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Abstract Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high‐throughput imaging experiments. VoltageFluors, a class of voltage‐sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. VoltageFluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data. © 2021 Wiley Periodicals LLC. This article was corrected on 20 July 2022. See the end of the full text for details. Basic Protocol 1: Preparation of dissociated rat hippocampal or cortical cultures Alternate Protocol: Preparation of microisland dissociated cultures Basic Protocol 2: Imaging of spontaneous activity in dissociated cultures using voltage‐sensitive dyes Basic Protocol 3: Analysis of spontaneous activity imaging data
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Laboratory Maintenance of the Chytrid Fungus Batrachochytrium dendrobatidis
Abstract The chytrid fungusBatrachochytrium dendrobatidis(Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impactBdis having on global ecosystems, much ofBd’s basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studyingBdcan shed light on the evolution of key fungal traits because chytrid fungi, includingBd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). StudyingBdin the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures ofBdfor use in the laboratory, how to growBdon solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving cryopreservedBdcultures Basic Protocol 2: Establishing synchronized liquid cultures ofBd Basic Protocol 3: Regular maintenance of synchronousBdin liquid culture Alternate Protocol 1: Regular maintenance of asynchronousBdin liquid culture Basic Protocol 4: Regular maintenance of synchronousBdon solid medium Alternate Protocol 2: Starting a culture on solid medium from a liquid culture Basic Protocol 5: Cryopreservation ofBd
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- Award ID(s):
- 1827257
- PAR ID:
- 10381139
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 12
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Batrachochytrium dendrobatidis(Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world.Bdbelongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids,Bddevelops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to studyBdcell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system forBd. We used electroporation to deliver exogenous DNA intoBdcells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of theBdlife cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms ofBdpathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.more » « less
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Symbiotic relationships between animals and microbes are important for a range of functions, from digestion to protection from pathogens. However, the impact of temperature variation on these animal-microbe interactions remains poorly understood. Amphibians have experienced population declines and even extinctions on a global scale due to chytridiomycosis, a disease caused by chytrid fungi in the genusBatrachochytrium. Variation in susceptibility to this disease exists within and among host species. While the mechanisms generating differences in host susceptibility remain elusive, differences in immune system components, as well as variation in host and environmental temperatures, have been associated with this variation. The symbiotic cutaneous bacteria of amphibians are another potential cause for variation in susceptibility to chytridiomycosis, with some bacterial species producing antifungal metabolites that prevent the growth ofBd. The growth of bothBdand bacteria are affected by temperature, and thus we hypothesized that amphibian skin bacteria may be more effective at preventingBdgrowth at certain temperatures. To test this, we collected bacteria from the skins of frogs, harvested the metabolites they produced when grown at three different temperatures, and then grewBdin the presence of those metabolites under those same three temperatures in a three-by-three fully crossed design. We found that both the temperature at which cutaneous bacteria were grown (and metabolites produced) as well as the temperature at whichBdis grown can impact the ability of cutaneous bacteria to inhibit the growth ofBd. While some bacterial isolates showed the ability to inhibitBdgrowth across multiple temperature treatments, no isolate was found to be inhibitive across all combinations of bacterial incubation orBdchallenge temperatures, suggesting that temperature affects both the metabolites produced and the effectiveness of those metabolites against theBdpathogen. These findings move us closer to a mechanistic understanding of why chytridiomycosis outbreaks and related amphibian declines are often limited to certain climates and seasons.more » « less
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Abstract Numerous species of amphibians declined in Central America during the 1980s and 1990s. These declines mostly affected highland stream amphibians and have been primarily linked to chytridiomycosis, a deadly disease caused by the chytrid fungusBatrachochytrium dendrobatidis(Bd). Since then, the majority of field studies on Bd in the Tropics have been conducted in midland and highland environments (>800 m) mainly because the environmental conditions of mountain ranges match the range of ideal abiotic conditions for Bd in the laboratory. This unbalanced sampling has led researchers to largely overlook host–pathogen dynamics in lowlands, where other amphibian species declined during the same period. We conducted a survey testing for Bd in 47 species (n = 348) in four lowland sites in Costa Rica to identify local host–pathogen dynamics and to describe the abiotic environment of these sites. We detected Bd in three sampling sites and 70% of the surveyed species. We found evidence that lowland study sites exhibit enzootic dynamics with low infection intensity and moderate to high prevalence (55% overall prevalence). Additionally, we found evidence that every study site represents an independent climatic zone, where local climatic differences may explain variations in Bd disease dynamics. We recommend more detection surveys across lowlands and other sites that have been historically considered unsuitable for Bd occurrence. These data can be used to identify sites for potential disease outbreaks and amphibian rediscoveries.more » « less
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Abstract Ecological niche models (ENMs) have been used frequently to predict the distribution and future spread of the pathogenic chytrid fungusBatrachochytrium dendrobatidis(Bd). Based on the assumption that chytridiomycosis outbreaks are most likely to occur where the conditions are ideal for Bd, many studies have identified high‐risk areas for chytridiomycosis and its associated mortality risk using only known Bd occurrences. However, the presence of a pathogen does not necessarily indicate high infection, disease or associated mortality.We used the BIOMOD2 package implemented in R, 19 bioclimatic variables, and 267 locality records, covering three levels of infection progress (occurrence, high infection loads and disease‐associated mortality), to calculate the potential areas where: (1) Bd is likely to be present, (2) amphibians are prone to harbour high infections and (3) chytridiomycosis‐related mortalities are likely to occur. We evaluate discrepancies among the three potential areas projected by the models, encompassing their spatial extent and associated environmental conditions.When all the Bd occurrences were used, the predicted area subjected to Bd risk covered 17% of the study area. However, when just mortality records were used, the predicted area decreased three‐fold. Notably, the three predicted areas only overlapped in 3% of the total study area, suggesting that the region at risk of mortality plus high infections constituted only one‐fifth of the predicted area for Bd presence. Mean temperature during the wettest and warmest 3 months of the year together with isothermality emerged as the most robust negative predictors in each of the three models.Synthesis and applications. Ecological niche models (ENMs) based on the presence data ofBatrachochytrium dendrobatidis(Bd) can overestimate the mortality risk of chytridiomycosis because the environmental conditions suitable for Bd presence do not always correspond to those conducive to significant host mortality. Distribution modelling can be a powerful tool when used correctly, and this study highlights the significance of careful data selection to ensure alignment with intended objectives. Considering the widespread use of ENMs to inform policy, meticulous design and comprehensive evaluation are imperative.more » « less
