Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientific Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer reviewedPublisher PD

Recent Asian origin of chytrid fungi causing global amphibian declines

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

© The Author(s) 2018.Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.T.W.J.G., M.C.F., D.S.S., A.L., E.C., F.C.C., J.B., A.A.C., C.M., F.S., B.R.S., S.O., were supported through the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity (NERC standard grant NE/K014455/1 and NE/E006701/1; ANR-08-BDVA-002-03). M.C.F., J.S., C.W., P.G. were supported by the Leverhulme Trust (RPG-2014-273), M.C.F., A.C., C.W. were supported by the Morris Animal Foundation. J.V. was supported by the Bolyai János Research Grant of the Hunagrian Academy of Sciences (BO/00597/14). F.G. and D.G. were supported by the Conservation Leadership Programme Future Conservationist Award. C.S.A. was supported by Fondecyt (No. 1181758). M.C.F. and A.C. were supported by. Mohamed bin Zayed Species Conservation Fund Project (152510704). GMR held a doctoral scholarship (SFRH/ BD/69194/2010) from Fundação para a Ciência e a Tecnologia. L.F.T., C.L., L.P.R. K.R.Z., T.Y.J., T.S.J. were supported by São Paulo Research Foundation (FAPESP #2016/25358-3), the National Counsel of Technological and Scientifc Development (CNPq #300896/2016–6) and a Catalyzing New International Collaborations grant from the United States NSF (OISE-1159513). C.S.A. was supported by Fondecyt (No. 1181758). T.M.D. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282).Peer Reviewe

The response in native wildlife to an invading pathogen: Swedish amphibians and Batrachochytrium dendrobatidis

Emerging infectious diseases are causing mortality and declines in wildlife populations globally. My thesis aims to get as clear a picture as possible of the effect the invasive chytrid fungus Batrachochytrium dendrobatidis has on the Swedish amphibian community. In Paper I I performed a large-scale survey testing for the presence of Bd in three regions in Sweden (Southern, Central and Northern). I sampled 1917 amphibians from 101 localities and found that Bd was widespread in southern and central Sweden, occurring in all nine investigated species and in 45.5 % of the sampled sites with an overall prevalence of 13.8%. I found a positive correlation between the temperature at spawning for each species and species prevalence. Species that require higher temperatures for egg-laying are distributed in the southern parts of the country, which led to a higher prevalence in the southern region. In Paper II, I investigated which local environmental factors in breeding habitats, landscape structure and amphibian community affect the occurrence and prevalence of Bd among breeding sites in southern Sweden. Bd prevalence in the four species with the highest prevalence (Bombina bombina, Bufotes variabilis, Epidalea calamita and Rana arvalis) was higher in ponds surrounded by less mature forest, few wetlands, and higher pH. In Papers III and IV, I looked at species and population differences in responses to Bd infection. I performed an infection experiment described in Paper III, where I exposed individuals from two common Swedish species (moor frog R. arvalis and common toad Bufo bufo) originating from two regions (north and south) with two different strains of Bd (from Sweden and the UK). I found that infection led to lower survival and growth in both species, more so in B. bufo than in R. arvalis. Small size proved to be a strong determinant of survival. As individuals from the northern population were significantly smaller than the southern ones, this may have led to the northern populations being more affected by Bd infection. In Paper IV, I studied variation in MHC Class IIB loci in B. bufo along a latitudinal gradient across Sweden. Variation in MCH genes decreased from south to north. Also, differences in survival from the experiment in Paper III could be explained by MHC haplotypes. I found that survival in the southern region was dependent on both Bd-strain and MHC haplotype

The response in native wildlife to an invading pathogen: Swedish amphibians and Batrachochytrium dendrobatidis

Emerging infectious diseases are causing mortality and declines in wildlife populations globally. My thesis aims to get as clear a picture as possible of the effect the invasive chytrid fungus Batrachochytrium dendrobatidis has on the Swedish amphibian community. In Paper I I performed a large-scale survey testing for the presence of Bd in three regions in Sweden (Southern, Central and Northern). I sampled 1917 amphibians from 101 localities and found that Bd was widespread in southern and central Sweden, occurring in all nine investigated species and in 45.5 % of the sampled sites with an overall prevalence of 13.8%. I found a positive correlation between the temperature at spawning for each species and species prevalence. Species that require higher temperatures for egg-laying are distributed in the southern parts of the country, which led to a higher prevalence in the southern region. In Paper II, I investigated which local environmental factors in breeding habitats, landscape structure and amphibian community affect the occurrence and prevalence of Bd among breeding sites in southern Sweden. Bd prevalence in the four species with the highest prevalence (Bombina bombina, Bufotes variabilis, Epidalea calamita and Rana arvalis) was higher in ponds surrounded by less mature forest, few wetlands, and higher pH. In Papers III and IV, I looked at species and population differences in responses to Bd infection. I performed an infection experiment described in Paper III, where I exposed individuals from two common Swedish species (moor frog R. arvalis and common toad Bufo bufo) originating from two regions (north and south) with two different strains of Bd (from Sweden and the UK). I found that infection led to lower survival and growth in both species, more so in B. bufo than in R. arvalis. Small size proved to be a strong determinant of survival. As individuals from the northern population were significantly smaller than the southern ones, this may have led to the northern populations being more affected by Bd infection. In Paper IV, I studied variation in MHC Class IIB loci in B. bufo along a latitudinal gradient across Sweden. Variation in MCH genes decreased from south to north. Also, differences in survival from the experiment in Paper III could be explained by MHC haplotypes. I found that survival in the southern region was dependent on both Bd-strain and MHC haplotype

Effects of host species and environmental factors on the prevalence of Batrachochytrium dendrobatidis in northern Europe

The fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a major threat to amphibian populations. To assist efforts to address such threats, we examined differences in Bd host infection prevalence among amphibian species and its relations to both local environmental factors in breeding habitats and landscape variables measured at three scales (500, 2000 and 5000 m radii) around breeding sites in southernmost Sweden. We sampled 947 anurans of six species in 31 ponds and assessed their infection status. We then examined correlations of infection prevalence with canopy cover, pond perimeter and pH (treated as local-scale pond characteristics), and the number of ponds, area of arable land, area of mature forest, number of resident people and presence of sea within the three radii (treated as landscape variables). The Bd infection prevalence was very low, 0.5-1.0%, in two of the six anuran species (Bufo bufo and Rana temporaria), and substantially higher (13-64%) in the other four (Bombina bombina, Bufotes variabilis, Epidalea calamita, Rana arvalis). In the latter four species Bd infection prevalence was positively associated with ponds' pH (site range: 5.3-8.1), and negatively associated with areas of mature forest and/or wetlands in the surroundings. Our results show that the infection dynamics of Bd are complex and associated with host species, local pond characteristics and several landscape variables at larger spatial scales. Knowledge of environmental factors associated with Bd infections and differences in species' susceptibility may help to counter further spread of the disease and guide conservation action plans, especially for the most threatened species

Data from: Effects of host species and environmental factors on the prevalence of Batrachochytrium dendrobatidis in northern Europe

The fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a major threat to amphibian populations. To assist efforts to address such threats, we examined differences in Bd host infection prevalence among amphibian species and its relations to both local environmental factors in breeding habitats and landscape variables measured at three scales (500, 2000 and 5000 m radii) around breeding sites in southernmost Sweden. We sampled 947 anurans of six species in 31 ponds and assessed their infection status. We then examined correlations of infection prevalence with canopy cover, pond perimeter and pH (treated as local-scale pond characteristics), and the number of ponds, area of arable land, area of mature forest, number of resident people and presence of sea within the three radii (treated as landscape variables). The Bd infection prevalence was very low, 0.5-1.0%, in two of the six anuran species (Bufo bufo and Rana temporaria), and substantially higher (13-64%) in the other four (Bombina bombina, Bufotes variabilis, Epidalea calamita, Rana arvalis). In the latter four species Bd infection prevalence was positively associated with ponds’ pH (site range: 5.3-8.1), and negatively associated with areas of mature forest and/or wetlands in the surroundings. Our results show that the infection dynamics of Bd are complex and associated with host species, local pond characteristics and several landscape variables at larger spatial scales. Knowledge of environmental factors associated with Bd infections and differences in species’ susceptibility may help to counter further spread of the disease and guide conservation action plans, especially for the most threatened species

Effects of host species and environmental factors on the prevalence of Batrachochytrium dendrobatidis in northern Europe

The fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a major threat to amphibian populations. To assist efforts to address such threats, we examined differences in Bd host infection prevalence among amphibian species and its relations to both local environmental factors in breeding habitats and landscape variables measured at three scales (500, 2000 and 5000 m radii) around breeding sites in southernmost Sweden. We sampled 947 anurans of six species in 31 ponds and assessed their infection status. We then examined correlations of infection prevalence with canopy cover, pond perimeter and pH (treated as local-scale pond characteristics), and the number of ponds, area of arable land, area of mature forest, number of resident people and presence of sea within the three radii (treated as landscape variables). The Bd infection prevalence was very low, 0.5-1.0%, in two of the six anuran species (Bufo bufo and Rana temporaria), and substantially higher (13-64%) in the other four (Bombina bombina, Bufotes variabilis, Epidalea calamita, Rana arvalis). In the latter four species Bd infection prevalence was positively associated with ponds' pH (site range: 5.3-8.1), and negatively associated with areas of mature forest and/or wetlands in the surroundings. Our results show that the infection dynamics of Bd are complex and associated with host species, local pond characteristics and several landscape variables at larger spatial scales. Knowledge of environmental factors associated with Bd infections and differences in species' susceptibility may help to counter further spread of the disease and guide conservation action plans, especially for the most threatened species

Acute caffeine differently affects risk-taking and the expression of BDNF and of adenosine and opioid receptors in rats with high or low anxiety-like behavior

Anxiety disorders are common psychiatric conditions with a partially elucidated neurobiology. Caffeine, an unspecific adenosine receptor antagonist, is a common psychostimulant with anxiogenic effects in sensitive individuals. High doses of caffeine produce anxiety-like behavior in rats but it is not known if this is specific for rats with high baseline anxiety-like behavior. Thus, the aim of this study was to investigate general behavior, risk-taking, and anxiety-like behavior, as well as mRNA expression (adenosine A2A and A1, dopamine D2, and, μ, κ, δ opioid, receptors, BDNF, c-fos, IGF-1) in amygdala, caudate putamen, frontal cortex, hippocampus, hypothalamus, after an acute dose of caffeine. Untreated rats were screened using the elevated plus maze (EPM), giving each rat a score on anxiety-like behavior based on their time spent in the open arms, and categorized into a high or low anxiety-like behavior group accordingly. Three weeks after categorization, the rats were treated with 50 mg/kg caffeine and their behavior profile was studied in the multivariate concentric square field (MCSF) test, and one week later in the EPM. qPCR was performed on selected genes and corticosterone plasma levels were measured using ELISA. The results demonstrated that the high anxiety-like behavior rats treated with caffeine spent less time in risk areas of the MCSF and resituated towards the sheltered areas, a behavior accompanied by lower mRNA expression of adenosine A2A receptors in caudate putamen and increased BDNF expression in hippocampus. These results support the hypothesis that caffeine affects individuals differently depending on their baseline anxiety-like behavior, possibly involving adenosine receptors. This highlights the importance of adenosine receptors as a possible drug target for anxiety disorders, although further research is needed to fully elucidate the neurobiological mechanisms of caffeine on anxiety disorders