Effects of the insecticide ß-endosulfan on tadpoles of Isthmohyla pseudopuma (Anura: Hylidae).

Conventional agriculture uses pesticides intensively. Once pesticides are released into the environment, they can be toxic to non-target organisms. Exposure of amphibians to pesticides can be lethal and affect their growth, development and behavior. ß-endosulfan is a persistent organochlorine that has been detected in environmental samples within protected sites in Costa Rica, far from agricultural areas. The aim of this study was to evaluate the lethal and sublethal effects, as well as changes in three biomarkers (Cholinesterase activity [ChE], glutathione S-transferase activity [GST] and lipid peroxidation [LPO]) in tadpoles of Isthmohyla pseudopuma exposed to ß-endosulfan. A 96-h acute test (20, 40, 60, 80, 100 and 200 µg/L) was performed in order to calculate the median lethal concentration (LC50), while effects on growth and development were assessed during a 4-weeks chronic test (10, 20, 30 and 50 µg/L). In addition, we measured the aforementioned biomarkers in tadpoles exposed to concentrations below the LC50. The 96-h LC50 for this species was 123.6 µg/L. We found no evidence of ß-endosulfan influencing any of the three biomarkers evaluated. At 50 µg/L, both length and total weight of tadpoles decreased with respect to the control. Also, at 30 and 50 µg/L we observed that individuals showed a slower development. Therefore, we demonstrated that at sublethal concentrations, ß-endosulfan negatively affects I. pseudopuma at early stages causing tadpoles to develop slower and smaller than normal.

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.

Habitat disturbance influences the skin microbiome of a rediscovered neotropical-montane frog.

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.

Combination of ketamine and xylazine with opioids and acepromazine in rats: Physiological changes and their analgesic effect analysed by ultrasonic vocalization.

In this study, the effect of four anaesthetic protocols that included the combination of xylazine (X) and ketamine (K) with acepromazine (A) and opioids (methadone (Me), morphine (Mo) or tramadol (T)) was evaluated in laboratory rats of both sexes. Ultrasonic vocalization (USV) was used as an indicator of pain during the recovery period. The objective was to evaluate the physiological parameters and the analgesic effect of each protocol to determine which protocol was the safest and fulfil the requirements of a balanced anaesthesia. The better protocols were the XKA protocol for both sexes and the XKMe protocol for females because the combinations achieve surgical plane of anaesthesia in rats. However, pain assessment during the formalin test revealed that rats anaesthetized with XKA produced more numbers of USV, suggesting that it is not a good protocol for the control of immediate postoperative pain. All protocols produced depression in body temperature and respiratory and heart rates, and had important effects, such as micturition and maintenance of open eyes. Only rats anaesthetized with XKA protocol did not present piloerection. These results demonstrated that good monitoring and care during anaesthesia must be included to prevent complications that compromise the life of the animal and to ensure a good recovery. The inclusion of analgesia in anaesthesia protocols must be used routinely, ensuring minimal presence of pain and thus more reliable results in the experimental procedures.

Batrachochytrium dendrobatidis infection in amphibians predates first known epizootic in Costa Rica.

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.

Quality of Adherence to the ARRIVE Guidelines in the Material and Methods Section in Studies Where Swine Were Used as Surgical Biomodels: A Systematic Review (2013-2018).

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.

Moving Beyond the Host: Unraveling the Skin Microbiome of Endangered Costa Rican Amphibians.

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.

Red seabream iridovirus associated with cultured Florida pompano Trachinotus carolinus mortality in Central America.

Mariculture of Florida pompano Trachinotus carolinus in Central America has increased over the last few decades and it is now a highly valued food fish. High feed costs and infectious diseases are significant impediments to the expansion of mariculture. Members of the genus Megalocytivirus (MCV), subfamily Alphairidovirinae, within the family Iridoviridae, are emerging pathogens that negatively impact Asian mariculture. A significant mortality event in Florida pompano fingerlings cultured in Central America occurred in October 2014. Affected fish presented with abdominal distension, darkening of the skin, and periocular hemorrhages. Microscopic lesions included cytomegalic 'inclusion body-bearing cells' characterized by basophilic granular cytoplasmic inclusions in multiple organs. Transmission electron microscopy revealed arrays of hexagonal virions (155-180 nm in diameter) with electron-dense cores within the cytoplasm of cytomegalic cells. Pathological findings were suggestive of an MCV infection, and the diagnosis was later confirmed by partial PCR amplification and sequencing of the viral gene encoding the myristylated membrane protein. The viral sequence revealed that the fingerlings were infected with an MCV genotype, red seabream iridovirus (RSIV), previously reported only from epizootics in Asian mariculture. This case underscores the threat RSIV poses to global mariculture, including the production of Florida pompano in Central America.

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.

Comparative leukocyte morphometric analysis between endemic Anurans from Brazil and the invasive species Lithobates catesbeianus / Análise morfométrica comparada entre Anuros endêmicos do Brasil e a espécie invasora Lithobates catesbeianus

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.(AU)

Anfíbios são indicadores ambientais potencialmente confiáveis e eficientes. Estudos referentes à morfologia de leucócitos de anuros são limitados, com poucos estudos morfométricos disponíveis em literatura. O presente estudo empregou técnicas morfométricas para caracterizar leucócitos de anuros Neotropicais brasileiros selecionados e compará-los com a espécie exótica rã-touro (Lithobates catesbeianus), família Ranidae. Esfregaços sanguíneos de 28 espécimes pertencentes a seis gêneros diferentes (Hyla, Phyllomedusa, Hypsiboas, Scinax, Physalaemus e Proceratophrys) foram comparados com amostras de esfregaços de L. catesbeianus. A média do diâmetro dos leucócitos foi calculada por um software de análise de imagens. One-way e teste de Bonferroni foram utilizados para avaliação estatística. Linfócitos, neutrófilos, eosinófilos e basófilos mostraram-se significativamente menores que os valores de referência reportados em outros gêneros de anfíbios, incluindo Lithobathes; por outro lado, a média do diâmetro dos monócitos não demonstrou variação significativa entre os gêneros. Esse é o primeiro estudo de avaliação morfométrica de leucócitos em espécies de anuros brasileiros. Nossos resultados sugerem que a separação geográfica possivelmente influencia a morfometria leucocitária.(AU)

Acute, chronic and biochemical effects of chlorothalonil on Agalychnis callidryas, Isthmohyla pseudopuma and Smilisca baudinii tadpoles.

Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. Efforts are being carried out to elucidate factors related to this phenomenon. Among these factors, pathogens, climate change, and environmental pollution have been suggested as possible causes. Regarding environmental pollutants, some pesticides are persistent in the environment and capable of being transported long distances from their release point. In Costa Rica, some pesticides have been detected in protected areas, at locations where amphibian populations have declined. Information about toxicity of pesticides used in Costa Rican agriculture to amphibians is still scarce, particularly for native species.Toxicity tests with chlorothalonil, a fungicide intensively used in Costa Rica, were carried out exposing tadpoles of three Costa Rican native species: Agalychnis callidryas, Isthmohyla pseudopuma, and Smilisca baudinii in order to evaluate acute and chronic toxicity as well as the biomarkers cholinesterase activity (ChE), glutathione-S transferase activity (GST), and lipid peroxidation (LPO).96-h LC50: 26.6 (18.9-35.8) µg/L to A. callidryas, 25.5 (21.3-29.7) µg/L to I pseudopuma and 32.3 (26.3-39.7) µg/L to S. baudinii were determined for chlorothalonil. These three species of anurans are among the most sensitive to chlorothalonil according to the literature. Besides, GST was induced in S. baudinii after exposure to sub-lethal concentrations of chlorothalonil while evisceration occurred in S. baudinii and A. callidryas tadpoles exposed to lethal concentrations of the fungicide. Chronic exposure to sub-lethal concentrations accelerated development in S. baudinii and caused lesions in tail of S. baudinii and I. pseudopuma tadpoles. Our results demonstrate that chlorothalonil is highly toxic to native amphibian species and that low concentrations can cause biochemical responses related to phase II of biotransformation and effects on development.

Survey of wild mammal hosts of cutaneous leishmaniasis parasites in panamá and costa rica.

The eco-epidemiology of American cutaneous leishmaniasis (ACL) is driven by animal reservoir species that are a source of infection for sand flies that serve as vectors infecting humans with Leishmania spp parasites. The emergence and re-emergence of this disease across Latin America calls for further studies to identify reservoir species associated with enzootic transmission. Here, we present results from a survey of 52 individuals from 13 wild mammal species at endemic sites in Costa Rica and Panama where ACL mammal hosts have not been previously studied. For Leishmania spp. diagnostics we employed a novel PCR technique using blood samples collected on filter paper. We only found Leishmania spp parasites in one host, the two-toed sloth, Choloepus hoffmanni. Our findings add further support to the role of two-toed sloths as an important ACL reservoir in Central America.

Ectoparasites of dogs in home environments on the Caribbean slope of Costa Rica.

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.

Ectoparasites of dogs in home environments on the Caribbean slope of Costa Rica / Ectoparasitos de cães no ambiente doméstico da vertente Caribe, Costa Rica

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.