Invasion by Cedrela odorata threatens long distance migration of Galapagos tortoises.

Invasive alien species are among the most pervasive threats to biodiversity. Invasive species can cause catastrophic reductions in populations of native and endemic species and the collapse of ecosystem function. A second major global conservation concern is the extirpation of large-bodied mobile animals, including long-distance migrants, which often have keystone ecological roles over extensive spatial extents. Here, we report on a potentially catastrophic synergy between these phenomena that threatens the endemic biota of the Galapagos Archipelago. We used GPS telemetry to track 140 migratory journeys by 25 Western Santa Cruz Island Galapagos tortoises. We plotted the spatial interaction between tortoise migrations and recently established non-native forest dominated by the invasive tree Cedrela odorata (Cedrela forest). We qualified (a) the proportion of migratory journeys that traversed Cedrela forest, and (b) the probability that this observed pattern occurred by chance. Tortoise migrations were overwhelmingly restricted to small corridors between Cedrela forest blocks, indicating clear avoidance of those blocks. Just eight of 140 migrations traversed extensive Cedrela stands. Tortoises avoid Cedrela forest during their migrations. Further expansion of Cedrela forest threatens long-distance migration and population viability of critically endangered Galapagos tortoises. Applied research to determine effective management solutions to mitigate Cedrela invasion is a high priority.

Novel herpesvirus in the critically endangered Galapagos pink land iguana.

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

HEMATOLOGY AND BLOOD CHEMISTRY VALUES IN CUBAN CROCODILES (CROCODYLUS RHOMBIFER) HOUSED AT THE ZAPATA SWAMP CROCODILE FARM, CUBA.

We report hematology and biochemistry reference intervals (RI) for the critically endangered Cuban crocodile (Crocodylus rhombifer). In November 2019, we sampled 43 adult crocodiles (6 male, 37 female) under human care at the Zapata Swamp Crocodile Farm in Matanzas, Cuba. These crocodiles are part of a breeding program for the species registered by the Convention on International Trade in Endangered Species (CITES). Visual health evaluations were performed immediately after manual restraint, and blood was collected from the postoccipital sinus. We performed packed cell volume (PCV), total solids (TS), complete blood counts (CBC), and biochemistry profiles for each crocodile on the day of sampling. Mean PCV (n = 42) was 21.1 ± 5.0% and TS (n = 42) 7.3 ± 1.2 mg/dl, respectively. Absolute white blood cell (WBC) (n = 40) was 9.6 ± 5.7 ｘ 109/L. Similar to other crocodilian species, the dominant leukocyte was lymphocytes (70.7 ± 10.4%), followed by heterophils (18.7 ± 9.7%). Two of the crocodiles had a high heterophil:lymphocyte ratio (0.87 and 0.74), although on visual exam they were both considered healthy. The range of creatine kinase was 41-1,482 U/L, and the higher values may be a reflection of muscle exertion at time of handling. Limitations to the study included skewed sex ratios and high lipemia and hemolysis in most samples collected. These are the first reference intervals reported for this species, including the first descriptions of WBC morphology. These data are valuable for the management of animals at the Zapata Swamp Crocodile Farm, for comparison with free-living Cuban crocodiles in Cuba, and for comparison with those managed under human care outside of Cuba.

Prevalence of Toxoplasma gondii in Galapagos birds: Inference of risk factors associated with diet.

Toxoplasma gondii is a zoonotic intracellular parasite of particular concern in the conservation of wildlife due to its ability to infect all homeotherms and potentially cause acute fatal disease in naive species. In the Galapagos (Ecuador), an archipelago composed of more than a hundred islets and islands, the presence of T. gondii can be attributed to human-introduced domestic cats, but little is known about its transmission in wildlife populations. We compared the prevalence of antibodies against T. gondii in sympatric Galapagos wild bird species that differ in diet and contact with oocyst-contaminated soil to determine the relative importance of trophic habits as an exposure factor. Plasma samples were obtained from 163 land birds inhabiting Santa Cruz, one of the cat-inhabited islands, and from 187 seabirds breeding in cat-free surrounding islands (Daphne Major, North Seymour, and South Plaza). These samples were tested for the presence of T. gondii antibodies using the modified agglutination test (MAT ≥ 1:10). All seven species of land birds and 4/6 species of seabirds presented seropositive results. All great frigatebirds (Fregata minor) (N = 25) and swallow-tailed gulls (Creagrus furcatus) (N = 23) were seronegative. Prevalence ranged from 13% in Nazca boobies (Sula granti) to 100% in Galapagos mockingbirds (Mimus parvulus). It decreased from occasional carnivores (63.43%) to granivores-insectivores (26.22%), and strict piscivores (14.62%). These results indicate that the consumption of tissue cysts poses the highest risk of exposure to T. gondii for Galapagos birds, followed by the ingestion of plants and insects contaminated by oocysts as important transmission pathways.

Temperature along an elevation gradient determines Galapagos tortoise sex ratios.

Climate change threatens endemic island ectothermic reptiles that display small population sizes and temperature-dependent sex determination (TSD). Studies of captive Galapagos tortoises demonstrate type A TSD with warmer incubation temperatures producing females. However, there are few published data from free-living Galapagos tortoises on incubation temperature regimes, and none on hatchling sex ratios in the wild or the potential impacts of climate change on future sex ratios. We sought to address these deficits by quantifying incubation temperatures of nests and sex ratios of juvenile tortoises along an elevation gradient on Santa Cruz Island. We focused on three geographically separated nesting zones with mean elevations of 14 m (lower), 57 m (middle), and 107 m (upper) above sea level. Nest temperatures in 54 nests distributed across the three nesting zones were measured every 4 h throughout the incubation period using iButton thermochrons. We used coelioscopy to conduct visual exams of gonads to determine the sex of 40 juvenile tortoises from the three nesting zones. During the middle trimester of incubation, the period during which sex is determined in turtles, mean nest temperatures were 25.75°C (SD = 1.08) in the upper zone, and 27.02°C (SD = 1.09), and 27.09°C (SD = 0.85) in the middle and lower zones, respectively. The proportion of juveniles that was male increased from 11.1% in the lower zone and 9.5% in the middle zone, to 80% in the upper zone. A ca. 50 m increase in elevation induced a decrease of >1.25°C in mean nest temperature during the second trimester of incubation. Over the same elevation change, the proportion of males in the juvenile tortoise population increased by ca. 70%. Temperatures on Galapagos are predicted to increase by 1-4°C over the next 50 years, which is likely to increase the frequency of female tortoises across the archipelago.

Health assessments uncover novel viral sequences in five species of Galapagos tortoises.

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.

Antimicrobial resistance in Galapagos tortoises as an indicator of the growing human footprint.

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.

Haematology and plasma biochemistry reference intervals for the critically endangered western Santa Cruz Galapagos tortoise (Chelonoidis porteri).

Reference intervals (RIs) are an increasingly valuable tool for monitoring captive and free-living wildlife populations. Galapagos tortoises are one of the most emblematic species on Earth with 9 of the 12 extant species considered endangered due to human activities. As part of a long-term health assessment within the Galapagos Tortoise Movement Ecology Programme, we sampled a total of 210 free-living Santa Cruz Galapagos tortoises (Chelonoidis porteri). We collected blood from the brachial vein and performed packed cell volume (PCV), total solids (TS), morphological evaluation, white blood cell (WBC) count estimates and differentials and a VetScan biochemistry panel for each individual. We calculated 95% RIs and 90% confidence intervals (CIs) using the Reference Value Advisor tool and following international standard guidelines. Tortoises were categorized by estimated age and sex, with RI and CI reported here for 164 adult tortoises and 46 sub-adult tortoises. We found significant differences between sexes, with adult females presenting a lower PCV and higher values for both calcium and potassium. Among age groups, adult tortoises presented higher PCV, TS and albumin and lower WBC counts, aspartate aminotransferase and creatine kinase than sub-adult tortoises. We also found that tortoises presented higher numbers of lymphocytes during the dry season, but higher basophils, eosinophils, phosphorus, potassium and TS during the humid season. The heterophil:lymphocyte ratio did not differ between groups. To the authors' knowledge, this is the first report of formal plasma biochemistry and haematology RI for free-living Galapagos tortoises. With the present study we provide an important diagnostic tool for captive-breeding programs in the Galapagos and zoological institutions globally that care for giant tortoises. The ultimate goal of this study is to contribute to the understanding of giant tortoise baseline health parameters and to inform local management decisions that help to conserve these emblematic species.

Antimicrobial resistance genes present in the faecal microbiota of free-living Galapagos tortoises (Chelonoidis porteri).

Antimicrobial resistance (AMR), encoded by plasmid-mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free-living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n = 28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi-resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large-scale distribution of these genes within the archipelago.