Detection of parasitic forms in feces of Chelonoidis carbonarius (Linnaeus, 1766) from Brazilian Cerrado.

Chelonoidis carbonarius is host to a great diversity of endoparasites. This research aimed to identify and taxonomically classify the parasitic forms of endoparasites found in the feces of C. carbonarius using coproparasitological methods. In total, 38 fecal samples were collected from animals captured in the municipalities of Cuiabá (n = 23) and Jataí (n = 15) in the Brazilian Ceraado. Fecal samples were submitted to at least one of the following techniques: Willis-Mollay flotation technique, zinc sulfate centrifugal flotation technique, and spontaneous sedimentation of Hoffman, and Pons, and Janer. Overall, 86.85% (33/38) of animals were positive in at least one of the diagnostic techniques. Of the 38 samples used, 50% of the animals were captive and 50% of the animals were free-living. Cysts, oocysts, and parasitic eggs were identified, as follows: Chapiniella spp. (65.78%; n = 24), Sauricola spp. (31.57%; n = 12), Ascaroidea (15.78%; n = 6), Oxyuridae (2.63%; n = 1), Trematoda (2.63%; n = 1), Acanthocephala (2.63%; n = 1), Balantidium spp. (39.47%; n = 15), Eimeria spp. (21.05%; n = 8), and Nyctotherus spp. (2.63%; n = 1). The study showed that both captive and free-living C. carbonarius are often parasitized by different endoparasite species, with nematode eggs being the most common.

Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa.

BACKGROUND: Hepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species' reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach. METHODS: Specimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database. RESULTS: Peripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi. CONCLUSIONS: The present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.).

Macro- and micro-anatomical investigation of the oropharyngeal roof of landform greek tortoise (Testudo graeca graeca) and semi-aquatic red-eared slider turtle (Trachemys scripta elegans).

The present investigation examined the oropharyngeal roof of two turtles having different feeding behaviors: the landform Greek tortoise (Testudo graeca graeca) primarily herbivores and the semi-aquatic red-eared slider turtle (Trachemys scripta elegans) lives in freshwater that opportunistic omnivorous grossly and by scanning and light microscopes. Grossly, the Greek tortoise had a V-shaped roof consisting of the upper rhamphotheca, peri-palatine region, upper alveolar ridge, peripheral palatine ridge, median palatine ridge, vomer, choanae, caudal palatine part, and pharynx. At the same time, the red-eared slider had a semilunar roof consisting of upper rhamphotheca, two peripheral palatine ridges, core of palatine ridges, upper alveolar band, vomer, choanae, caudal palatine part, and pharynx. SEM revealed that the red-eared slider roof appeared more straightforward. The upper rhamphotheca is sharp, with a median premaxillary notch in the red-eared slider that gives a powerful bite for cutting to compensate absence of the teeth. Additionally, the red-eared slider's upper alveolar band is interrupted by a single upper alveolar ridge that appears spiky, pointed, and longer as it needs powerful chewing of prey and there are two types of teeth-like projections at its peri-palatine area for food-crushing and chewing. The Greek tortoise palatine region had numerous ridges and folds to provide roughness for food processing. Greek tortoises had small-sized choanae with two choanal folds to minimize choanal openings when eating dusty grasses. Histologically, Greek tortoise palate was rostrally thicker and more keratinized than caudally, and the caudal palatine region was characterized by a single pair of circumvallate-like papilla with multiple mucous openings and secretions, while red-eared slider palate was slightly keratinized at the peri-choanal region, and the rest of the palate was non-keratinized with few mucous openings. The current investigation found various structural oropharyngeal roof adaptations to feeding behavior in the omnivore red-eared slide compared to the herbivorous Greek turtle.

Genetic diversity and host specificity of Blastocystis in reptiles, Eastern Thailand.

Blastocystis inhabits the digestive tracts of a diverse range of hosts. Transmission patterns, including host specificity, and the clinical and public health significance of Blastocystis in humans remain poorly understood. This study aimed to investigate the distribution and genetic diversity of Blastocystis in herbivorous and carnivorous reptiles in Eastern Thailand. A total of 501 faecal samples were collected from 363 iguanas, 79 bearded dragons, 50 tortoises, and nine snakes in an animal breeding farm in Chonburi Province, Eastern Thailand. Detection and differentiation of Blastocystis was based on amplification, sequencing, and phylogenetic analysis of specific small subunit (SSU) ribosomal RNA genes from faecal DNA extracted from the samples. Altogether 101/501 samples (20 %) were polymerase chain reaction (PCR) and sequencing-positive for Blastocystis, 90 (89 %) of which were from iguanas; the remaining positive samples were from African spurred tortoise (n=6), Bearded dragon (n=3), Leopard tortoise (n=1), and Red-footed tortoise (n=1). Phylogenetic analysis revealed that most of the Blastocystis sequences from iguanas were largely similar, and they were distinct from those of the tortoises. Subtype 17 was found in the three bearded dragons and likely reflected Blastocystis from prey animals. This is the largest survey of Blastocystis in reptiles to date. Remarkable differences in Blastocystis colonization rates and genetic diversity were observed between iguanas and other reptile orders, and what was considered Blastocystis colonization was only observed in herbivorous reptiles.

Pathogenic Potential of Opportunistic Gram-Negative Bacteria Isolated from the Cloacal Microbiota of Free-Living Reptile Hosts Originating from Bulgaria.

Reptiles are known to be asymptomatic carriers of various zoonotic pathogens. A number of Gram-negative opportunistic commensals are causative agents of bacterial infections in immunocompromised or stressed hosts and are disseminated by reptiles, whose epidemiological role should not be neglected. Since most studies have focused on exotic species, in captivity or as pet animals, the role of wild populations as a potential source of pathogens still remains understudied. In the present study, we isolated a variety of Gram-negative bacteria from the cloacal microbiota of free-living lizard and tortoise hosts (Reptilia: Sauria and Testudines) from the Bulgarian herpetofauna. We evaluated their pathogenic potential according to their antibiotic susceptibility patterns, biofilm-forming capacity, and extracellular production of some enzymes considered to play roles as virulence factors. To our knowledge, the phenotypic manifestation of virulence factors/enzymatic activity and biofilm formation in wild reptile microbiota has not yet been widely investigated. All isolates were found to be capable of forming biofilms to some extent and 29.6% of them could be categorized as strong producers. Two strains proved to be excellent producers. The majority of the isolated strains showed extracellular production of at least one exoenzyme. The most pronounced pathogenicity could be attributed to the newly isolated Pseudomonas aeruginosa strain due to its multiresistance, excellent biofilm formation, and expression of exoenzymes.

Digital skull anatomy of the Oligocene North American tortoise Stylemys nebrascensis with taxonomic comments on the species and comparisons with extant testudinids of the Gopherus-Manouria clade.

The anatomy of North American tortoises is poorly understood, despite a rich fossil record from the Eocene and younger strata. Stylemys nebrascensis is a particularly noteworthy turtle in this regard, as hundreds of specimens are known from Oligocene deposits, and as this species is one of the earliest fossil turtles to have been described in the scientific literature. Since its initial description based on a shell, many specimens with more complete material have been referred to Stylemys nebrascensis. Here, we review and confirm the referral of an important historic specimen to Stylemys nebrascensis, which includes shell, non-shell postcranial, and skull material. This allows us to document unique skull features of Stylemys nebrascensis (e.g., an unusual 'poststapedial canal' that connects the posterior skull surface with the cavum acustico-jugulare) and to refer another well-preserved skull to the species. Based on computed-tomography scanning of these two skulls, we provide a detailed description of the cranial and mandibular osteology of Stylemys nebrascensis. Stylemys nebrascensis has a combination of plesiomorphic skull characteristics (e.g., retention of a medial jugal process) and derived traits shared with extant gopher tortoises (e.g., median premaxillary ridge) that suggest it may be a stem-representative of the gopher tortoise lineage. This supports the hypothesis that extant and fossil tortoises from North America form a geographically restricted clade that split from Asian relatives during the Paleogene.

Automated hematological cell count using sysmex XN-1000V in Testudo hermanni: Agreement with manual count.

Mediterranean area represents the main habitat of Testudo hermanni. Clinical signs of disease of these tortoises are non-specific, making the hematology results crucial in revealing underlying pathological conditions. However, accurate automated identification of blood cell populations is hampered by the presence of nucleated erythrocytes (NRBC) and thrombocytes (Thr), necessitating manual methods such as counting chambers. The aim of the study was to assess the performance of the novel automated hematology analyzer Sysmex XN-1000 V, which includes a a specific channel (WNR) for counting NRBC, in accurately identify and quantify the different blood cell populations of Testudo hermanni. Additionally, its agreement with manual counts was evaluated. Fifty heparinized blood samples were initially counted using the Neubauer improved chamber and then analysed twice with Sysmex XN-1000 V. Thirteen out of 50 samples were instrumentally counted again after 48 h to assess the inter-assay precision. All WNR scattergrams were re-analysed using an ad hoc gate panel to differentiate two populations: NRBCs (weak fluorescence signal) and WBC + Thr (high fluorescence signal). Sysmex XN-1000 V demonstrated optimal intra- and inter-assay precision for NRBCs (CV 0.98% ± 1.96; 1.31% ± 2.98) and moderate precision for WBC + Thr (CV 9.24% ± 16.61; 12.69% ± 10.35). No proportional nor constant errors were observed between the methods for both the populations. The instrumental NRBC counts were consistently slightly lower, while WBC + Thr counts were slightly higher compared to manual counts. These findings suggest that Sysmex XN-1000 V can be used for analyzing cell populations in heparinized blood of Testudo hermanni. However, specific instrumental reference intervals are suggested.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroys side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach / Sexagem por eversão do falo em Phrynops geoffroanus (Testudines: Chelidae): uma nova abordagem não invasiva

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroy's side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Efficacy of the combination of milbemycin oxime and afoxolaner for the control of myiasis by Musca domestica in desert tortoises (Gopherus berlandieri).

The efficacy of the combination of milbemycin oxime and afoxolaner was evaluated in desert tortoises infested with fly larvae. Oral administration of the combination of milbemycin oxime and afoxolaner eliminated the infestation without generating any evident adverse effects on the tortoises.

L'efficacité de la combinaison de milbémycine oxime et d'afoxolaner a été évaluée chez des tortues du désert infestées par des larves de mouches. L'administration orale de la combinaison de milbémycine oxime et d'afoxolaner traite l'infestation sans générer d'effets indésirables évidents sur les tortues.

A eficácia da associação de milbemicina oxima e afoxolaner foi avaliada em tartarugas do deserto infestadas por larvas de moscas. A administração oral da combinação de afoxolaner e milbemicina oxima eliminou a infestação sem gerar nenhum efeito adverso evidente às tartarugas.

Se evaluó la eficacia de la combinación de milbemicina oxima y afoxolaner en tortugas del desierto infestadas con larvas de mosca. La administración oral de la combinación de milbemicina oxima y afoxolaner eliminó la infestación sin generar efectos adversos evidentes en las tortugas.

Mycotic Diseases in Chelonians.

Turtles and 'tortoises' populations are declining in number, the factors driving extinction risks being related to habitat loss and degradation, climate change, introduction of invasive plant and animal species, consumption by humans for food and medicinal use, and collection for international pet trade. Fungal infections represent one of the main threats for ecosystem health. The present narrative review deals with conventional and emerging mycoses of Chelonians. Although conventional mycoses in captive and pet reptiles would depend on poor husbandry, being the agents mostly opportunistic pathogens, some fungal species were reported to occur more frequently, such as the entomopathogen Purpureocillium lilacinum. Furthermore, emerging agents such as the Fusarium solani species complex have been recognized as a real threat for the surviving of some aquatic species, acting as primary pathogens. This complex has been recently included within pathogens in One Health issues. Emydomyces testavorans is recognized as an emerging threat, although, due its recent identification, information about its epidemiology is limited. Data about treatments and outcomes of mycoses in Chelonians are also referred.

PREVALENCE AND PARASITE INTENSITY OF HAEMOGREGARINES IN AFRICAN BELL HINGE-BACK (KINIXYS BELLIANA) AND AFRICAN HOME'S HINGE-BACK (KINIXYS HOMEANA) TORTOISES IN IBADAN, NIGERIA.

Haemogregarines are a group of intracellular blood parasites reported in reptiles, other vertebrate taxa including mammals and fish, and haematophagous invertebrates. Information on prevalence, biodiversity, and host-specificity of haemogregarines of tortoises in Nigeria are scarce. A total of 120 African hinge-back tortoises (Kinixys belliana, n=70, and K. homeana, n=50) were bought from the Wildlife and Herbal Market in Ibadan, Nigeria. Blood was withdrawn via the subcarapacial sinuses of each tortoise. The prevalence and parasite intensity of haemogregarine infection was determined using a light microscope, and parasite species were determined by PCR using HepF300 and HepR900 primers. A sequence of 654 base pairs (bp; Hepatozoon cf. fitzsimonsi) from the study aligned with published 18SrRNA and closely related with a similarity of 97.38% to Hepatozoon fitzsimonsi sequenced from Kinixys zobensis in South Africa. Overall prevalence of haemogregarines was 53.33% from light microcopy and 75.83% from PCR, which is considered very high. Higher prevalence and mean±SD parasite intensity were recorded for K. belliana (82.89%, 0.29%) than for K. homeana (66.00%, 0.26%). Prevalence rates and parasite intensities of haemogregarines were significantly higher in wet than in dry seasons. The differences in prevalence and intensity between and within species may be due to habitat characteristics, feeding habits of vectors and hosts, seasons, and vector abundance.

From STRs to SNPs via ddRAD-seq: Geographic assignment of confiscated tortoises at reduced costs.

Assigning individuals to their source populations is crucial for conservation research, especially for endangered species threatened by illegal trade and translocations. Genetic assignment can be achieved with different types of molecular markers, but technical advantages and cost saving are recently promoting the shift from short tandem repeats (STRs) to single nucleotide polymorphisms (SNPs). Here, we designed, developed, and tested a small panel of SNPs for cost-effective geographic assignment of individuals with unknown origin of the endangered Mediterranean tortoise Testudo hermanni. We started by performing a ddRAD-seq experiment on 70 wild individuals of T. hermanni from 38 locations. Results obtained using 3182 SNPs are comparable to those previously obtained using STR markers in terms of genetic structure and power to identify the macro-area of origin. However, our SNPs revealed further insights into the substructure in Western populations, especially in Southern Italy. A small panel of highly informative SNPs was then selected and tested by genotyping 190 individuals using the KASP genotyping chemistry. All the samples from wild populations of known geographic origin were genetically re-assigned with high accuracy to the original population. This reduced SNPs panel represents an efficient molecular tool that enables individuals to be genotyped at low cost (less than €15 per sample) for geographical assignment and identification of hybrids. This information is crucial for the management in-situ of confiscated animals and their possible re-allocation in the wild. Our methodological pipeline can easily be extended to other species.

Linking animal migration and ecosystem processes: Data-driven simulation of propagule dispersal by migratory herbivores.

Animal migration is a key process underlying active subsidies and species dispersal over long distances, which affects the connectivity and functioning of ecosystems. Despite much research describing patterns of where animals migrate, we still lack a framework for quantifying and predicting how animal migration affects ecosystem processes. In this study, we aim to integrate animal movement behavior and ecosystem functioning by developing a predictive modeling framework that can inform ecosystem management and conservation.We propose a framework to model individual-level migration trajectories between populations' seasonal ranges as well as the resulting dispersal and fate of propagules carried by the migratory animals, which can be calibrated using empirical data at every step of the modeling process. As a case study, we applied our framework to model the spread of guava seeds, Psidium guajava, by a population of migratory Galapagos tortoises, Chelonoidis porteri, across Santa Cruz Island. Galapagos tortoises are large herbivores that transport seeds and nutrients across the island, while Guava is one of the most problematic invasive species in the Galapagos archipelago.Our model can predict the pattern of spread of guava seeds alongside tortoises' downslope migration range, and it identified areas most likely to see establishment success. Our results show that Galapagos tortoises' seed dispersal may particularly contribute to guava range expansion on Santa Cruz Island, due to both long gut retention time and tortoise's long-distance migration across vegetation zones. In particular, we predict that tortoises are dispersing a significant amount of guava seeds into the Galapagos National Park, which has important consequences for the native flora.The flexibility and modularity of our framework allow for the integration of multiple data sources. It also allows for a wide range of applications to investigate how migratory animals affect ecosystem processes, including propagule dispersal but also other processes such as nutrient transport across ecosystems. Our framework is also a valuable tool for predicting how animal-mediated propagule dispersal can be affected by environmental change. These different applications can have important conservation implications for the management of ecosystems that include migratory animals.

A comparison of non-surgical methods for sexing young gopher tortoises (Gopherus polyphemus).

Many turtle species have temperature-dependent sex determination (TSD), raising the prospect that climate change could impact population dynamics by altering sex ratios. Understanding how climate change will affect populations of animals with TSD requires a reliable and minimally invasive method of identifying the sexes of young individuals. This determination is challenging in many turtles, which often lack conspicuous external sexual dimorphism until years after hatching. Here, we explore four alternatives for sexing three age classes of captive-reared young gopher tortoises (Gopherus polyphemus), a terrestrial turtle of conservation concern native to the southeastern United States: (1) naive testosterone levels, (2) testosterone levels following a follicle stimulating hormone (FSH) challenge, (3) linear morphological measurements, and (4) geometric morphometrics. Unlike some other turtle species, male and female neonatal gopher tortoises have overlapping naive testosterone concentration distributions, justifying more complicated methods. We found that sex of neonates (<7 days old) is best predicted by a "random forest" machine learning model with naive testosterone levels and morphological measurements (8% out-of-bag error). Sex of hatchlings (4-8 months old) was predicted with 11% error using a simple threshold on naive testosterone levels, or with 4% error using a simple threshold on post-FSH testosterone levels. Sex of juveniles (approximately 3.5 years old) was perfectly predicted using a simple threshold on naive testosterone levels. Sexing hatchlings at >4 months of age is the easiest and most reliable non-surgical method for sex identification. Given access to a rearing facility and equipment to perform hormone assays, these methods have the potential to supplant laparoscopic surgery as the method of choice for sexing young gopher tortoises.

Antimicrobial Resistant Salmonella in Chelonians: Assessing Its Potential Risk in Zoological Institutions in Spain.

Salmonella is mostly noted as a food-borne pathogen, but contact with chelonians has also been reported as a source of infection. Moreover, high levels of antimicrobial resistance (AMR) have been reported in Salmonella isolated from wild and captive reptiles. The aim of this study was to assess the occurrence of Salmonella AMR carriage by chelonians admitted to two zoological institutions in Spain, characterizing the isolates to assess the Salmonella AMR epidemiology in wildlife. To this end, 152 chelonians from nine species were sampled upon their arrival at the zoological nuclei. Salmonella identification was based on ISO 6579-1:2017 (Annex D), isolates were serotyped and their AMR analysed according to the EU Decision 2013/652. Moreover, the genetic relationship of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). Results showed 19% (29/152) of the chelonians positive to Salmonella, all of them tortoises. For all isolates, 69% (20/29) were resistant and 34% (10/29) multidrug-resistant (MDR) strains. PFGE clustered isolates according to the serovar, confirming a low genetic diversity. In conclusion, this study shows a high presence of MDR Salmonella strains in tortoises at their entry into zoological nuclei. This condition highlights the need to establish Salmonella detection protocols for the entry of animals into these centres.

Low-coverage reduced representation sequencing reveals subtle within-island genetic structure in Aldabra giant tortoises.

Aldabrachelys gigantea (Aldabra giant tortoise) is one of only two giant tortoise species left in the world and survives as a single wild population of over 100,000 individuals on Aldabra Atoll, Seychelles. Despite this large current population size, the species faces an uncertain future because of its extremely restricted distribution range and high vulnerability to the projected consequences of climate change. Captive-bred A. gigantea are increasingly used in rewilding programs across the region, where they are introduced to replace extinct giant tortoises in an attempt to functionally resurrect degraded island ecosystems. However, there has been little consideration of the current levels of genetic variation and differentiation within and among the islands on Aldabra. As previous microsatellite studies were inconclusive, we combined low-coverage and double-digest restriction-associated DNA (ddRAD) sequencing to analyze samples from 33 tortoises (11 from each main island). Using 5426 variant sites within the tortoise genome, we detected patterns of within-island population structure, but no differentiation between the islands. These unexpected results highlight the importance of using genome-wide genetic markers to capture higher-resolution genetic structure to inform future management plans, even in a seemingly panmictic population. We show that low-coverage ddRAD sequencing provides an affordable alternative approach to conservation genomic projects of non-model species with large genomes.

Diversity of microorganisms in Hyalomma aegyptium collected from spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia.

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health.

The "right" side of sleeping: laterality in resting behaviour of Aldabra giant tortoises (Aldabrachelys gigantea).

Although some studies investigated lateralization in reptiles, little research has been done on chelonians, focusing only on few behaviours such as righting response and escape preference. The aim of this study was to investigate lateralization in Aldabra giant tortoises (Aldabrachelys gigantea), focusing on asymmetrical positioning of the limbs and the head during resting behaviour, called sleep-like behaviour, involving both wild tortoises and individuals under human care. Subjects of the study were 67 adult Aldabra tortoises (54 free ranging on Curieuse, 13 under human care in Mahè Botanical Garden). For each tortoise observed during sleep-like behaviour, we recorded the position of the head (on the left, on the right or in line with the body midline) and we collected which forelimb and hindlimb were kept forward. Moreover, the number of subjects in which limbs were in a symmetrical position during the sleep-like behaviour was recorded. Based on our results, the number of tortoises with asymmetrical position of head and limb was higher (head: 63%; forelimbs: 88%; hindlimbs: 70%) than the number of tortoises with symmetrical position of the head and the limb. Regarding the head, throughout the subjects found with the asymmetrical position of the head during sleep-like behaviour, tortoises positioning the head on the right (42%) were more than those sleeping with the head on the left (21%). We found a relationship between the position of the forelimbs and hindlimbs during sleep-like behaviour. We reported no differences between Mahè (under human care) and Curieuse (wild) tortoises. Findings of this preliminary study underlined traces of group-level lateralization in head positioning during the sleep-like behaviour, possibly due to a left-eye/right-hemisphere involvement in anti-predatory responses and threatening stimuli as reported in reptiles and other vertebrates. This study aims at adding data on brain lateralization, often linked to lateralized behaviours, in reptiles, especially in chelonians.

Molecular screening of ticks of the genus Amblyomma (Acari: Ixodidae) infesting South African reptiles with comments on their potential to act as vectors for Hepatozoon fitzsimonsi (Dias, 1953) (Adeleorina: Hepatozoidae).

In South Africa, the role of reptilian ticks in the transmission of haemoparasites is lacking, in part, due to limited information on tick diversity and their associated haemoparasites. The aim of this research was to identify tick species parasitizing reptiles and to molecularly screen these ectoparasites for species of the blood apicomplexan genus Hepatozoon. Samples were collected from Ndumo Game Reserve, KwaZulu-Natal, and the Cape Columbine region, Western Cape. Reptiles collected included 2 snakes, 2 monitor lizards of a single species respectively, as well as 17 tortoises of four species. Ticks collected from these were morphologically identified as Amblyomma latum (n = 2) and Amblyomma marmoreum (n = 98), this identification was molecularly confirmed using 16S rRNA and CO1 genes. Screening for Hepatozoon was done by amplifying the 18S rRNA gene. A species of Hepatozoon, Hepatozoon fitzsimonsi, was identified from A. marmoreum ticks, with an overall prevalence of 10%. This Hepatozoon species, has been described parasitizing tortoises from southern Africa, and has been reported from ticks infesting tortoises from Kenya, East Africa. Even though ticks have been suggested to be the likely vector of this Hepatozoon species, with this supported by the findings of Hepatozoon-like developmental stages in ticks collected off of infected tortoises, a recent systematic revision placed this species in a newly erected genus Bartazoon, a genus vectorised by biting insects. The present study thus provides further support for ticks acting as the potential vectors of H. fitzsimonsi.