Biology subjects

Bosch, J.

Publications and source records attributed to Bosch, J..

18 recordsLinked to original sources

bioRxiv2026

Landscape-mediated spread dynamics and anticipatory surveillance of African swine fever in wild boar: insights from the 2025-2026 Catalonia utbreak in Spain

African swine fever (ASF) in wild boar poses major surveillance and control challenges, particularly in periurban and human-modified landscapes where ecological complexity, delayed detection, and heterogeneous host movements complicate out-break interpretation. In late 2025, ASF was detected in wild boar in Catalonia, Spain, creating a high-priority epidemiological scenario at the wildlife-urban interface. In this study, we analysed the 2025-2026 Catalonia outbreak using scenario-based spatial modelling with the WIMBOARD (Wild Integrated Movement Boar Outbreak and Risk Dynamics) framework. Simulations under baseline control conditions were used to interpret spread dynamics and support anticipatory surveillance. The results indicate that outbreak expansion was structured and directional rather than isotropic. Temporal outputs suggested delayed detectability between infection prevalence and mortality signals, whereas cumulative spatial risk maps, time-to-infection surfaces, and monthly infection-risk dynamics identified directional asymmetries and differentiated phases of spread. An early southward dispersal signal was consistent with initial field observations, while the north-northwest sector emerged as the most consequential expansion scenario because of its stronger functional connectivity and higher potential for regional amplification and persistence, while a later northeast-ward phase remained comparatively less influential within the 500-day simulation horizon. Monthly risk surfaces further showed that ASF spread behaved as a moving eco-epidemiological wavefront, allowing identification of shifting surveillance windows before mortality became apparent. These findings support the interpretation of the Catalonia outbreak as a landscape-mediated epidemiological process shaped by functional connectivity, wildlife behavioural adaptation, peri-urban ecological structure, and delayed detection. This study shows how spatially explicit eco-epidemiological modelling can support outbreak interpretation, surveillance prioritisation, and proactive wildlife disease management under complex field conditions.

scientific communication and education↗

bioRxiv2026

Latitudinal Patterns in Immunogenetic and Microbiome Diversity in two anuran species: Bufo bufo and Bufo spinosus

Evolutionary and demographic processes such as selection, drift and migration shape the genetic variation of populations. Genetic diversity is often lower in populations toward higher latitudes. This decrease potentially threatens their survival as several factors are putting more pressure on the populations, including the spread of infectious diseases. In this study, we combined whole-genome re-sequencing with MHC class II genotyping and skin microbiome profiling in Bufo bufo and B. spinosus, two closely related European toad species. We investigated the underlying immunogenetic and microbial variation resulting from different demographic histories and environmental conditions to identify their potential impact on infection outcomes in these two species. We found lower immunogenetic diversity in B. bufo compared to B. spinosus, with highly significant differences in genes related to adaptive and innate immunity. We found lower overall MHC class II diversity and skin microbiome diversity at the species level in B. bufo, compared with B. spinosus. In contrast, at the individual level, B. bufo showed higher MHC allelic diversity and greater diversity in the core skin microbiota than B. spinosus. Together, our findings suggest that divergence in immunogenetic background and host-associated microbial communities may underlie differences in susceptibility to emerging infectious diseases. This integrative framework provides new insight into how host genetics and microbial communities jointly influence disease outcomes across environmental gradients.

evolutionary biology↗

bioRxiv2025

Climate-driven specialisation in plant-pollinator networks peaks outside the tropics

Pollination is a key ecological process sustaining biodiversity and food security, yet global patterns of plant-pollinator specialisation have remained unresolved. Using the largest global dataset of quantitative networks (>3,400 networks, >110,000 interactions), we show that the latitudinal specialisation gradient (LSG) exists, but it is non-linear, hemispherically asymmetric, and strongly taxon-dependent. Network-level and pollinator specialisation were lowest in the tropics and peaked at northern mid-latitudes, whereas plants tended to become more specialised toward higher latitudes. Climate consistently outperformed latitude, species richness, and environmental productivity as a predictor of these patterns. Specialisation declined with increasing temperature, rose with moderate rainfall before declining at the wettest sites, and increased with temperature seasonality, but plants and pollinators responded differently to these drivers. Functional groups diverged strongly: ectothermic insects were most specialised in cooler, seasonal climates, while birds showed weaker links to latitude but reduced specialisation in wetter regions. These findings demonstrate that climate, rather than latitude or species richness, structures global variation in specialisation. Because warmer and less seasonal climates promote generalisation, climate change is likely to disrupt the most specialised pollination systems, unevenly across taxa and regions, with important consequences for biodiversity and ecosystem stability.

ecology↗

bioRxiv2025

Function-Based Selection of Synthetic Communities Enables Mechanistic Microbiome Studies

Understanding the complex interactions between microbes and their environment requires robust model systems such as synthetic communities (SynComs). We developed a functionally directed approach to generate SynComs by selecting strains that encode key functions identified in metagenomes. This approach enables the rapid construction of SynComs tailored to any ecosystem. To optimize community design, we implemented genome-scale metabolic models, providing in silico evidence for cooperative strain coexistence prior to experimental validation. Using this strategy, we designed multiple host-specific SynComs, including those for the rumen, mouse, and human microbiomes. By weighting functions differentially enriched in diseased versus healthy individuals, we constructed SynComs that capture complex host-microbe interactions. Notably, we designed an inflammatory bowel disease SynCom of 10 members that successfully induced colitis in gnotobiotic IL10-/- mice, demonstrating the potential of this method to model disease-associated microbiomes. Our study establishes a targeted framework for designing SynComs to advance mechanistic insights into host-microbe interactions. HighlightsO_LIAutomated functional selection of SynComs based on metagenomic data C_LIO_LIEcosystem-specific SynComs capture the functional landscape of microbiota C_LIO_LIColitis-inducing SynCom developed as a model for inflammatory bowel diseases C_LI

microbiology↗

bioRxiv2025

A phototaxis assay to measure sublethal effects of pesticides on bees

Pesticides are considered a main driver of world-wide bee declines. In agricultural areas, bees are exposed to combinations of pesticides at low concentrations. The extent to which these low levels may cause sublethal effects remains unknown. Laboratory methods to detect sublethal effects are needed as a first step in assessing the potential hazards of pesticides at low concentrations. Current bee risk assessment schemes rely on a single species, the highly social Apis mellifera, and provide insufficient coverage of sublethal effects. Due to fundamental life history differences, available tests cannot be applied to solitary bees. We provide a simple phototaxis assay to detect sublethal pesticide effects on bees. The assay is highly effective (only 6.63% of the bees failed to respond) and provides an unambiguous binary response (bees either walk straight to the light source or walk erratically across the arena). We validate the assay by conducting two experiments. In the first one, we estimate dose-response curves and calculate ED50 and benchmark dose (BMD) values of an insecticide on Osmia bicornis and O. tricornis. In the second one, we assess the effects of three insecticide doses, alone and in combination with a fungicide, in O. cornuta and A. mellifera. These experiments show that our assay can detect effects of field-realistic levels of acetamiprid exposure as low as 1-30 ng/bee. The phototaxis assay can be used to obtain relevant ecotoxicological endpoints at low sublethal concentrations in both solitary and honey bees, thus contributing to fill an important gap in bee risk assessment.

ecology↗

bioRxiv2025

First occurrence of Ranavirus in Scandinavian peninsula

Emerging infectious diseases (EIDs) pose a major threat to global amphibian populations, contributing to widespread mortality and species extinctions. Among EIDs, those caused by fungal infection pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and viral infections of the genus Ranavirus (Rv), represent the most significant threats to amphibian biodiversity. Here, we test for occurrence of Rv infection in three different locations in southeastern Sweden. Using a quantitative PCR (qPCR) assay, complemented by a secondary PCR-based validation method targeting the viral major capsid protein gene (MCP) and additional five partial sequences, we detected Rv infection in two of three locations and in five out of 43 individuals tested. This is the first confirmed record of Rv occurrence reported at such high latitudes in north Europe and the first one from Scandinavian peninsula, contributing important insights into infection prevalence in northern amphibian populations. These findings establish a basis for further research for the conservation of these vulnerable populations.

ecology↗

bioRxiv2024

Lineage-specific trait variation generates widespread, contemporaneous coexistence and competitive exclusion dynamics in an invasive, multihost wildlife parasite

Invasive and highly virulent parasites are emerging worldwide, transported to new locations and into novel hosts by anthropogenic activities. Multiple introduction events lead to interactions amongst genetically dissimilar genotypes that can result in either competitive exclusion, coexistence, or cycling through a combination of the two. Here, we report how intra-lineage trait variation of the multihost amphibian parasite Batrachochytrium dendrobatidis drives contrasting outcomes of inter-lineage interactions on two continents, Europe and Africa. Through field surveys, competition experiments and mathematical modelling we show that interactions between the same two lineages in the two continents and in different hosts demonstrate both ends of the exclusion/coexistence continuum. Trait variation in one of the two predominating lineages, BdGPL, is responsible for these contrasting outcomes: In Europe, BdGPL is highly competitive and has constrained the distribution of the other, BdCAPE, to two locations. In Africa, BdGPL and BdCAPE can mutually invade host populations when the other lineage is already resident, potentially leading to coinfections and recombination. That these contrasting outcomes are prolonged and contemporaneous for the same two lineages shows that epidemiological models of invasive parasites need to account for trait variation both within and across lineages.

ecology↗

bioRxiv2024

Broad diversity of human gut bacteria accessible via a traceable strain deposition system

Numerous bacteria in the human gut microbiome remain unknown and/or have yet to be cultured. While collections of human gut bacteria have been published, few strains have been made publicly available. A major hurdle in making strains publicly available is their deposition to public culture collections. We propose a framework for the bulk-deposition of strains to culture collections, which removes many of the barriers previously identified (www.dsmz.de/bulk-deposit). Using this bulk-deposition system we have created a publicly available collection of human gut isolates. The Human intestinal Bacteria Collection (HiBC) (www.hibc.rwth-aachen.de) contains 340 strains representing 198 species within 29 families and 7 phyla, of which 29 previously unknown species are taxonomically described and named. These included two butyrate-producing species of Faecalibacterium and new dominant species associated with health and inflammatory bowel disease, Ruminococcoides intestinale and Blautia intestinihominis, respectively. Plasmids were prolific within the HiBC isolates, with almost half (46%) of strains containing plasmids, with a maximum of six within a strain. This included a broadly occurring plasmid (pBAC) that exists in three diverse forms across Bacteroidales species. Megaplasmids were identified within two strains, the pMMCAT megaplasmid is globally present within multiple Bacteroidales species. This collection of easily searchable and publicly available gut bacterial isolates will facilitate functional studies of the gut microbiome.

microbiology↗

bioRxiv2024

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 - 3.7 {micro}g/mL). It also significantly reduced liver infection of chimeric FRG-humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a new therapy for Pv.

immunology↗

bioRxiv2023

Expanding the Drosophila toolkit for dual control of gene expression

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA-system or QF-system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterizsed GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue-specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

genetics↗

bioRxiv2023

The Major Histocompatibility Complex modulates Batrachochytrium dendrobatidis and Ranavirus infections in three amphibian species

Genetic variation of immune genes is an important component of genetic diversity. Major histocompatibility complex (MHC) genes have been put forward as a model for studying how genetic diversity is maintained and geographically distributed in wild populations. Pathogen-mediated selection processes (i.e., heterozygosity advantage, rare-allele advantage or fluctuating selection) and demography are believed to generate and maintain the extreme diversity of MHC genes observed. However, establishing the relative importance of the different proposed mechanisms has proved extremely difficult, but heterozygote advantage is expected to be more detectable when multiple pathogens are considered simultaneously. Here, we test whether MHC diversity in three amphibian species (Ichthyosaura alpestris, Pleurodeles waltl, and Pelophylax perezi) is driven by pathogen-mediated selection. We examined the relationship between the individual MHC class II exon variability with individual infection status (infected or not), infection intensity, and co-infection of two main amphibian pathogens: Batrachochytrium dendrobatidis (Bd) and Ranavirus sp. (Rv). We found higher MHC class II exon 2 allelic diversity in I.alpestris and P. perezi than in P.waltl but no significant differences in allele frequencies between infection groups. We also observed significant differences in Bd infection intensity between Bd infected individuals and co-infected individuals depending on the number of MHC loci that an individual carries. For I. alpestris, we show stronger evidence for MHC associations with infection intensity and status when individuals carry specific alleles and supertypes. Our results suggest that studying the association between MHC genes and single and co-infected individuals might provide new insights into host-parasite evolution and a better understanding of evolutionary mechanisms driven by MHC diversity.

evolutionary biology↗

bioRxiv2023

Naturally acquired and Vaccine-induced Human Monoclonal Antibodies to Plasmodium vivax Duffy Binding Protein Inhibit Invasion of Plasmodium knowlesi (PvDBPOR) Transgenic Parasites

The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to Plasmodium vivax (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and their protein-protein interaction is central to vivax blood stage malaria. Here we compared the functional activity of humAbs derived from naturally exposed and vaccinated individuals for the first time using easily cultured P. knowlesi (Pk) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. Using this model, we evaluated the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 g/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 g/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10-100 g/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 g/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. This PkPvDBPOR model system enables efficient assessment of NA and VI humAbs individually and in combination. IMPORTANCEGiven the importance of Duffy blood group antigen and P. vivax Duffy binding protein (PvDBP) interaction leading to blood stage vivax malaria, development of vaccines or therapeutic human monoclonal antibodies (humAbs) targeting PvDBP are key strategies for treating and controlling Pv. The P. knowlesi-based PkPvDBPOR transgenic model system enables efficient assessment of NA and VI humAbs individually and in combination. As such, this model could prioritize specific humAb combinations ahead of clinical trials of these reagents.

microbiology↗

bioRxiv2022

Contrasting patterns of genetic admixture explain the phylogeographic history of Iberian high mountain populations of midwife toads

Multiple Quaternary glacial refugia in the Iberian Peninsula, commonly known as "refugia within refugia", allowed diverging populations to come into contact and admix, potentially boosting substantial mito-nuclear discordances. In this study, we employ a comprehensive set of mitochondrial and nuclear markers to shed light onto the drivers of geographical differentiation in Iberian high mountain populations of the midwife toads Alytes obstetricans and A. almogavarii from the Pyrenees, Picos de Europa and Guadarrama Mountains. In the three analysed mountain regions, we detected evidence of extensive mito-nuclear discordances and/or admixture between taxa. Clustering analyses identified three major divergent lineages in the Pyrenees (corresponding to the eastern, central and central-western Pyrenees), which possibly recurrently expanded and admixed during the succession of glacial- interglacial periods that characterised the Late Pleistocene, and that currently follow a ring-shaped diversification pattern. On the other hand, populations from the Picos de Europa mountains (NW Iberian Peninsula) showed a mitochondrial affinity to central-western Pyrenean populations and a nuclear affinity to populations from the central Iberian Peninsula, suggesting a likely admixed origin for Picos de Europa populations. Finally, populations from the Guadarrama Mountain Range (central Iberian Peninsula) were depleted of genetic diversity, possibly as a consequence of a recent epidemic of chytridiomycosis. This work highlights the complex evolutionary history that shaped the current genetic composition of high mountain populations, and underscores the importance of using a multilocus approach to better infer the dynamics of population divergence.

genetics↗

bioRxiv2021

High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

biochemistry↗

bioRxiv2021

Development of Small Molecule Inhibitors Targeting PBX1 Transcription Signaling as a Novel Cancer Therapeutic Strategy

PBX1 (pre-B cell leukemia transcription factor 1) is a transcription factor involved in diverse cellular functions including organ development, stem cell renewal, and tumorigenesis. PBX1 is localized at chr1q23.3, a frequently amplified chromosomal region, and it is overexpressed in many human malignancies including breast, lung, melanoma, and ovarian carcinomas. Cancer cells with elevated PBX1 signaling are particularly vulnerable to PBX1-withdrawal. We designed a series of small molecule compounds capable of docking to the interface between PBX1 and its cognate DNA target sequence and identified a lead compound, T417, which efficiently hindered the formation of the PBX1 transcriptional complex and affected the transcription of PBX1 target genes. In cell-based assays, T417 significantly suppressed long-term self-renewal and proliferation of cancer cells expressing high levels of PBX1 but not of those expressing low levels of PBX1. T417 also re-sensitized platinum-resistant ovarian tumor cells to carboplatin and produced synergistic anti-tumorigenic effects in vivo in combination with carboplatin. Normal tissues were spared, likely due to the lower PBX1 expression levels. Since PBX1 functions as a molecular hub in developing cancer recurrence and treatment resistance, our data highlight the potential of targeting the PBX-DNA interface as a therapeutic strategy for patients whose tumors rely on PBX1 activation for survival.

pathology↗

bioRxiv2020

Segregation, connectivity, and gradients of deactivation in neural correlates of evidence in social decision making

Functional imaging studies of sensory decision making have detected a signal associated with evidence for decisions that is consistent with data from single-cell recordings in laboratory animals. However, the generality of this finding and its implications on our understanding of the organization of the fMRI signal are not clear. In the present functional imaging study, we investigated decisions in an elementary social cognition domain to identify the neural correlates of evidence, their segregation, connectivity, and their relationship to task deactivations. Besides providing data in support of an evidence-related signal in a social cognition task, we were interested in embedding these neural correlates in models of supramodal associative cortex placed at the top of a hierarchy of processing areas. Participants were asked to decide which of two depicted individuals was saddest based on information rich in sensory features (facial expressions) or through contextual cues suggesting the mental state of others (stylized drawings of mourning individuals). The signal associated with evidence for the decision was located in two distinct networks differentially recruited depending on the information type. Using the largest peaks of the signal associated with evidence as seeds in a database of connectivity data, these two networks were retrieved. Furthermore, the hubs of these networks were located near or along a ribbon of cortex located between task activations and deactivations between areas affected by perceptual priming and the deactivated areas of the default network system. In associative cortex, these findings suggest gradients of progressive relative deactivation as a possible neural correlate of the cortical organization envisaged by structural models of cortical organization and by predictive coding theories of cortical function.

neuroscience↗

bioRxiv2019

Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids

Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. This method is a simple and fast alternative to HDR for certain strategies such as C-terminal tagging and gene disruption. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-RFP into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Finally, we apply this fast knock-in method to uncharacterized small open reading frame (smORF) genes. Therefore, homology-independent insertion is a useful genome editing technique in Drosophila that will better enable researchers to dissect gene function.\n\nArticle summaryWe report a fast and simple genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. We demonstrate its usefulness in cultured cells to fluorescently tag endogenous proteins and in the fly germ line to generate heritable insertions that disrupt gene function and can act as expression reporters.

genetics↗

bioRxiv2018

Development and worldwide use of a non-lethal and minimal population-level impact protocols for the isolation of chytrids from amphibians

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile 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 interested researchers worldwide 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 widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. 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 important group of emerging fungal pathogens.

ecology↗