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Normal reference intervals (RI) of hematologic and biochemical parameters are important for assessing and monitoring the health status of captive and free-living chelonians; however, such information is not available for most wildlife species. Giant Galapagos tortoises are one of the most iconic animals on earth and health information can make an important contribution to their conservation and management. This study provides formal RI of haematology and plasma biochemistry parameters and describes cell morphology along with morphometrics of free-living Eastern Santa Cruz (Chelonoidis donfaustoi), Española (Chelonoidis hoodensis) and San Cristóbal tortoises (Chelonoidis chathamensis). We explored differences in blood parameters between sexes, across the tortoise species in this study and with previously published RI of the Western Santa Cruz tortoise (Chelonoidis porteri). Biochemistry parameters of both Santa Cruz species were overall more similar to each other than to Española and San Cristobal tortoises. This research constitutes the first RI for these three Galapagos tortoise species and may be of value for advising captive-breeding and conservation plans. We recommend further research to establish RI in additional tortoise species so we may better understand and interpret haematology and biochemistry parameters as a valuable conservation tool for species of this critically endangered taxon.

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Emerging infectious diseases (EIDs) have been reported as causes of morbidity and mortality in free-living animal populations, including turtles and tortoises, and they have even resulted in species extinctions, with human activities contributing to the spread of many of these diseases. In the Galapagos, giant tortoises are endangered due to habitat change, invasive species, and other human impacts; however, the impact of EIDs on Galapagos tortoise conservation remains understudied. To fill this gap, we conducted health assessments of five tortoise species from the islands of Santa Cruz, Isabela and Española. We performed health evaluations of 454 animals and PCR testing for pathogens known to be relevant in other tortoise species. We identified two novel sequences of adenoviruses and four of herpesviruses. Based on alignments of the DNA polymerase gene and maximum likelihood phylogenetic analyses, we found both novel adenoviruses to be most closely related to red footed tortoise adenovirus 2, by nucleotide sequence and red footed tortoise adenovirus 1, based on amino acid sequence. Three of the herpesvirus sequences translated into the same deduced amino acid sequence; therefore, they may be considered the same viral species, closely related to terrapene herpesvirus 2. The fourth herpesvirus sequence was highly divergent from any sequence previously detected and is related to an eagle owl herpesvirus based on nucleotide sequence and to loggerhead oro-cutaneous herpesvirus based on amino acids. These novel viruses may be pathogenic for giant tortoises under specific conditions (e.g., stress). Continued screening is crucial to determine if these viruses play a role in tortoise fitness, morbidity and survival. This information allows us to provide recommendations to the Galapagos National Park Directorate and other institutions to improve the management of these unique species both in Galapagos and globally, and for tortoise reintroduction plans throughout the archipelago.

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Antimicrobial resistance has become one of the main public health threats worldwide with anthropogenic activities driving the spread of resistance. Understanding and combatting the spread of resistant bacteria is a top priority for global health institutions, and it is included as one of the main goals of the One Health initiative. Giant tortoises (Chelonoidis spp.), some of the most iconic species on Earth, are widely distributed across the Galapagos archipelago and are thus perfect candidates to test the hypothesis that wildlife species in the Galapagos carry antimicrobial resistant genes (ARGs) associated with human activities. We sampled a total of 200 free-living Galapagos tortoises from western Santa Cruz Island (C. porteri), the most human-populated island of the archipelago, and 70 tortoises (C. vandenburghi) from the isolated Alcedo Volcano on Isabela Island, a natural area with minimal human presence. Fecal samples were analyzed by quantitative PCR for a panel of 21 ARGs conferring resistance for eight antimicrobial classes. We found ARGs in both Santa Cruz and Alcedo Volcano giant tortoises; however, both qualitative and quantitative results showed higher loads of ARGs in tortoises inhabiting the human modified environments of Santa Cruz. Moreover, Santa Cruz tortoises sampled in higher human-modified landscapes (i.e., farmlands and urban areas) presented a higher number of ARGs, antimicrobial classes, and multi-resistant microbiomes than those from less anthropized areas within the same island. Our findings suggest that human activities in Galapagos have a negative impact on ecosystem health through ARG dispersal. This research highlights a new threat for the health and conservation of the unique wildlife of the Galapagos, their ecosystems, and the humans inhabiting this World Heritage Site. Our recommendation to local policy makers is to control and reduce the use of antibiotics in both human and animal health, thus helping enforce antimicrobial regulations.

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Galápagos giant tortoises are an essential component of their ecosystem and evaluation of parasites in their populations is essential for the management of conservation processes. Coccidiosis is the most common intestinal infection in free-living and captive reptiles. The aim of this study was to characterize molecularly the presence of Eimeria sp. in captive reared giant tortoises from Santa Cruz, Santiago, Española, and Pinzon Islands hatched and housed at the tortoise rearing center on Santa Cruz Island, Galápagos, by sequencing of the 18S rRNA gene. Galápagos. All samples were previously analyzed by coproparasitoscopic flotation technique and PCR for molecular identification. The results obtained by microscopy examination showed oocysts in all samples. PCR and sequencing indicated the presence Eimeria sp., showing a similarity percentage of 98% with Eimeria environmental. In conclusion, we identified a group of coccidia of the genus Eimeria sp. (MK909931) in Galápagos tortoises.

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Understanding how individual movement scales with body size is of fundamental importance in predicting ecological relationships for diverse species. One-dimensional movement metrics scale consistently with body size yet vary over different temporal scales. Knowing how temporal scale influences the relationship between animal body size and movement would better inform hypotheses about the efficiency of foraging behaviour, the ontogeny of energy budgets, and numerous life-history trade-offs. We investigated how the temporal scaling of allometric patterns in movement varies over the course of a year, specifically during periods of motivated (directional and fast movement) and unmotivated (stationary and tortuous movement) behaviour. We focused on a recently diverged group of species that displays wide variation in movement behaviour - giant Galapagos tortoises (Chelonoidis spp.) - to test how movement metrics estimated on a monthly basis scaled with body size. We used state-space modelling to estimate seven different movement metrics of Galapagos tortoises. We used log-log regression of the power law to evaluate allometric scaling for these movement metrics and contrasted relationships by species and sex. Allometric scaling of movement was more apparent during motivated periods of movement. During this period, allometry was revealed at multiple temporal intervals (hourly, daily and monthly), with values observed at daily and monthly intervals corresponding most closely to the expected one-fourth scaling coefficient, albeit with wide credible intervals. We further detected differences in the magnitude of scaling among taxa uncoupled from observed differences in the temporal structuring of their movement rates. Our results indicate that the definition of temporal scales is fundamental to the detection of allometry of movement and should be given more attention in movement studies. Our approach not only provides new conceptual insights into temporal attributes in one-dimensional scaling of movement, but also generates valuable insights into the movement ecology of iconic yet poorly understood Galapagos giant tortoises.

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Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

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Loss of key plant-animal interactions (e.g., disturbance, seed dispersal, and herbivory) due to extinctions of large herbivores has diminished ecosystem functioning nearly worldwide. Mitigating for the ecological consequences of large herbivore losses through the use of ecological replacements to fill extinct species' niches and thereby replicate missing ecological functions has been proposed. It is unknown how different morphologically and ecologically a replacement can be from the extinct species and still provide similar functions. We studied niche equivalency between 2 phenotypes of Galápagos giant tortoises (domed and saddlebacked) that were translocated to Pinta Island in the Galápagos Archipelago as ecological replacements for the extinct saddlebacked giant tortoise (Chelonoidis abingdonii). Thirty-nine adult, nonreproductive tortoises were introduced to Pinta Island in May 2010, and we observed tortoise resource use in relation to phenotype during the first year following release. Domed tortoises settled in higher, moister elevations than saddlebacked tortoises, which favored lower elevation arid zones. The areas where the tortoises settled are consistent with the ecological conditions each phenotype occupies in its native range. Saddlebacked tortoises selected areas with high densities of the arboreal prickly pear cactus (Opuntia galapageia) and mostly foraged on the cactus, which likely relied on the extinct saddlebacked Pinta tortoise for seed dispersal. In contrast, domed tortoises did not select areas with cactus and therefore would not provide the same seed-dispersal functions for the cactus as the introduced or the original, now extinct, saddlebacked tortoises. Interchangeability of extant megaherbivores as replacements for extinct forms therefore should be scrutinized given the lack of equivalency we observed in closely related forms of giant tortoises. Our results also demonstrate the value of trial introductions of sterilized individuals to test niche equivalency among candidate analog species.

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