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Abstract Biodiversity loss is extreme in amphibians. Despite ongoing conservation action, it is difficult to determine where we stand in overcoming their extinction crisis. Among the most threatened amphibians are the 131 Neotropical harlequin toads. Many of them declined since the 1980s with several considered possibly extinct. Recently, more than 30 species have been rediscovered, raising hope for a reversing trend in the amphibian extinction crisis. We use past and present data available for harlequin toads (Atelopus), to examine whether the amphibian extinction crisis is still in an emergency state. Since 2004 no species has improved its population status, suggesting that recovery efforts have not been successful. Threats include habitat change, pathogen spread and climate change. More mitigation strategies need implementation, especially habitat protection and disease management, combined with captive conservation breeding. With harlequin toads serving as a model, it is clear that the amphibian extinction crisis is still underway.

The Puerto Rican Common coqui frog (Eleutherodactylus coqui) has a long history as an invasive species in places such as Hawaii. Since its introduction in Costa Rica, scarce information is available to understand why and how the habitat in the Turrialba town is suitable for the species. Our goal was to analyze the habitat selection of E. coqui to identify if there are key habitat features that explained its success there. We measured 9 site variables that may affect the habitat selection of E. coqui in 92 survey units of 10 m radius distributed over a 500 m radius from its introduction point. We registered the presence/pseudo-absence data of E. coqui and environmental variables in each survey unit during eight surveys. We ran occupancy models to determine the influence of the variables on the habitat selection and to estimate its detection probability. We found that sites near the introduction point, containing abundant vegetation, bromeliads, and palms have a higher probability to be occupied by E. coqui. The habitat selection in Costa Rica shares characteristics with the populations of Puerto Rico and Hawaii. But, unlike the case in Hawaii, in Costa Rica this species has maintained a limited dispersal because the potentially higher biotic resistance, as well a sedentary behavior. However, the microhabitat conditions used by E. coqui in the study site are common throughout the country. Therefore, active management in new populations and environmental education programs to avoid human transportation of the species is critical to reduce its dispersal.

Abstract Background The skin microbiome serves as a first line defense against pathogens in vertebrates. In amphibians, it has the potential to protect against the chytrid fungus Batrachochytrium dendrobatis (Bd), a likely agent of amphibian declines. Alteration of the microbiome associated with unfavorable environmental changes produced by anthropogenic activities may make the host more susceptible to pathogens. Some amphibian species that were thought to be “extinct” have been rediscovered years after population declines in the late 1980s probably due to evolved Bd-resistance and are now threatened by anthropogenic land-use changes. Understanding the effects of habitat disturbance on the host skin microbiome is relevant for understanding the health of these species, along with its susceptibility to pathogens such as Bd. Here, we investigate the influence of habitat alteration on the skin bacterial communities as well as specifically the putative Bd-inhibitory bacterial communities of the montane frog Lithobates vibicarius. This species, after years of not being observed, was rediscovered in small populations inhabiting undisturbed and disturbed landscapes, and with continuous presence of Bd. Results We found that cutaneous bacterial communities of tadpoles and adults differed between undisturbed and disturbed habitats. The adults from disturbed habitats exhibited greater community dispersion than those from undisturbed habitats. We observed a higher richness of putative Bd-inhibitory bacterial strains in adults from disturbed habitats than in those from undisturbed habitats, as well as a greater number of these potential protective bacteria with a high relative abundance. Conclusions Our findings support the microbial “Anna Karenina principle”, in which disturbance is hypothesized to cause greater microbial dispersion in communities, a so-called dysbiosis, which is a response of animal microbiomes to stress factors that decrease the ability of the host or its microbiome to regulate community composition. On the positive side, the high richness and relative abundance of putative Bd-inhibitory bacteria may indicate the development of a defense mechanism that enhances Bd-protection, attributed to a co-occurrence of more than 30-years of host and pathogen in these disturbed habitats. Our results provide important insight into the influence of human-modified landscapes on the skin microbiome and health implications of Bd-survivor species.

Introducción: El sapo arlequín, Atelopus varius, se encuentra en peligro crítico y fue declarada “aparentemente extinta” en Costa Rica en 1996. Una de las posibles causas fue el hongo patógeno Batrachochytrium dendrobatidis. En 2008 se redescubrió una población viva en Las Tablas y aquí analizamos la situación con este hongo. Objetivo: Evaluar prevalencia y probabilidad de infección del sapo por B. dendrobatidis. Métodos: Se recolectaron hisopados de piel de individuos vivos (para pruebas de ADN) y trozos histológicos de individuos muertos (para observación microscópica directa). Resultados: De 363 animales, solo el 6% tenía el hongo, particularmente los subadultos. Conclusión: La población de sapos parece estable, pero se debe tener cuidado de no propagar el hongo, aunque este sea actualmente escaso.

Some neotropical amphibians, including a few species in Costa Rica, were presumed to be “extinct” after dramatic population declines in the late 1980s but have been rediscovered in isolated populations. Such populations seem to have evolved a resistance/tolerance to Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes a deadly skin disease and is considered one of the main drivers of worldwide amphibian declines. The skin microbiome is an important component of the host’s innate immune system and is associated with Bd-resistance. However, the way that the bacterial diversity of the skin microbiome confers protection against Bd in surviving species remains unclear. We studied variation in the skin microbiome and the prevalence of putatively anti-Bd bacterial taxa in four co-habiting species in the highlands of the Juan Castro Blanco National Park in Costa Rica using 16S rRNA amplicon sequencing. Lithobates vibicarius, Craugastor escoces, and Isthmohyla rivularis have recently been rediscovered, whereas Isthmohyla pseudopuma has suffered population fluctuations but has never disappeared. To investigate the life stage at which the protective skin microbiome is shaped and when shifts occur in the diversity of putatively anti-Bd bacteria, we studied the skin microbiome of tadpoles, juveniles and adults of L. vibicarius. We show that the skin bacterial composition of sympatric species and hosts with distinct Bd-infection statuses differs at the phyla, family, and genus level. We detected 94 amplicon sequence variants (ASVs) with putative anti-Bd activity pertaining to distinct bacterial taxa, e.g., Pseudomonas spp., Acinetobacter johnsonii, and Stenotrophomonas maltophilia. Bd-uninfected L. vibicarius harbored 79% more putatively anti-Bd ASVs than Bd-infected individuals. Although microbiome composition and structure differed across life stages, the diversity of putative anti-Bd bacteria was similar between pre- and post-metamorphic stages of L. vibicarius. Despite low sample size, our results support the idea that the skin microbiome is dynamic and protects against ongoing Bd presence in endangered species persisting after their presumed extinction. Our study serves as a baseline to understand the microbial patterns in species of high conservation value. Identification of microbial signatures linked to variation in disease susceptibility might, therefore, inform mitigation strategies for combating the global decline of amphibians.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), are implicated in the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd presence in 1016 anuran museum specimens collected throughout Costa Rica. The earliest specimen that tested positive for Bd was collected in 1964. Across all time periods, we found an overall infection rate (defined as the proportion of Bd-positive individuals) of 4%. The number of infected individuals remained relatively low across all species tested and the range of Bd-positive specimens was shown to be geographically constrained up until the 1980s; when epizootics are hypothesized to have occurred. After that time, infection rate increased three-fold, and the range of specimens tested positive for Bd increased, with Bd-positive specimens collected across the entire country. Our results suggest that Bd dynamics in Costa Rica are more complicated than previously thought. The discovery of Bd's presence in the country preceding massive declines leads to a number of different hypotheses: 1) Bd invaded Costa Rica earlier than previously known, and spread more slowly than previously reported; 2) Bd invaded multiple times and faded out; 3) an endemic Bd lineage existed; 4) an earlier Bd lineage evolved into the current Bd lineage or hybridized with an invasive lineage; or 5) an earlier Bd lineage went extinct and a new invasion event occurred causing epizootics. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine the most plausible spatial-temporal, host-pathogen dynamics that could best explain the epizootics resulting in amphibian declines in Costa Rica and throughout Central America.

Over the last two decades, pigs have become animal biomodels widely used for the investigation and practice of surgical techniques because of their great physiological and anatomical similarities to humans. Even though many of these studies must be carried out later in humans, the description of basic information is limited, making exact repetitions of the reported experimental methods impossible. In this review, 108 studies from 2013 to 2018 were considered to determine the quality of adherence to the ARRIVE guidelines in the reports of the methodologies. The majority of the studies lacked the details recommended in the ARRIVE guidelines regarding data directly related to the welfare of animals undergoing surgery and those about anesthetic protocols and analgesics. Information related to sample size calculation and housing and husbandry conditions was also very limited. We believe that the ARRIVE guidelines are an excellent tool for good-quality reporting. We encourage scientists to consistently use them as a tool to improve the quality of their scientific reports and, consequently, ensure animal welfare.

Amphibians are potentially reliable and efficient bioindicators. Existing anuran white blood cell morphology studies are limited, with only a few morphometric studies available. We employed morphometric techniques to characterize leukocytes of selected Neotropical anurans from Brazil and compare our findings with the exotic American Bullfrog (Lithobates catesbeianus), genus Ranidae. We compared blood smears of 28 specimens from six different genera (Hyla, Phyllomedusa, Hypsiboas, Scinax, Physalaemus, and Proceratophrys) with samples from L. catesbeianus. Leukocyte average diameter was calculated by an image analysis software. One-way analyses of variance and Bonferroni tests were used on statistical analyses. Lymphocytes, neutrophils, eosinophils, and basophils were significantly smaller than the reference ranges reported for other amphibian genera, including Lithobathes, whereas monocyte diameters did not differ significantly between genera. This is the first study to evaluate leukocyte morphometrics of Brazilian anuran species. Our findings suggest that geographical separation could possibly influence leukocyte morphometry.

Reports on ectoparasites on dogs in Central America are scarce. The aim of this study was to identify flea, louse and tick species infesting dogs in home environments on the Caribbean slope of Costa Rica, and determine their frequency and coexistence. Ectoparasites were collected from dogs in 83 rural homes at five study sites. Specimens were identified and separated according to species. Fleas were the most common ectoparasite (G = 22,217, DF = 8, p = 0.004). Ctenocephalides felis and Pulex simulans were found in 83% and 55% of the homes with ectoparasites, respectively. Trichodectes canis (13%), Heterodoxus spiniger (10%), Rhipicephalus sanguineus (18%), Rhipicephalus (Boophilus) microplus (5%) and Amblyomma ovale (8%) were also present. More than one species was collected in most cases (66%), and the most common combination was C. felis and P. simulans (59% of homes with fleas). The high frequency of P. simulans emphasizes the need for adequate identification. This was the first study involving different ectoparasites of dogs in Costa Rica, as well as the first report of T. canis in this country. The relative frequency and coexistence of these ectoparasites in the home environment may have implications for animal and human health.Relatos de ectoparasitos em cães da América Central são escassos. O objetivo deste estudo foi identificar espécies de pulgas, piolhos e carrapatos que infestam os cães no ambiente doméstico da vertente Caribe da Costa Rica, determinando sua frequência e coexistência. Foram coletados ectoparasitos de cães em 83 casas rurais de cinco sítios do estudo. Os indivíduos foram separados e identificados. Os ectoparasitos mais comuns foram as pulgas (G = 22.217, DF = 8, p = 0.004). Ctenocephalides felis e Pulex simulans foram observados em 83% e 55% das moradias com ectoparasitos, respectivamente. Da mesma forma também foram observados Trichodectes canis (13%), Heterodoxus spiniger (10%), Rhipicephalus sanguineus (18%), Rhipicephalus (Boophilus) microplus (5%) e Amblyomma ovale (8%). Na maioria dos casos, mais de uma espécie foi achada (66%), sendo que a combinação mais comum foi C. felis e P. simulans (59% de moradias com pulgas). A frequência alta de P. simulans ressalta a necessidade de sua adequada identificação. Este é o primeiro estudo que envolve diferentes ectoparasitas de cães na Costa Rica, da mesma forma, é o primeiro relato de T. canis no país. A frequência relativa e coexistência destes ectoparasitos no ambiente domiciliar poderiam ter implicações na saúde animal e humana.