Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized - genetically and morphologically - several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas, Streptomyces, Enterobacter, Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Ranavirus is widespread in Costa Rica and co-occurs with threatened amphibians.

Amphibians are globally threatened by emerging infectious diseases, and ranaviruses are among the most concerning pathogens to threaten species in the wild. We sampled for ranaviruses in wild amphibians at 8 sites in Costa Rica, spanning broad climatic zones and taxonomic associations. Seven of these sites are inhabited by highly threatened amphibian species that persist at low global population sizes after population declines due to amphibian chytridiomycosis. One of the surveyed sites is occupied by an introduced amphibian species, which is relatively rare in Central America but may be an important pathway for long-distance transport of ranaviruses. We detected ranavirus using quantitative polymerase chain reaction in 16.3% of the 243 individuals and among 5 of our 8 sites, but not at the site with the introduced species. Infection prevalence varied among species and sites, but not with mean annual temperature or mean annual precipitation. Infection intensity did not vary with species, site, temperature, or precipitation. Our results show that ranavirus infection is spatially widespread in Costa Rica, affecting a broad range of host species, and occurs across climatic zones-though we encountered no mortality or morbidity in our sampled species. Ranaviruses are known to cause intermittent mass mortality in amphibian populations, and the threatened species sampled here are likely vulnerable to population impacts from emerging ranaviruses. Therefore, we believe the potential impacts of ranaviruses on amphibian populations in tropical regions have likely been underestimated, and that they should be viewed as a potential major stressor to threatened amphibians in tropical regions.

Solid fuel use is associated with anemia in children.

Over 3 billion people use solid fuels as a means of energy and heating source, and ~ 50% of households burn them in inefficient, poorly ventilated stoves. In 2010, ~ 43% of the 640 million preschool children in 220 countries suffered from a certain degree of anemia, with iron deficiency as the main cause in developed countries whereas its causes remained multifactorial in the undeveloped group. In this study, we explore the relations of country-wide variables that might affect the people's health status (from socioeconomic status to more specific variables such as water access). We found independent relationship between solid fuel use and anemia in children under five years old (p < 0.0001), taking into account the prevalence of anemia in pregnant woman and the access to improved water sources. Countries in which the population uses solid fuel the most have over three times higher anemia rates in children than countries with the lowest prevalence of solid fuels use. There is still a complex relationship between solid fuels use and anemia, as reflected in its worldwide significance (p < 0.05) controlled for measles immunization, tobacco consumption, anemia in pregnant mothers, girl's primary education, life expectancy and improved water access but not (p > 0.05) when weighing for sanitation access or income per capita.

Ecological study on solid fuel use and pneumonia in young children: A worldwide association.

BACKGROUND AND OBJECTIVE: Pneumonia constitutes one of the major causes of worldwide mortality in young children. Poverty has been traditionally assigned as the underlying factor accounting for these trends. However, the independent role of solid fuel use yielding biomass pollution on pneumonia rates among young children has not been extensively examined. METHODS: Independent socio-economic variables, and the percentage of solid fuel use, tobacco consumption, improved water access source and sanitation facilities were extracted for each country from the available public databases. Multivariate regression analyses were performed to assess potential associations between these recognized risk factors and country pneumonia incidence in young children <5 years of age. RESULTS: Multivariate linear regression analyses yielded two models that accounted for approximately 87% of the variance, and included solid fuel use, tobacco consumption, sanitation access, measles immunization, life expectancy, access to electricity and the Human Development Index (HDI) as being independently associated with the number of annual pneumonia cases per child <5 years of age. CONCLUSION: In this ecological study, current country rates of pneumonia among young children are independently associated with the use of solid fuels. We postulate that interventions aimed at reducing indoor solid fuel biomass pollution through implementation of efficient stoves will translate into meaningful decreases in child mortality and morbidity.

Differential patterns of Batrachochytrium dendrobatidis infection in relict amphibian populations following severe disease-associated declines.

Global amphibian biodiversity has declined dramatically in the past 4 decades, and many amphibian species have declined to near extinction as a result of emergence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). However, persistent or recovering populations of several amphibian species have recently been rediscovered, and such populations may illustrate how amphibian species that are highly susceptible to chytridiomycosis may survive in the presence of Bd. We conducted field surveys for Bd infection in 7 species of Costa Rican amphibians (all species that have declined to near extinction but for which isolated populations persist) to characterize infection profiles in highly Bd-susceptible amphibians post-decline. We found highly variable patterns in infection, with some species showing low prevalence (~10%) and low infection intensity and others showing high infection prevalence (>80%) and either low or high infection intensity. Across sites, infection rates were negatively associated with mean annual precipitation, and infection intensity across sites was negatively associated with mean average temperatures. Our results illustrate that even the most Bd-susceptible amphibians can persist in Bd-enzootic ecosystems, and that multiple ecological or evolutionary mechanisms likely exist for host-pathogen co-existence between Bd and the most Bd-susceptible amphibian species. Continued monitoring of these populations is necessary to evaluate population trends (continuing decline, stability, or population growth). These results should inform efforts to mitigate impacts of Bd on amphibians in the field.

A new species of brilliant green frog of the genus Tlalocohyla (Anura, Hylidae) hiding between two volcanoes of northern Costa Rica.

A new species of hylid frog is described from Tapir Valley Nature Reserve, located on the Caribbean slope of Tenorio Volcano in Bijagua, Alajuela Province, Costa Rica. A molecular phylogenetic analysis supports its inclusion in the genus Tlalocohyla. Morphological, morphometric, larval, and acoustic characteristics further distinguish it from other species in the genus and support its uniqueness. The new species is closely related to T. picta and T. smithii, and is separated by at least 500 kilometers from the nearest known occurrence of a population of T. picta in southern Honduras. The new species is readily distinguished from all other Tlalocohyla by its brilliant green coloration marked with a pronounced, incomplete light dorsolateral stripe that is bordered above by a diffuse reddish-brown stripe. Its dorsum is marked with bold reddish brown spots and its ventral skin is fully transparent. This new Tlalocohyla is currently only known from the type locality, where it inhabits a lentic wetland system with an emergent herbaceous vegetation-dominated benthic zone, surrounded by tropical rainforest. A description of its bioacoustic repertoire and information on natural history, reproduction and habitat preference of this new species are provided.

The activation of hypothalamic AMP-activated protein kinase by oxidative stress is related to hyperphagia in diabetic rats.

OBJECTIVE: During diabetes, there are increased blood glucose levels and oxidative stress. The relationship between oxidative stress and the phosphorylation of AMP-activated protein kinase at the hypothalamic level has been little studied. The objective of this study was to analyze the relationship between oxidative stress and AMP-activated protein kinase activation in Wistar rats with hyperphagia and hyperglycemia. METHODS: Rats at 7, 14, and 28 days with diabetes were used. Control rats were included. Food intake was calculated to determine hyperphagia. The hypothalamus was extracted to evaluate oxidative stress markers by spectrophotometry; phosphorylation of AMP-activated protein kinase, growth hormone receptor 1a, and neuropeptide Y expression were determined by Western blot. RESULTS: There was a significant increase in the consumption of food in the experimental groups. The level of malondialdehyde decreased in the 7-day group (33%) and increased significantly in the 28-day group (90%), glutathione peroxidase activity increased in the 7-day group (70%) and decreased in the 28-day group (34%), and the phosphorylation of AMP-activated protein kinase increased significantly in the 28-day group (86%). Under ex-vivo conditions in animals with 28 days of hyperglycemia, glutathione peroxidase activity increased 195%, the malondialdehyde level decreased 87%, phosphorylation of AMP-activated protein kinase decreased 53%, and growth hormone receptor 1a expression decreased 66%, when treating hyperglycemic hypothalamic tissue with an antioxidant. NPY expression increased in hyperglycemia, and antioxidant treatment did not regulate its expression. CONCLUSIONS: The activation of AMP-activated protein kinase is related with an increase in oxidative stress markers in hyperglycemic and hyperphagic rats.

Brain Metastases in Patients With Soft-Tissue Sarcomas: Management and Survival-A SEER Population-Based Cohort Study.

INTRODUCTION: Brain metastases (BM) in soft-tissue sarcomas (STS) is associated with poor prognosis. This large population analysis presents (1) demographic and clinical variables of these patients, (2) potential risk factors, (3) impact of BM on overall survival, and (4) treatment strategies. METHODS: Patients with STS and BM were identified from the Surveillance, Epidemiology, and End Results database. Demographic and clinical variables, as well as treatment modalities, were analyzed. Overall 5-year survival was calculated using the Kaplan-Meier method, and the survival difference was assessed using the log-rank test. A multivariate analysis was performed using the Cox proportional hazard regression to determine the risk factors. RESULTS: Twenty-two patients (22/8,433) with STS presented BM at diagnosis. A multivariate analysis showed that women and American Indians/Alaska Natives had a greater risk of presenting BM. The most common histological subtype to metastasize to the brain was alveolar soft part sarcoma (4/22). In 54.5% of patients with BM, the tumor had also metastasized to the lung, although having synchronous bone, liver, and lung metastases showed the greatest increase in risk for presenting BM (odd ratio [OR] = 1,857.7, confidence interval [CI] 95%, 88.4 to 3,9046.6, P < 0.0001). Individually, bone metastasis increased the risk of presenting BM the most (OR = 205.0, CI 95%, 30.5 to 1,379.6, P < 0.0001). The mean survival of patients with BM was 10.22 months. The standard treatment approach included surgery, radiation therapy, and chemotherapy. CONCLUSION: BM in patients with STS represents an infrequent but lethal event. Women and American Indians/Alaska Natives are at a higher risk of presenting BM, as well as patients with synchronous metastases. Patients are mainly managed with systemic therapy.

Hydroxychloroquine / azithromycin in COVID-19: The association between time to treatment and case fatality rate.

BACKGROUND: Currently, there is no formally accepted pharmacological treatment for COVID-19. MATERIALS AND METHODS: We included COVID-19 outpatients of a Peruvian primary care center from Lima, Peru, who were treated between April 30 - September 30, 2020, with hydroxychloroquine and azithromycin. Logistic regression was applied to determine factors associated with case-fatality rate. RESULTS: A total of 1265 COVID-19 patients with an average age of 44.5 years were studied. Women represented 50.1% of patients, with an overall 5.9 symptom days, SpO2 97%, temperature of 37.3 °C, 41% with at least one comorbidity and 96.1% one symptom or sign. No patient treated within the first 72 h of illness died. The factors associated with higher case fatality rate were age (OR = 1.06; 95% CI 1.01-1.11, p = 0.021), SpO2 (OR = 0.87; 95% CI 0.79-0.96, p = 0.005) and treatment onset (OR = 1.16; 95% CI 1.06-1.27, p = 0.002), being the latter the only associated in the multivariate analysis (OR = 1.18; 95% CI 1.05-1.32, p = 0.005). 0.6% of our patients died. CONCLUSIONS: The case fatality rate in COVID-19 outpatients treated with hydroxychloroquine/azithromycin was associated with the number of days of illness on which treatment was started.

Diabetes increases the risk of COVID-19 in an altitude dependent manner: An analysis of 1,280,806 Mexican patients.

AIMS: The objective of this study is to analyze how the impact of Diabetes Mellitus [DM] in patients with COVID-19 varies according to altitudinal gradient. METHODS: We obtained 1,280,806 records from adult patients with COVID-19 and DM to analyze the probability of COVID-19, development of COVID-19 pneumonia, hospitalization, intubation, admission to the Intensive Care Unit [ICU] and case-fatality rates [CFR]. Variables were controlled by age, sex and altitude of residence to calculate adjusted prevalence and prevalence ratios. RESULTS: Patients with DM had a 21.8% higher prevalence of COVID-19 and an additional 120.2% higher prevalence of COVID-19 pneumonia. The adjusted prevalence was also higher for these outcomes as well as for hospitalization, intubation and ICU admission. COVID-19 and pneumonia patients with DM had a 97.0% and 19.4% higher CFR, respectively. With increasing altitudes, the probability of being a confirmed COVID-19 case and the development of pneumonia decreased along CFR for patients with and without DM. However, COVID-19 patients with DM were more likely to require intubation when residing at high altitude. CONCLUSIONS: The study suggests that patients with DM have a higher probability of being a confirmed COVID-19 case and developing pneumonia. Higher altitude had a protective relationship against SARS-CoV-2 infection; however, it may be associated with more severe cases in patients with and without DM. High altitude decreases CFR for all COVID-19 patients. Our work also shows that women are less affected than men regardless of altitude.

Frequency of depression and quality of life in patients with diabetes mellitus in public health facilities in Metropolitan Lima.

INTRODUCTION: Diabetes is one of the main pandemics in recent years. Its association with depression increases the risk of mortality and morbidity. The coexistence of both diseases leads to poor management of diabetes, which leads to a worse quality of life. OBJECTIVE: To determine the frequency of depression in patients with diabetes mellitus and the effect of both pathologies on the quality of life in patients who attend outpatient appointments at public health facilities in Lima and Callao. METHODOLOGY: Secondary analysis of the Epidemiological Study of Mental Health of depression in diabetic adults. The instrument used to determine the depressive episode was the MINI (Mini-International Neuropsychiatric Interview) while quality of life was measured using the Mezzich Quality of Life Index. Diagnosis information of type 1 or 2 diabetes was obtained from the daily medical record (HIS) of care. RESULTS: The frequency of depression in the 471 patients with diabetes was 5.8% in the last two weeks. While the annual frequency was 8.6% and 31.8% at some point in life. Being a woman was associated with a greater frequency of depression. Quality of life was lower in patients with diabetes and depression (p < 0.005). CONCLUSIONS: The frequency of depression in patients with diabetes who are treated on an outpatient basis in public health centres is higher than the general population and their quality of life is significantly reduced, which raises the need for considering depression as an additional factor to the burden of morbidity of this condition.

Age and altitude of residence determine anemia prevalence in Peruvian 6 to 35 months old children.

BACKGROUND: A Demographic and Family Health Survey (ENDES, for Encuesta Demográfica y de Salud Familiar in Spanish) is carried out annually in Peru. Based on it, the anemia prevalence was 43.6% in 2016 and 43.8% in 2017 using the WHO cutoff value of 11 g/dL and the altitude-correction equation. OBJECTIVE: To assess factors contributing to anemia and to determine its prevalence in Peruvian children 6 to 35 months old. METHODS: We used the MEASURE DHS-based ENDES survey to obtain representative data for11364 children from 6 to 35 months old on hemoglobin and health determinants. To evaluate normal hemoglobin levels, we used the original WHO criterion of the 5th percentile in children without chronic malnutrition and then applied it to the overall population. Relationships between hemoglobin and altitude levels, usage of cleaning methods to sanitize water safe to drink, usage of solid fuels and poverty status were tested using methodology for complex survey data. Percentile curves were made for altitude intervals by plotting hemoglobin compared to age. The new anemia rates are presented in graphs by Peruvian political regions according to the degree of public health significance. RESULTS: Hemoglobin increased as age and altitude of residence increased. Using the 5th percentile, anemia prevalence was 7.3% in 2016 and 2017. Children from low altitudes had higher anemia prevalence (8.5%) than those from high altitudes (1.2%, p<0.0001). In the rainforest area of Peru, anemia prevalence was highest (13.5%), while in the highlands it was lowest (3.3%, p<0.0001). With access to safe drinking water and without chronic malnutrition, anemia rates could be reduced in the rainforest by 45% and 33%, respectively. CONCLUSION: Anemia prevalence in Peruvian children from 6 to 35 months old was 7.3% in 2016 and 2017.

The impact of COVID-19 in diabetic kidney disease and chronic kidney disease: A population-based study.

BACKGROUND: The spectrum of pre-existing renal disease is known as a risk factor for severe COVID-19 outcomes. However, little is known about the impact of COVID-19 on patients with diabetic nephropathy in comparison to patients with chronic kidney disease. METHODS: We used the Mexican Open Registry of COVID-19 patients 11 to analyze anonymized records of those who had symptoms related to COVID-19 to analyze the rates of SARS-CoV-2 infection, development of COVID-19 pneumonia, admission, intubation, Intensive Care Unit admission and mortality. Robust Poisson regression was used to relate sex and age to each of the six outcomes and find adjusted prevalences and adjusted prevalence ratios. Also, binomial regression models were performed for those outcomes that had significant results to generate probability plots to perform a fine analysis of the results obtained along age as a continuous variable. RESULTS: The adjusted prevalence analysis revealed that that there was a a 87.9% excess probability of developing COVID-19 pneumonia in patients with diabetic nephropathy, a 5% excess probability of being admitted, a 101.7% excess probability of intubation and a 20.8% excess probability of a fatal outcome due to COVID-19 pneumonia in comparison to CKD patients (p<0.01).  Conclusions: Patients with diabetic nephropathy had nearly a twofold rate of COVID-19 pneumonia, a higher probability of admission, a twofold probability of intubation and a higher chance of death once admitted compared to patients with chronic kidney disease alone. Also, both diseases had higher COVID-19 pneumonia rates, intubation rates and case-fatality rates compared to the overall population.

Frequency of Depression and Quality of Life in Patients with Diabetes Mellitus in Public Health Facilities in Metropolitan Lima. / Frecuencia de depresión y calidad de vida en pacientes con diabetes mellitus en establecimientos de salud pública de Lima Metropolitana.

INTRODUCTION: Diabetes is one of the main pandemics in recent years. Its association with depression increases the risk of mortality and morbidity. The coexistence of both diseases leads to poor management of diabetes, which leads to a worse quality of life. OBJECTIVE: To determine the frequency of depression in patients with diabetes mellitus and the effect of both pathologies on the quality of life in patients who attend outpatient appointments at public health facilities in Lima and Callao. METHODOLOGY: Secondary analysis of the Epidemiological Study of Mental Health of depression in diabetic adults. The instrument used to determine the depressive episode was the MINI (Mini-International Neuropsychiatric Interview) while quality of life was measured using the Mezzich Quality of Life Index. Diagnosis information of type 1 or 2 diabetes was obtained from the daily medical record (HIS) of care. RESULTS: The frequency of depression in the 471 patients with diabetes was 5.8% in the last two weeks. While the annual frequency was 8.6% and 31.8% at some point in life. Being a woman was associated with a greater frequency of depression. Quality of life was lower in patients with diabetes and depression (p <0.005). CONCLUSIONS: The frequency of depression in patients with diabetes who are treated on an outpatient basis in public health centres is higher than the general population and their quality of life is significantly reduced, which raises the need for considering depression as an additional factor to the burden of morbidity of this condition.

Histamine H1 and H3 receptor activation increases the expression of Glucose Transporter 1 (GLUT-1) in rat cerebro-cortical astrocytes in primary culture.

Astrocytes take up glucose via the 45 kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1 h incubation) significantly increased GLUT-1 mRNA to 153 ±â€¯7, 163 ±â€¯2 and 168 ±â€¯13% of control values, respectively. In immunoblot assays, incubation (3 h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224 ±â€¯12, 305 ±â€¯11 and 193 ±â€¯13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) → diacylglycerol (DAG)/Ca2+→ PKC and PLC → DAG/Ca2+ → PKC → MAPK pathways.

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Assessment of Bacterial Communities Associated With the Skin of Costa Rican Amphibians at La Selva Biological Station.

Amphibian skin is a suitable environment for rich communities of microorganisms, both beneficial and detrimental to the host. The amphibian cutaneous microbiota has been hypothesized to play an important role as symbionts, protecting their hosts against disease. Costa Rica has one of the most diverse assemblages of amphibians in the world and we know very little about the microbiota of these tropical animals. For comparison with other studies, we explore the diversity of the skin bacterial communities employing16S rRNA amplicon sequencing of swab samples from twelve species of frogs at La Selva Biological Station in Sarapiquí, Heredia province. The predominant phylum detected in our studies was Proteobacteria, followed by Bacteroidetes and Actinobacteria, with these three phyla representing 89.9% of the total bacterial taxa. At the family level, Sphingobacteriaceae and Comamonadaceae were highly represented among samples. Our results suggest that host species and host family are significant predictors of the variation in microbiota composition. This study helps set the foundation for future research about microbiota composition and resilience to unfavorable conditions, leading to improvement in managing strategies for endangered amphibian species.

Characterization of Batrachochytrium dendrobatidis Inhibiting Bacteria from Amphibian Populations in Costa Rica.

Global amphibian declines and extinction events are occurring at an unprecedented rate. While several factors are responsible for declines and extinction, the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been cited as a major constituent in these events. While the effects of this chytrid fungus have been shown to cause broad scale population declines and extinctions, certain individuals and relict populations have shown resistance. This resistance has been attributed in part to the cutaneous bacterial microbiome. Here, we present the first study characterizing anti-Bd bacterial isolates from amphibian populations in Costa Rica, including the characterization of two strains of Serratia marcescens presenting strong anti-Bd activity. Transcriptome sequencing was utilized for delineation of shifts in gene expression of the two previously uncharacterized strains of S. marcescens grown in three different treatments comprising Bd, heat-killed Bd, and a no Bd control. These results revealed up- and down-regulation of key genes associated with different metabolic and regulatory pathways. This information will be valuable in continued efforts to develop a bacterial-based approach for amphibian protection as well as providing direction for continued mechanistic inquiries of the bacterial anti-Bd response.

Characterization of the Skin Microbiota of the Cane Toad Rhinella cf. marina in Puerto Rico and Costa Rica.

Rhinella marina is a toad native to South America that has been introduced in the Antilles, likely carrying high loads of microorganisms, potentially impacting local community diversity. The amphibian skin is involved in pathogen defense and its microbiota has been relatively well studied, however, research focusing on the cane toad microbiota is lacking. We hypothesize that the skin microbial communities will differ between toads inhabiting different geographical regions in Central America and the Caribbean. To test our hypothesis, we compared the microbiota of three populations of R. cf. marina toads, two from Costa Rican (native) and one Puerto Rican (exotic) locations. In Costa Rica, we collected 11 toads, 7 in Sarapiquí and 4 from Turrialba while in Puerto Rico, 10 animals were collected in Santa Ana. Separate swab samples were collected from the dorsal and ventral sites resulting in 42 samples. We found significant differences in the structure of the microbial communities between Puerto Rico and Costa Rica. We detected as much as 35 different phyla; however, communities were dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Alpha diversity and richness were significantly higher in toads from Puerto Rico and betadiversity revealed significant differences between the microbiota samples from the two countries. At the genus level, we found in Santa Ana, Puerto Rico, a high dominance of Kokuria, Niabella, and Rhodobacteraceae, while in Costa Rica we found Halomonas and Pseudomonas in Sarapiquí, and Acinetobacter and Citrobacter in Turrialba. This is the first report of Niabella associated with the amphibian skin. The core microbiome represented 128 Operational Taxonomic Units (OTUs) mainly from five genera shared among all samples, which may represent the symbiotic Rhinella's skin. These results provide insights into the habitat-induced microbial changes facing this amphibian species. The differences in the microbial diversity in Puerto Rican toads compared to those in Costa Rica provide additional evidence of the geographically induced patterns in the amphibian skin microbiome, and highlight the importance of discussing the microbial tradeoffs in the colonization of new ecosystems.