rates. Food deprivation suppressed corticosterone rates of adult cells by 43% but not those of juvenile cells. In a reciprocal manner, food deprivation had no significant effect on aldosterone rates of adult cells, but it suppressed those of juvenile cells by 52%. A short re-feeding period normalized most rates in both adult and juvenile cells and further augmented the adult aldosterone rate by 54%. The effect of the feeding regimens on ACTH sensitivity varied with life stage and with steroid. The overall sensitivity of adult cells to ACTH was nearly three times that of juvenile cells. Collectively, the data presented here and data from previous work indicate that food restriction/deprivation in Sceloporus lizard species causes a functional remodeling of the adrenocortical tissue. Furthermore, life stage adds more complexity to this remodeling.

COMPARISON OF PROTEIN ELECTROPHORESIS AND BIOCHEMICAL ANALYSIS FOR THE QUANTIFICATION OF PLASMA ALBUMIN IN HEALTHY BEARDED DRAGONS (POGONA VITTICEPS).

While electrophoresis is considered the standard method for evaluation of protein concentrations as a result of its direct measurement, albumin is often quantified with biochemical assays. Many laboratory-based chemistry analyzers and clinic-based point-of-care analyzers use the dye bromocresol green (BCG) for the quantitation of albumin. Several studies have shown that albumin concentrations obtained by the standard (BCG) dye-binding method are significantly different from those obtained by protein electrophoresis in avian species and chelonia. The goal of this study was to compare plasma albumin concentrations obtained by the BCG method with those derived from electrophoresis in bearded dragons (Pogona vitticeps). Thirty-six heparinized plasma samples were obtained from 13 clinically healthy male bearded dragons. Albumin was quantified by protein electrophoresis and by the BCG dye-binding method. The two methods were significantly different (P < 0.0001, paired t-test; P < 0.0001, Wilcoxon signed-rank test), with the BCG measurement always equal to or higher than the electrophoretic result. The measurements from both methods were significantly correlated (r = 0.8634, P < 0.0001), but concordance between the two techniques was poor. The Bland-Altman plot appeared to show a greater difference between the two measurements with lower albumin values and lesser difference with higher values. These results indicate that bearded dragon plasma albumin concentration measurements obtained by the BCG dye-binding method are unreliable when compared to those obtained with electrophoresis, suggesting that albumin should be measured by protein electrophoresis for health assessment in bearded dragons.

Cytogenetic analysis of nuclear abnormalities in the erythrocytes of gecko lizards (Phyllopezus periosus) collected in a semi-arid region of northeast Brazil: Possible effects of natural background radioactivity.

High natural-background radioactivity levels occur in the semi-arid region of the State of Rio Grande do Norte, northeastern Brazil. We have studied the lizard Phyllopezus periosus, an endemic species of the Brazilian caatinga with saxicolous habitat, as a bioindicator of environmental quality. Specimens were collected in three areas, an environmental protection area and two areas recognized as having high natural background radiation, one of these being a mining area. Level of metals and gamma radiation emitters present in the water sources potentially used by the lizards were measured. The biological endpoints assessed were micronuclei and nuclear abnormalities in blood samples. Significant differences in background radioactivity levels were found among the assessed areas. Statistically significant differences in micronuclei and nuclear abnormality frequencies were seen, among the study areas and a relationship between radioactivity level and genetic damage was observed.

Effects of temperature on plasma corticosterone in a native lizard.

The glucocorticoid stress response is frequently used to indicate vertebrate response to the environment. Body temperature may affect glucocorticoid concentrations, particularly in ectotherms. We conducted lab manipulations and field measurements to test the effects of body temperature on plasma corticosterone (predominant glucocorticoid in reptiles) in eastern fence lizards (Sceloporus undulatus). First, we acclimated lizards to one of 4 treatments: 22 °C, 29 °C, 33 °C, or 36 °C, and measured cloacal temperatures and plasma corticosterone concentrations at baseline and after exposure to a standardized stressor (cloth bag). Both baseline and stress-induced corticosterone concentrations were lower in lizards with lower body temperatures. Second, we acclimated lizards to 22 °C or 29 °C and exposed them to a standardized (cloth bag) stressor for 3 to 41 min. Lizards acclimated to 29 °C showed a robust increase in plasma corticosterone concentrations with restraint stress, but those at 22 °C showed no such increases in corticosterone concentrations. Third, we measured lizards upon capture from the field. There was no correlation between body temperature and baseline plasma corticosterone in field-caught lizards. These results suggest body temperature can significantly affect plasma corticosterone concentrations in reptiles, which may be of particular concern for experiments conducted under laboratory conditions but may not translate to the field.

The nocturnal leopard gecko (Eublepharis macularius) uses UVb radiation for vitamin D3 synthesis.

Vitamin D is an important regulator of calcium and phosphorus homeostasis in animals. It can be acquired from the diet or synthesised de novo when skin is exposed to UVb. Vitamin D deficiency can lead to a complex of diseases collectively called metabolic bone disease (MBD). Diurnal lizards without access to UVb are prone to develop vitamin D deficiency, even when dietary vitamin D3 is provided. A trial was conducted to determine whether juvenile nocturnal lizards require access to UVb to prevent vitamin D deficiency. All leopard geckos (Eublepharis macularius) were supplemented with dietary vitamin D3. One group was exposed to low level UVb radiation (33-51 µW/cm2) from hatching until 6 months of age and a second group remained unexposed. Animals were fed ad libitum and their growth and weight gain compared with non-exposed controls. At the end of the trial, blood samples were analysed for vitamin D3 metabolites. The concentration of the vitamin D3 metabolite, 25(OH)D3, was higher in UVb exposed animals (61 ± 20 vs. 38 ± 8 nmol/L), confirming cutaneous synthesis with UVb exposure. Growth and weight gain were similar in both groups, and this, together with the absence of clinical symptoms, suggests that dietary vitamin D3 alone can meet the vitamin D requirements for growth of this nocturnal gecko, during the first six months of life. It remains to be investigated whether the higher vitamin D metabolite levels holds other health benefits for this species, such as improved bone density or immune response.

Plasticity of haemoglobin concentration and thermoregulation in a mountain lizard.

The plastic capability of species to cope with the new conditions created by climate change is poorly understood. This is particularly relevant for organisms restricted to high elevations because they are adapted to cold temperatures and low oxygen availability. Therefore, evaluating trait plasticity of mountain specialists is fundamental to understand their vulnerability to environmental change. We transplanted mountain lizards, Iberolacerta cyreni, 800 m downhill to evaluate the plastic response in body condition, thermoregulation traits, haemoglobin level, and haemoparasite load. Initial measurements of body mass, total haemoglobin concentration ([Hb]), hematic parasite intensities, dorsal luminance, and thermoregulatory behaviour were resampled after two and four weeks of acclimation. We also tested whether an anti-parasitic drug reduced haemoparasite intensity. After only two weeks of acclimation to a lower elevation, lizards decreased 42% in [Hb], had 17% less parasite intensities, increased body condition by 25%, and raised by ~3% their mean preferred temperatures and their voluntary thermal maximum. The anti-parasitic treatment had no significant effect on the intensity of hematic parasites, but our results suggest that negative effects of haemoparasites on [Hb] are relaxed at lower elevation. The rapid plastic changes observed in thermal preferences, body condition, [Hb], and parasite intensity of I. cyreni demonstrate a potential plastic response of a mountain specialist. This may be adaptive under the climatic extremes typical of mountain habitats. However, there is uncertainty in whether the observed plasticity can also help overcome long term environmental changes.

Plasma estradiol and progesterone concentrations during the female reproductive cycle in a highly placentotrophic viviparous lizard, Mabuya sp.

The neotropical genus Mabuya are obligate placentotrophic viviparous lizards, which have a short vitellogenesis that produces microlecithal oocytes and a prolonged time of gestation (9 to 10 months). The hormonal control of female reproductive activity during follicular growth and pregnancy has not been studied, although it is known that the corpus luteum can produce progesterone, but regresses early in pregnancy, being replaced in this function by the placenta. Through enzyme immunoassay (EIA) we measured the plasma concentrations of estradiol (E2) and progesterone (P4) in females of a population of Mabuya sp at different stages of their reproductive cycle. Previously, we confirmed the presence of P4 in plasma by high-performance liquid chromatography methods with diode-array detector ultraviolet (HPLC-DAD-UV). The average concentration values of E2 and P4 were compared among reproductive stages and their dynamics were related to what is known in other oviparous and viviparous amniotes. The plasma E2 concentrations of Mabuya sp. are below the levels found in other viviparous reptiles, probably related to the substantial reduction of its follicular growth phase. Its highest concentration was detected during vitellogenesis, related to its function in the growth and maturation of the ovarian follicles and oviduct preparation for pregnancy; lower levels were observed during pregnancy, but they increase at the end when a new vitellogenesis event begins and massive placental maternal-fetal nutrient transfer occurs. High concentrations of P4 were found during pregnancy, related to its function in the maintenance of the developing embryos within the oviduct. The highest levels of P4 were found at early gestation, then they descend from mid-gestation to the end of gestation. Although some characteristics of hormonal control related to the high level of placentotrophy were observed in this species, the changes in plasma sex steroid concentrations during the reproductive cycle in females of Mabuya sp. follow patterns seen in other viviparous amniotes.

Anticancer Properties of Asian Water Monitor Lizard (Varanus salvator), Python (Malayopython reticulatus) and Tortoise (Cuora kamaroma amboinensis).

BACKGROUND: Cancer contributes to significant morbidity and mortality despite advances in treatment and supportive care. There is a need for the identification of effective anticancer agents. Reptiles such as tortoise, python, and water monitor lizards are exposed to heavy metals, tolerate high levels of radiation, feed on rotten/germ-infested feed, thrive in unsanitary habitat and yet have prolonged lifespans. Such species are rarely reported to develop cancer, suggesting the presence of anticancer molecules/mechanisms. METHODS: Here, we tested effects from sera of Asian water monitor lizard (Varanus salvator), python (Malayopython reticulatus) and tortoise (Cuora kamaroma amboinensis) against cancer cells. Sera were collected and cytotoxicity assays were performed using prostate cancer cells (PC3), Henrietta Lacks cervical adenocarcinoma cells (HeLa) and human breast adenocarcinoma cells (MCF7), as well as human keratinized skin cells (Hacat), by measuring lactate dehydrogenase release as an indicator for cell death. Growth inhibition assays were performed to determine the effects on cancer cell proliferation. Liquid chromatography mass spectrometry was performed for molecular identification. RESULTS: The findings revealed that reptilian sera, but not bovine serum, abolished viability of Hela, PC3 and MCF7 cells. Samples were subjected to liquid chromatography mass spectrometry, which detected 57 molecules from V. salvator, 81 molecules from Malayopython reticulatus and 33 molecules from C. kamaroma amboinensis and putatively identified 9 molecules from V. salvator, 20 molecules from Malayopython reticulatus and 9 molecules from C. kamaroma amboinensis when matched against METLIN database. Based on peptide amino acid composition, binary profile, dipeptide composition and pseudo-amino acid composition, 123 potential Anticancer Peptides (ACPs) were identified from 883 peptides from V. salvator, 306 potential ACPs from 1074 peptides from Malayopython reticulatus and 235 potential ACPs from 885 peptides from C. kamaroma amboinensis. CONCLUSION: To our knowledge, for the first time, we reported comprehensive analyses of selected reptiles' sera using liquid chromatography mass spectrometry, leading to the identification of potentially novel anticancer agents. We hope that the discovery of molecules from these animals will pave the way for the rational development of new anticancer agents.

Effects of glyphosate, cypermethrin, and chlorpyrifos on hematological parameters of the tegu lizard (Salvator merianae) in different embryo stages.

Different studies have demonstrated effects of pesticides during embryo development in vertebrates and stage-dependent effects, but there is no information concerning this for Salvator merianae. We evaluated the effects of the herbicides Glyphosate Roundup (GLY-RU) and Glyphosate Panzer Gold (GLY-PZ); and the insecticides Chlorpyrifos (CPF) and Cypermethrin (CYP), and their complex mixtures, at different concentrations in hematological parameters of S. merianae embryos at two different development stages. The analyzed parameters were Total and Differential White Blood Cells Count, Heterophils/Lymphocytes index (H/L), Lobularity index, and Natural Antibodies (Nabs titres), as well as growth, embryo mortality and birth delay. Heterophils decreased in the intermediate concentrations tested of CYP and GLY-RU, in animals exposed at 33-days development. Lymphocytes increased in the intermediate concentration tested of GLY-RU, and the H/L index decreased in the maximum concentration tested of GLY-RU. NAbs titres increased in those animals exposed to the maximum CYP concentration tested. However, animals exposed at 3/5-days development showed no differences among treatments in most of the analyzed parameters, suggesting a stage-dependent response. Nevertheless, those animals exposed to GLY-PZ showed lower Nabs titres in relation to negative control. These results suggest effects on different hematological parameters related to the immune system of S. merianae, according to the used pesticide (herbicide or insecticide), its concentration and commercial formulation (GLY-RU or GLY-PZ), and the stages of development of the exposed animals. Our results reveal the importance of carrying out studies that evaluate the effects of permanent exposure of living beings and their environments to these toxics.

Identification of Antibacterial Molecule(s) from Animals Living in Polluted Environments.

BACKGROUND: Snakes feed on germ-infested rodents, while water monitor lizards thrive on rotten matter in unhygienic conditions. We hypothesize that such creatures survive the assault of superbugs and are able to fend off disease by producing antimicrobial substances. In this study, we investigated the potential antibacterial activity of sera/lysates of animals living in polluted environments. METHODS: Snake (Reticulatus malayanus), rats (Rattus rattus), water monitor lizard (Varanus salvator), frog (Lithobates catesbeianus), fish (Oreochromis mossambicus), chicken (Gallus gallus domesticus), and pigeon (Columba livia) were dissected and their organ lysates/sera were collected. Crude extracts were tested for bactericidal effects against neuropathogenic E. coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus cereus and Klebsiella pneumoniae. To determine whether lysates/sera protect human cells against bacterialmediated damage, cytotoxicity assays were performed by measuring lactate dehydrogenase release as an indicator of cell death. Lysates/sera were partially characterized using heat-treatment and pronasetreatment and peptide sequences were determined using the Liquid Chromatography Mass Spectrometry (LC-MS). RESULTS: Snake and water monitor lizard sera exhibited potent broad-spectrum bactericidal effects against all bacteria tested. Heat inactivation and pronase-treatment inhibited bactericidal effects indicating that activity is heat-labile and pronase-sensitive suggesting that active molecules are proteinaceous in nature. LCMS analyses revealed the molecular identities of peptides. CONCLUSION: The results revealed that python that feeds on germ-infested rodents and water monitor lizards that feed on rotten organic waste possess antibacterial activity in a heat-sensitive manner and several peptides were identified. We hope that the discovery of antibacterial activity in the sera of animals living in polluted environments will stimulate research in finding antibacterial agents from unusual sources as this has the potential for the development of novel strategies in the control of infectious diseases.

Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards).

Pathogenic Leishmania spp. detected in lizards from Northwest China using molecular methods.

BACKGROUND: Leishmaniosis, a disease caused by pathogenic Leishmania parasites, remains an unresolved health problem in the New World and the Old World. It is well known that lizards can be infected by a subgenus of Leishmania parasites, i.e. Sauroleishmania, which is non-pathogenic to humans. However, evidence suggests that lizards may also harbor pathogenic Leishmania species including the undetermined Leishmania sp., discovered in our previous work. Leishmania DNA in lizard blood can be detected by using molecular methods, such as the polymerase chain reaction (PCR). RESULTS: Three hundred and sixteen lizards, representing 13 species of four genera, were captured for blood samples collection in Northwest China. Two reliable molecular markers (cytochrome b and heat shock protein 70 genes) were used for detection in the lizard blood samples, to confirm a widespread presence of pathogenic Leishmania parasites and the distribution pattern of Leishmania spp. in lizards from Northwest China. The PCR data indicated positive detection rate for Leishmania in all the tested lizards with an overall prevalence of 57.91% (183/316). Apart from lizard parasites like Leishmania tarentolae and Leishmania sp., several pathogenic Leishmania including L. turanica, L. tropica and L. donovani complex were identified by using phylogenetic analysis. Co-existence of different haplotypes was observed in most Leishmania DNA-positive lizards with an overall rate of 77.6% (142/183). Even mixed infections with different Leishmania species appeared to occur in the lizards with an overall rate of 37.7% (69/183). CONCLUSIONS: Lizards can harbor pathogenic Leishmania spp. Co-existence of different haplotypes or even species of Leishmania indicates mixed infections in natural lizard host. Lizards may contribute to the spread of Leishmania parasites. The pathogenic Leishmania species detected in lizards from Northwest China may be of great eco-epidemiological importance.

Impact of immunological state on eco-physiological variables in one of the southernmost lizards in the world.

The immune state is an essential component of survival as it directly influences physiological performance and health status. Variation in the leukocyte profile, a significantly increase in body temperature, and a detriment of the eco-physiological performance are among the possible consequences of an unhealthy state. In this study we analyse and discuss how field body temperature, preferred body temperature, the speed for sprint and long runs, locomotor stamina, and body condition can be affected by the immunological state (i.e. leukocyte profile) in a wild population of Liolaemus sarmentoi. Juveniles and adult males with a high percentage of eosinophils, basophils, and a low percentage of monocytes preferred higher body temperatures in a thermal gradient, while pregnant females maintained thermal preferences independently of leukocyte profile. Although juveniles with a high percentage of heterophils showed less locomotor stamina, adult males and pregnant females showed no differences in locomotor performance in relation to leukocyte profile. This study represents a starting point in eco-immunology of a wild lizard population of Liolaemus in cold and temperate environments of Patagonia where the southward shift in the geographic ranges of pathogen populations due to global warming represents a threat to resident host populations.

Corticosterone in Lizard Egg Yolk Is Reduced by Maternal Diet Restriction but Unaltered by Maternal Exercise.

When females face adverse environmental conditions, physiological changes, such as elevated corticosterone levels, to cope with the stressors may also impact their offspring. Such maternal effects are often considered adaptive and may "prime" the offspring for the same adverse environment, but maternal corticosterone levels do not always match that of the eggs produced. We examined how diet restriction and increased locomotor activity, via exercise training, affected steroid hormone levels of female green anole lizards, as well as the hormone levels in the yolk of their eggs. Diet restriction did not affect female hormone levels, but training increased corticosterone levels. Despite this, training did not affect yolk steroid levels, but eggs from females with diet restriction had lower corticosterone levels in yolk. This suggests that two common stressors, food shortage and increased locomotor activity, impact female physiology in a way that is not translated to her offspring.

Gonadal disruption after single dose exposure of prothioconazole and prothioconazole-desthio in male lizards (Eremias argus).

Prothioconazole (PTC) is a widely used triazole fungicide with low toxicity, and its desulfurization metabolite, prothioconazole-desthio (PTC-d), is reported to have higher reproductive toxicity to mammals. However, little is known about the reproductive toxicity, much less endocrine disrupting effect, of these two chemicals on reptiles. In this study, we investigated the effects of single dose of PTC/PTC-d (100 mg kg-1 body weight) exposure on the pathomorphism of testes and epididymides, serum sex steroid hormones (testosterone and 17ß-estradiol) and transcription of steroidogenic-related genes (STARD, cyp11A, cyp17, cyp19A, 17ß-HSD, 3ß-HSD, AR and ER-α) in gonads of male lizards (Eremias argus). Although structural disorder existed in PTC-d exposure group, severe gonadal disruption, especially suppression of spermatogenesis was only observed in testis after PTC treatment, which consequently led to the lack of spermatozoa in epididymal ducts. Consistent with this result, T/E2 value in PTC exposure was elevated to a significant higher level compared with control and continually increased over time, while T/E2 value in the PTC-d exposure group slightly increased only at 12 h. These results demonstrated a more serious disruption of PTC on male lizard gonads than PTC-d. In addition, the expression of cyp17 gene was inhibited at 6 h, however, was induced at 12 h, and exhibited negative correlations with STARD, cyp11A and 3ß-HSD after PTC exposure at each timepoint. In PTC-d group, the expression of STARD and 3ß-HSD were significantly down-regulated, in contrast, cyp11A and cyp17 were up-regulated, and each gene showed consistent changes over time. For 17ß-HSD, no significance was observed in both treated groups. This study was the first to compare the gonadal disruption of PTC and PTC-d in male lizards and elucidated that these two chemicals influenced the physiological function of male gonads through differential transcriptional modulation.

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BACKGROUND: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. . RESULTS: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple ß-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon ß-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 ß-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. CONCLUSIONS: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.

Seasonal changes in plasma concentrations of the thyroid, glucocorticoid and reproductive hormones in the tegu lizard Salvator merianae.

The tegu lizard Salvator merianae is a large, widely distributed teiid lizard endemic to South America that exhibits annual cycles of high activity during the spring and summer, and hibernation during winter. This pattern of activity and hibernation is accompanied by profound seasonal changes in physiology and behavior, including endothermy during the austral spring. The unusual combination of seasonal endothermy, hibernation and oviparity, in a non-avian, non-mammalian species, makes S. merianae an interesting subject for study of comparative aspects of endocrine regulation of seasonal changes in physiology. In the present study, we first validated commercially available immunoassay kits for quantification of hormone concentrations of the reproductive (testosterone, estradiol and progesterone), adrenal (corticosterone), and thyroid [thyroxine (T4) and triiodothyronine (T3)] axes in plasma of an outdoor, captive adult male and female S. merianae in southeastern Brazil. All assays exhibited parallelism and accuracy with S. merianae plasma. We next assessed patterns of concentration of these hormones across the annual cycle of S. merianae. Testosterone in males and estradiol in females peaked in spring coincident with the peak in reproductive behavior. Progesterone in females was significantly elevated in October coincident with putative ovulation when gravid females build nests. Thyroid hormones, known for regulating energy metabolism, varied seasonally with some sex-dependent differences. T4 gradually increased from an annual nadir during pre-hibernation and hibernation to high concentrations during spring in both sexes. In contrast, T3 did not vary seasonally in males, but females showed a two-fold increase in T3 during the spring reproductive season. T3 may be involved in energy investment during the seasonal production of large clutches of eggs. Corticosterone was significantly elevated during the active season in both sexes, suggesting its involvement in mobilization of energy stores and modulation of behavior (territoriality) and physiology. Ours is the first investigation of concurrent changes in reproductive, thyroid and adrenal hormone concentrations in this endemic and physiologically unique South American lizard. Our findings set the stage for future investigations to determine the extent to which these hormones influence activity and thermoregulation in S. merianae.

Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu).

Several geographical areas where Salvator merianae is distributed in Argentina are included in regions with agricultural activity and exposed to pesticide formulations. Some pesticides could affect defense mechanisms being able alter structures of some components of immune and endocrine systems. To assess the potential effects of pesticides in this reptile under seminatural conditions, on the immune system and endocrine responses in S. merianae we analyzed several blood parameters. Total (TWBCC), differential (DWBCC) white blood cells count, heterophils/lymphocytes index (H/L), lobularity index (LI), natural antibodies (NAbs) titres, complement system (CS), and corticosterone concentration were analyzed in animals exposed to a mixture of cypermethrin (25%), glyphosate (66.2%) and chlorpyrifos (48%) formulations. In addition, body size was considered in these analyzes. TWBCC and NAbs revealed lower values in organisms exposed to pesticides respect to a control indicating a possible immunosuppression effect. Besides, the LI showed a greater number of lobes in organism exposed demonstrating symptoms of chronic infection. In addition, we observed a reduced growth in these animals possibly related to a less energy investment in body mass to maintain an active defense against pesticides. Finally, we found high levels of plasma corticosterone in animals exposed to mix formulation that could demonstrate neuroendocrine axis activation. Other parameters like DWBCC, H/L index and activity of CS showed no differences in treated animals respect to control group, which could indicate low sensibility of these parameters to the concentration of pesticides used. Our results provide evidence of the toxic effects of pesticides on different immune system parameters, but also a trade-off among these parameters, corticosterone levels and growth. In this way, we can conclude that the formulated pesticides applied widely and constantly in the areas occupied by S. merianae, would be affecting its immune and endocrine systems and therefore its ability to defend against external agents. This kind of studies is of great interest to know the possible responses of wild species to anthropogenic disturbances such as pesticide contamination.

Baseline morphometric, haematological and plasma biochemical parameters in free-ranging eastern water dragons (Intellagama lesueurii lesueurii).

OBJECTIVE: The aim of this study was to determine baseline reference data for morphometric measurements and haematological and plasma biochemical parameters in clinically healthy eastern water dragons (Intellagama lesueurii lesueurii), accounting for the variables of season and sex. The clinical objective was to provide clinicians and researchers with baseline reference intervals (RIs) in order to assess accurately the health of a population or individual animals. METHODS: The study group comprised 39 free-ranging eastern water dragons. To monitor seasonal changes in data two study periods were assigned: September-October 2009 (spring) and January-March 2010 (summer). The 18 males and 21 females were captured in the grounds of Taronga Zoo, Mosman (33°50'45'' S, 151°15'20'' E) in Sydney, New South Wales, Australia. Basic morphometric, haematological and plasma biochemical parameters were measured. RIs were generated in accordance with the American Society of Veterinary Clinical Pathology guidelines. Data were analysed for the effects of sex, season and sex-season interactions using restricted maximal likelihood modelling. RESULTS: Reference values were established in eastern water dragons for morphometric data and RIs were generated for haematological and plasma biochemical levels. The study showed seasonal variation in uric acid. Weight, total length, snout-vent length and basophil % showed significant differences according to sex. Sex-season interactions were detected for calcium, monocyte % and total protein (refractometry). CONCLUSION: Multiple parameters exhibited normal physiological variation between season and sex and an interaction between the two factors. These variables need to be considered in the clinical context when interpreting results of haematological and biochemical analyses in eastern water dragons. The RIs established from this population will provide a basis for further studies in both free-living and captive eastern water dragons, and for comparison in clinical cases.