New Data on the Larval Stages of Leptophallus nigrovenosus (Digenea, Plagiorchiata).

(1) Background: Leptophallus nigrovenosus, an esophageal parasite that primarily affects water snakes of the genus Natrix, has a known life cycle that involves snail and amphibian hosts. However, the biological aspects, chaetotaxic patterns, and pathogeny of this parasite in its hosts have not been fully elucidated. (2) Methods: Snails (Planorbarius metidjensis) were collected in Spain and examined for cercaria emergence. The larvae were used to experimentally infect Salamandra salamandra, and metacercariae were isolated. Their chaetotaxy was studied using microscopy and scanning electron microscopy. The eye histology was also examined. (3) Results: The cercariae displayed distinctive morphological characteristics. The results of this study revealed three types of ciliated sensory papillae on the cercarial teguments, suggesting an adaptation for host detection and orientation. The metacercariae isolated from subcutaneous tissues showed oval bodies covered in spines. The chaetotaxy patterns matched those of Leptophallinae species. This is the first report of the presence of L. nigrovenosus in the snail P. metidjensis. Additionally, this study detected metacercariae in the eyes of S. salamandra, emphasizing the need for further research on trematode infections in amphibian eyes. (4) Conclusions: Members of the genus Salamandra can serve as secondary intermediate hosts for L. nigrovenosus, and the presence of metacercariae in amphibian eyes may have implications for the survival and habitat management of these amphibians. Understanding this parasite's prevalence, transmission dynamics, and impacts on host populations is crucial for conservation strategies.

Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

Embryonic and juvenile snakes (Natrix maura, Linnaeus 1758) compensate for high elevation hypoxia via shifts in cardiovascular physiology and metabolism.

The colonization of novel environments requires a favorable response to conditions never, or rarely, encountered in recent evolutionary history. For example, populations colonizing upslope habitats must cope with lower atmospheric pressure at elevation, and thus reduced oxygen availability. The embryo stage in oviparous organisms is particularly susceptible, given its lack of mobility and limited gas exchange via diffusion through the eggshell and membranes. Especially little is known about responses of Lepidosaurian reptiles to reduced oxygen availability. To test the role of physiological plasticity during early development in response to high elevation hypoxia, we performed a transplant experiment with the viperine snake (Natrix maura, Linnaeus 1758). We maintained gravid females originating from low elevation populations (432 m above sea level [ASL]-normoxia) at both the elevation of origin and high elevation (2877 m ASL-extreme high elevation hypoxia; approximately 72% oxygen availability relative to sea level), then incubated egg clutches at both low and high elevation. Regardless of maternal exposure to hypoxia during gestation, embryos incubated at extreme high elevation exhibited altered developmental trajectories of cardiovascular function and metabolism across the incubation period, including a reduction in late-development egg mass. This physiological response may have contributed to the maintenance of similar incubation duration, hatching success, and hatchling body size compared to embryos incubated at low elevation. Nevertheless, after being maintained in hypoxia, juveniles exhibit reduced carbon dioxide production relative to oxygen consumption, suggesting altered energy pathways compared to juveniles maintained in normoxia. These findings highlight the role of physiological plasticity in maintaining rates of survival and fitness-relevant phenotypes in novel environments.

Mobile Zoos and Other Itinerant Animal Handling Events: Current Status and Recommendations for Future Policies.

Mobile zoos are events in which non-domesticated (exotic) and domesticated species are transported to venues such as schools, hospitals, parties, and community centres, for the purposes of education, entertainment, or social and therapeutic assistance. We conducted literature searches and surveyed related government agencies regarding existing provisions within laws and policies, number of mobile zoos, and formal guidance issued concerning operation of such events in 74 countries or regions. We also examined governmental and non-governmental guidance standards for mobile zoos, as well as websites for mobile zoo operations, assessed promotional or educational materials for scientific accuracy, and recorded the diversity of species in use. We used the EMODE (Easy, Moderate, Difficult, or Extreme) algorithm, to evaluate identified species associated with mobile zoos for their suitability for keeping. We recorded 14 areas of concern regarding animal biology and public health and safety, and 8 areas of false and misleading content in promotional or educational materials. We identified at least 341 species used for mobile zoos. Mobile zoos are largely unregulated, unmonitored, and uncontrolled, and appear to be increasing. Issues regarding poor animal welfare, public health and safety, and education raise several serious concerns. Using the precautionary principle when empirical evidence was not available, we advise that exotic species should not be used for mobile zoos and similar itinerant events.

Coinfection of Chytrid Fungi in Urodeles during an Outbreak of Chytridiomycosis in Spain.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), has had an unprecedented impact on amphibian biodiversity. Although Bd is globally widespread, Bsal is currently spreading, increasing the probability that these pathogens will co-occur in individual amphibians. Interactions among coinfecting parasites can have significant outcomes on disease dynamics and impact and, therefore, may have important consequences for amphibian conservation. We analyzed the patterns of Bd-Bsal coinfections in two species of free-ranging urodeles during an outbreak of chytridiomycosis in Spain. Our goals were to assess 1) the probability of co-occurrence of both chytrid species and 2) the correlation of pathogen loads in coinfected hosts. We detected coinfections in 81.58% of Triturus marmoratus (n=38) and in 18.75% of Ichthyosaura alpestris (n=16). Histopathologic lesions of chytridiomycosis were observed only in T. marmoratus. Our results demonstrate a positive relationship between Bd and Bsal loads in T. marmoratus, whereas the co-occurrence analysis showed a random association among pathogens in both urodele species. Overall, we show that Bd-Bsal coinfections intensify pathogen load in T. marmoratus and could, therefore, increase disease severity and have important consequences for the conservation of some amphibian species.

Symmetry-Breaking Stabilities in Carapace Curvature on Testudo (Reptilia, Testudinidae).

The aim of this research was to contribute to the study of the doming geometry of Testudo carapace as an unstable point of equilibrium when animals are overturned. We performed this research using geometric morphometric using a sample of 64 Testudo individuals belonging to different species (T. hermannin = 30, T. graecan = 3, T. marginata n = 13 and T. horsfieldii n = 18), sexes and ages. A set of four sagittal landmarks (discrete homologous points) and 15 pairs of semi-landmarks, on the frontal doming of the carapace, were digitized on individual carapace pictures. Significative fluctuating asymmetry was detected, defined as small, completely random departures from bilateral symmetry, but much less than directional asymmetry, which appeared highly significative. Anti-symmetry did not appear. Carapace asymmetry was dominated by a clear right directionality. A possible biological speculation could be that this asymmetry more that easing the self-righting potential ("kinematic instability", understood as the ability to self-right without effort), makes stable ventral turning difficult ("static stability", understood as the ability to resist passively turning the body produced by destabilizing forces). This asymmetry is present among both sexes but more marked among males. An explanation for this sexually differentiated pattern could be the higher locomotion and the fight for mating in males, making them consequently more prone to losing their balance and falling on their back. These data may be useful in studying adaptative traits in Testudo species as well as establishing a seminal base for future studies. This research is the first attempt to explore a suitable method to assess doming asymmetry which could be useful in future, more extensive investigations, on a larger interspecific sample.

Climate warming triggers the emergence of native viruses in Iberian amphibians.

The number of epizootics in amphibian populations caused by viruses of the genus Ranavirus is increasing worldwide. Yet, causes for pathogen emergence are poorly understood. Here, we confirmed that the Common midwife toad virus (CMTV) and Frog virus 3 (FV3) are responsible for mass mortalities in Iberia since the late 1980s. Our results illustrate the Iberian Peninsula as a diversity hotspot for the highly virulent CMTV. Although this pattern of diversity in Europe is consistent with spread by natural dispersal, the exact origin of the emergence of CMTV remains uncertain. Nevertheless, our data allow hypothesizing that the Iberian Peninsula might harbor the ancestral population of CMTVs that could have spread into the rest of Europe. In addition, we found that climate warming could be triggering the CMTV outbreaks, supporting its endemic status in the Iberian Peninsula.

Getting It Straight: Accommodating Rectilinear Behavior in Captive Snakes-A Review of Recommendations and Their Evidence Base.

Snakes are sentient animals and should be subject to the accepted general welfare principles of other species. However, they are also the only vertebrates commonly housed in conditions that prevent them from adopting rectilinear behavior (ability to fully stretch out). To assess the evidence bases for historical and current guidance on snake spatial considerations, we conducted a literature search and review regarding recommendations consistent with or specifying ≥1 × and <1 × snake length enclosure size. We identified 65 publications referring to snake enclosure sizes, which were separated into three categories: peer-reviewed literature (article or chapter appearing in a peer-reviewed journal or book, n = 31), grey literature (government or other report or scientific letter, n = 18), and opaque literature (non-scientifically indexed reports, care sheets, articles, husbandry books, website or other information for which originating source is not based on scientific evidence or where scientific evidence was not provided, n = 16). We found that recommendations suggesting enclosure sizes shorter than the snakes were based entirely on decades-old 'rule of thumb' practices that were unsupported by scientific evidence. In contrast, recommendations suggesting enclosure sizes that allowed snakes to fully stretch utilized scientific evidence and considerations of animal welfare. Providing snakes with enclosures that enable them to fully stretch does not suggest that so doing allows adequate space for all necessary normal and important considerations. However, such enclosures are vital to allow for a limited number of essential welfare-associated behaviors, of which rectilinear posturing is one, making them absolute minimum facilities even for short-term housing.

Evolutionary history of mental glands in turtles reveals a single origin in an aquatic ancestor and recurrent losses independent of macrohabitat.

Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.

Turning Negatives into Positives for Pet Trading and Keeping: A Review of Positive Lists.

The trading and keeping of exotic pets are associated with animal welfare, conservation, environmental protection, agricultural animal health, and public health concerns and present serious regulatory challenges to legislators and enforcers. Most legislation concerning exotic pet trading and keeping involves restricting or banning problematic species, a practice known as "negative listing". However, an alternative approach adopted by some governments permits only the keeping of animals that meet certain scientifically proven criteria as suitable in respect of species, environmental, and public health and safety protections. We conducted an evaluation of positive lists for the regulation of pet trading and keeping within the context of the more prevalent system of restricting or prohibiting species via negative lists. Our examination of international, national, and regional regulations in Europe, the United States, and Canada found that criteria used for the development of both negative and positive lists were inconsistent or non-specific. Our online surveys of governments received limited responses, although telephone interviews with officials from governments either considering or developing positive lists provided useful insights into their attitudes and motivations towards adopting positive lists. We discuss key issues raised by civil servants including perceived advantages of positive lists and anticipated challenges when developing lists of suitable species. In addition, we compare functions of negative and positive lists, and recommend key principles that we hope will be helpful to governments concerning development and implementation of regulations based on positive lists.

High-elevation hypoxia impacts perinatal physiology and performance in a potential montane colonizer.

Climate change is generating range shifts in many organisms, notably along the elevational gradient in mountainous environments. However, moving up in elevation exposes organisms to lower oxygen availability, which may reduce the successful reproduction and development of oviparous organisms. To test this possibility in an upward-colonizing species, we artificially incubated developing embryos of the viperine snake (Natrix maura) using a split-clutch design, in conditions of extreme high elevation (hypoxia at 2877 m above sea level; 72% sea-level equivalent O2 availability) or low elevation (control group; i.e. normoxia at 436 m above sea level). Hatching success did not differ between the two treatments. Embryos developing at extreme high elevation had higher heart rates and hatched earlier, resulting in hatchlings that were smaller in body size and slower swimmers compared to their siblings incubated at lower elevation. Furthermore, post-hatching reciprocal transplant of juveniles showed that snakes which developed at extreme high elevation, when transferred back to low elevation, did not recover full performance compared to their siblings from the low elevation incubation treatment. These results suggest that incubation at extreme high elevation, including the effects of hypoxia, will not prevent oviparous ectotherms from producing viable young, but may pose significant physiological challenges on developing offspring in ovo. These early-life performance limitations imposed by extreme high elevation could have negative consequences on adult phenotypes, including on fitness-related traits.

Multiple glacial refugia and contemporary dispersal shape the genetic structure of an endemic amphibian from the Pyrenees.

Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species' present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

The chemistry and histology of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa.

Despite evidence from anatomy, behavior and genomics indicating that the sense of smell in turtles is important, our understanding of chemical communication in this group is still rudimentary. Our aim was to describe the microanatomy of mental glands (MGs) in a freshwater turtle, Mauremys leprosa (Geoemydidae), and to assess the chemical composition of their secretions with respect to variation among individuals and between sexes. MGs are paired sac-like organs on the gular region of the neck and are dimorphic in this species with males having fully functional holocrine glands while those of females appear non-secretory and vestigial. In adult males, the glandular epithelium of the inner portion of the gland provides exocytotic products as well as cellular debris into the lumen of the gland. The contents of the lumen can be secreted through the narrow duct portion of the gland ending in an orifice on the surface of the skin. Females have invaginated structures similar in general outline to male glands, but lack a glandular epithelium. Using gas chromatography coupled to mass spectrometry, we identified a total of 61 compounds in mental gland secretions, the most numerous being carboxylic acids, carbohydrates, alkanes, steroids and alcohols. The number of compounds per individual varied widely (mean (median) ± SD = 14.54 (13) ± 8.44; min = 3; max = 40), but only cholesterol was found in all samples. We found that the relative abundances of only six chemicals were different between the sexes, although males tended to have larger amounts of particular compounds. Although the lipid fraction of mental gland secretions is rich in chemical compounds, most occur in both sexes suggesting that they are metabolic byproducts with no role in chemical signaling. However, the relative amounts of some compounds tended to be higher in males, with significantly larger amounts of two carboxylic acids and one steroid, suggesting their putative involvement in chemical communication.

Ancient mitogenomics clarifies radiation of extinct Mascarene giant tortoises (Cylindraspis spp.).

The five extinct giant tortoises of the genus Cylindraspis belong to the most iconic species of the enigmatic fauna of the Mascarene Islands that went largely extinct after the discovery of the islands. To resolve the phylogeny and biogeography of Cylindraspis, we analysed a data set of 45 mitogenomes that includes all lineages of extant tortoises and eight near-complete sequences of all Mascarene species extracted from historic and subfossil material. Cylindraspis is an ancient lineage that diverged as early as the late Eocene. Diversification of Cylindraspis commenced in the mid-Oligocene, long before the formation of the Mascarene Islands. This rejects any notion suggesting that the group either arrived from nearby or distant continents over the course of the last millions of years or had even been translocated to the islands by humans. Instead, Cylindraspis likely originated on now submerged islands of the Réunion Hotspot and utilized these to island hop to reach the Mascarenes. The final diversification took place both before and after the arrival on the Mascarenes. With Cylindraspis a deeply divergent clade of tortoises became extinct that evolved long before the dodo or the Rodrigues solitaire, two other charismatic species of the lost Mascarene fauna.

Variation of preferred body temperatures along an altitudinal gradient: A multi-species study.

Temperature affects the physiological functions of ectotherms. To maintain optimal body temperature and ensure physiological performance, these organisms can use behavioral adjustments to keep the body temperature in their specific temperature range, so-called preferred temperature (Tpref). It is therefore crucial to describe and understand how Tpref vary within and amongst populations to predict the effects of climate change of altitudinal range shifts in organisms. We aimed at determining the altitudinal variations in Tpref in three ectothermic species (the Pyrenean brook salamander - a semi-aquatic and thigmothermic amphibian - the European common lizard and the wall lizard - both heliothermic species). Using an experimental approach where Tpref were measured along a temperature gradient in laboratory conditions, we used a cross-sectional approach to compare the variation of Tpref measured in populations sampled along the altitudinal gradient in the Pyrenees. We hypothesized a complex and highly variable intra-specific response of Tpref along geographical clines, with a positive relationship between Tpref and altitude (as predicted by the countergradient variation), the reverse pattern (referring to the adaptation of local optima hypothesis), or no relationship at all. Our results corroborated partially the countergradient hypothesis in the salamander (middle to high elevation part). At high altitude level, individuals may compensate for lower opportunities of favorable conditions by choosing a high temperature which maximizes their activities. However, populations from low elevation level hence better supported the adaptation of local optima hypothesis, such as both lizard species, for which Tpref tended instead to decrease with altitude. Lizards from cold climates may be physiologically adapted to low temperature, with the ability to reach optimal functioning at lower temperature than individuals from low altitude. Our findings suggest that predicting future niche models should therefore take into account the variability of Tpref, where species might be differently affected by global warming.

Gender Identification by Cloacoscopy and Cystoscopy in Juvenile Chelonians.

Cloacoscopy and cystoscopy are simple, noninvasive to minimally invasive techniques that provide excellent visualization, and result in fast recovery. General or intrathecal anesthesia is sufficient. They can be performed in free-ranging turtles under field conditions. Cloacoscopic gender identification of external genitalia is not reliable because of the high degree of misinterpretation between phallus and clitoris, especially in juveniles. However, saline-infusion or air insufflation cystoscopy through the urinary bladder (or accessory vesicles/bladders) is often effective for the visualization of gonads and to identify the sex. Visualization of gonads is feasible through the urinary bladder or accessory vesicle wall in many species.

Inside the Redbox: applications of haematology in wildlife monitoring and ecosystem health assessment.

Blood analyses have great potential in studies of ecology, ecotoxicology and veterinary science in wild vertebrates based on advances in human and domestic animal medicine. The major caveat for field researchers, however, is that the 'rules' for human or domestic animal haematology do not always apply to wildlife. The present overview shows the strengths and limitations of blood analyses in wild vertebrates, and proposes a standardisation of pre-analytical procedures plus some suggestions for a more systematic examination of blood smears to increase the diagnostic value of blood data. By discussing the common problems that field researchers face with blood variables, we also aim to highlight common ground enabling new researchers in the field to accurately collect blood samples and interpret and place their haematological findings into the overall picture of an ecological or eco-toxicological study. Besides showing the practicality and ecological relevance of simple blood variables, this study illustrates the suitability of blood samples for the application of cutting-edge analytical procedures for expanding the current repertoire of diagnostic tools in wildlife monitoring and ecosystem health assessment.

Incidence of pigmented skin tumors in a population of wild Montseny brook newt (Calotriton arnoldi).

We report the presence of pigmented skin tumors in three populations of the endangered amphibian Montseny brook newt, Calotriton arnoldi, one of the European amphibian species with the smallest distribution range (40 km(2) in the Montseny Natural Park, Catalonia, Spain). Examination of one of the tumors by light microscopy was consistent with chromatophoroma and was most suggestive of a melanophoroma. Tumors were not found in juveniles. In adults, only two of three populations were affected. The proportions of males and females affected were not significantly different, but there was a positive correlation between body size and presence of tumors in both sexes. The etiology of chromatophoromas remains unknown but, in our study, they do not appear to have been caused by water quality or Ultraviolet B.

Pancreatitis associated with the helminth Serpinema microcephalus (Nematoda: Camallanidae) in exotic red-eared slider turtles (Trachemys scripta elegans).

Pancreatitis associated with the helminth Serpinema microcephalus was found in three of 19 free-ranging red-eared slider turtles (Trachemys scripta elegans) captured between March 2003 and September 2004 in southern Spain. Microscopic changes were associated with parasite migrations and were characterized by central areas of necrosis surrounded by leukocytes and resulted in destruction of exocrine tissue. The blood profile of one of the three female turtles revealed eosinophilia and hyperglycemia, common in helminth infections and pancreatic disorders respectively. These are the first reported cases of pancreatitis caused by the nematode S. microcephalus in the exotic and newly colonized host T. s. elegans.

Molecular characterization of a new serovar of Salmonella bongori 13,22:z39:- isolated from a lizard.

Three Salmonella strains isolated from a lizard (Gallotia simoni) in the "Isla del Hierro" (Canary Islands, Spain) were serotyped as Salmonella bongori serotype 13,22:z39:-, which has not been described in the Kauffmann-White scheme of Salmonella serovars. In order to shed light on the assignment of those strains to the S. bongori species, several genes were amplified and/or sequenced. The iroB gene has been reported to be present only in S. enterica, while the invA gene has been described as being a helpful tool in distinguishing Salmonella from other bacterial species. Both genes were amplified and, as expected, only invA could be amplified. The fliC gene, encoding the phase 1 flagellin fljB gene, encoding phase 2 flagellin, and the gapA gene, which is believed to present polymorphic alleles among different subspecies, were amplified and sequenced. The sequence obtained from fliC(z39) matched with the sequences fliC(z39) obtained from other serovars. The sequence obtained from gapA clustered into the S. bongori group when it was compared to others previously described. We conclude that these three isolates are members of the S. bongori species representing a new serovar that will be described in the next supplement to the Kauffmann-White scheme.