Emerging infectious disease in lentic environments : the ecology and biogeography of the amphibian chytrid fungus Batrachochytrium dendrobatidis, with perspectives on water quality, limnology, and chemical contaminants

Biodiversity losses in terrestrial, freshwater, and marine systems are accelerating at a global scale and the most threatened vertebrate taxa are those associated with freshwater habitats. The causes of biodiversity losses are often complex and include synergistic effects of natural and human-induced stressors, such as habitat loss and fragmentation, urbanization, invasive species, contaminants, global climate change, and emerging infectious diseases. In the last 35 years, the amphibian extinction rate has been estimated to exceed 105 times the baseline expected rate for all species and in the USA, the number of occupied amphibian sites has been reported to be declining by 3.7% per year. Among the many threats to amphibians, the role of disease in population declines has been recognized increasingly over the last two decades. Numerous amphibian diseases have been identified and attributed to mass mortality events. Chytridiomycosis, the emerging infectious disease caused by the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is implicated as a causal agent in many recent global amphibian population declines and extinctions. To understand the pathology and conservation implications of Bd, a greater understanding of its ecology, life history, and distribution in the wild is of paramount importance. Although it has an impact on the persistence of selected amphibian populations around the world, the full scope of the effects of chytridiomycosis on global amphibian population declines are not well understood. Most Bd research efforts have focused on Bd in amphibian hosts per se, with little attention to understand the environmental associations and dynamics of free-living Bd outside of the amphibian host. In particular, information on Bd responses to climatic variation outside of hosts is a research gap. Furthermore, as a microorganism within an aquatic environment, studies are lacking of potential water quality associations, how Bd may interact with other members of their biological communities, and how Bd responses to chemical contaminants found in aquatic environments. My research begins to fill these gaps by studying the basic ecology of free-living Bd in field settings, and investigating factors that may influence its distribution at a landscape scale, occurrence at a regional scale, and detection at a site scale. Herein, I describe spatial and temporal patterns in the detection and density of free-living Bd in aquatic habitats in two different geographic regions of the United States, Alaska (Chapter 2) and Oregon (Chapter 3). The Alaska work examines Bd ecology at the northernmost extent of amphibian occurrence in North America, where climate associations may be particularly relevant and where Bd occurrence may be representative of one of the most novel pathogen-host systems in the world. I also describe (Chapter 2) experimental results of Bd and amphibian response to extreme cold temperatures they may experience in continental settings, at high elevations, and at high latitudes. My Oregon studies (Chapter 3) focus on multivariate associations of free-living Bd occurrences with a suite of aquatic environmental factors, both abiotic and biotic in nature. In Chapter 4, I describe how amphibians and Bd respond to agricultural chemicals (fungicides) that they may be exposed to in field settings. These results are specific to Bd, but might also warrant consideration as fungicidal treatments for a newly described chytrid affecting salamanders; both of these amphibian chytrids have been detected in captive animals and solutions to treat trade animals for the pathogen are gaining relevancy. Finally, in Chapter 5, I reflect upon the journey of conservation biologists and herpetologists for 25 years of amphibian decline research, with global losses becoming widely recognized in 1989. In this context, my research significantly advances understanding of the geographic distribution and ecology of one potential threat factor to amphibian populations on Earth, Batrachochytrium dendrobatidis. The factors that I report to both promote or limit free-living Bd distribution and abundance will further inform pathogen dynamics research

Does the agricultural fungicide Tebuconazole inhibit the growth of the amphibian chytrid fungus?

In the US, significant research is ongoing regarding the interactions of pesticides with species and processes within wetland ecosystems. For example, the effect of such pollutants on amphibian survival and physiology has been an area of focused research. Negative impacts of pesticides to non-target organisms (e.g. amphibians) have been observed. The effects of the fungal disease to amphibians that are also exposed to pesticides is complex, however the effects of pesticides on the free-living fungal pathogen has received little attention. We hypothesized that Tebuconazole would inhibit the growth of the amphibian chytrid fungus in culture. To test this hypothesis, we measured the growth of the fungus exposed to a high concentration of Tebuconazole (0.02mg/ml) over a 12 day period. We observed a rapid increase in fungal density in the first two days of the experiment, followed by a steady decrease in the density. We conclude that a high concentration (0.02 mg/ml) amphibian chytrid fungus growth appears to be inhibited by Tebuconazole, although exposure time appears to be an important factor in the response

Bat Monitoring in North Coast and Cascades Network Summer 2019

The detection of white-nose syndrome (WNS) in King County, WA in 2016 prompted the National Park Service to mobilize region-wide bat monitoring and disease surveillance. One of the goals of the Pacific West Region WNS response plan was to implement acoustic monitoring to determine the distribution of bat species and evaluate changes in distribution as WNS spreads. From May to September 2019, we conducted acoustic monitoring at 61 unique sites at two North Coast and Cascades Network (NCCN) parks, Mount Rainier National Park and North Cascades National Park Service Complex). We report results from one of seven elevational transects, Nisqually to Paradise at Mount Rainier National Park. We detected 8 of 11 species known to western Washington. We also detected more bat calls and higher species diversity in riparian habitats compared to forest openings

Prey of reintroduced fishers and their habitat relationships in the Cascades T Range, Washington

Conservation and recovery of forest carnivores requires an understanding of their habitat requirements, as well as requirements of their prey. In much of the western United States, trapping and habitat loss led to extirpations of fishers (Pekania pennanti) by the mid-20th century, and reintroductions are ongoing to restore fishers to portions of their former range. Fisher recovery in Washington State has been limited by isolation from other populations, but other potentially important factors, such as diet of fishers in this region and prey availability, have not been thoroughly investigated. We collected hair samples from potential prey and fishers for stable isotope analysis to identify important prey items for fishers within a reintroduction area in southern Washington. We then estimated the abundance of prey species at 21 sites across a gradient of forest structural classes within the fisher reintroduction area, and assessed the effects of forest age and vegetation on the prey community using permutational multivariate analysis of variance and non-metric multidimensional scaling. Stable isotopes revealed that larger prey items, including snowshoe hares (Lepus americanus) and/or mountain beavers (Aplodontia rufa), were the most important prey item(s) for fishers in the southern Cascades. We found distinct but equally diverse prey communities in old-growth (unmanaged) and young (heavily managed) forest stands, with snowshoe hares and mountain beavers most common in young forests, while chipmunks (Neotamius spp.) and small mammals were more common in older forests. Our results suggest a discrepancy between the habitats where important fisher prey are most abundant and habitat requirements of fishers. Snowshoe hares and mountain beavers were most abundant in young forests, whereas fishers are associated with landscapes dominated by older forest stands or those that provide large woody structures, which fishers use for denning and resting. Our results add to growing evidence that forest landscape mosaics provide valuable habitat for fishers in the Pacific Northwest, suggesting that both mature and younger forest stands are important for fishers and fisher recovery

Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America

Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L⁻¹. The highest density observed was ~3 million zoospores L⁻¹. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time

Range-wide sources of variation in reproductive rates of northern spotted owls

We conducted a range-wide investigation of the dynamics of site-level reproductive rate of northern spotted owls using survey data from 11 study areas across the subspecies geographic range collected during 1993–2018. Our analytical approach accounted for imperfect detection of owl pairs and misclassification of successful reproduction (i.e., at least one young fledged) and contributed further insights into northern spotted owl population ecology and dynamics. Both nondetection and state misclassification were important, especially because factors affecting these sources of error also affected focal ecological parameters. Annual probabilities of site occupancy were greatest at sites with successful reproduction in the previous year and lowest for sites not occupied by a pair in the previous year. Site-specific occupancy transition probabilities declined over time and were negatively affected by barred owl presence. Overall, the site-specific probability of successful reproduction showed substantial year-to-year fluctuations and was similar for occupied sites that did or did not experience successful reproduction the previous year. Site-specific probabilities for successful reproduction were very small for sites that were unoccupied the previous year. Barred owl presence negatively affected the probability of successful reproduction by northern spotted owls in Washington and California, as predicted, but the effect in Oregon was mixed. The proportions of sites occupied by northern spotted owl pairs showed steep, near-monotonic declines over the study period, with all study areas showing the lowest observed levels of occupancy to date. If trends continue it is likely that northern spotted owls will become extirpated throughout large portions of their range in the coming decades

March Mammal Madness and the power of narrative in science outreach.

March Mammal Madness is a science outreach project that, over the course of several weeks in March, reaches hundreds of thousands of people in the United States every year. We combine four approaches to science outreach - gamification, social media platforms, community event(s), and creative products - to run a simulated tournament in which 64 animals compete to become the tournament champion. While the encounters between the animals are hypothetical, the outcomes rely on empirical evidence from the scientific literature. Players select their favored combatants beforehand, and during the tournament scientists translate the academic literature into gripping "play-by-play" narration on social media. To date ~1100 scholarly works, covering almost 400 taxa, have been transformed into science stories. March Mammal Madness is most typically used by high-school educators teaching life sciences, and we estimate that our materials reached ~1% of high-school students in the United States in 2019. Here we document the intentional design, public engagement, and magnitude of reach of the project. We further explain how human psychological and cognitive adaptations for shared experiences, social learning, narrative, and imagery contribute to the widespread use of March Mammal Madness

Spatial and Temporal Patterns of Amphibian Chytrid Fungus \u3ci\u3eBatrachochytrium Dendrobatidis\u3c/i\u3e Occupancy in Amphibian Habitats

Chytrid fungi are the most ancestral of the fungi and are global in distribution. There are over 1200 species of Chytridiomycota described from freshwater, marine and terrestrial systems in temperate, tropical and tundra environments. Chytridiales are characterized by a range of morphologies and share the flask- or pot-like shape of the zoosporangia, within which motile zoospores develop. Chytrids function primarily as plant saprobes and parasites, but some also parasitize animals. Chytrids are observed in conjunction with the decline of freshwater and marine algal blooms, they decompose excess pollen, and comprise the fungal flora in gut of herbivores. Some chytrids also parasitize micro-invertebrates, insects and amphibians. The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd) is the only chytrid known to infect a vertebrate host. Bd exists free-living in the aquatic environment. Lab experiments have demonstrated Bd survival on sterilized moist sand for up to three months and it remained infective in lake water for up to seven weeks. Bd cultures can be maintained under lab conditions for several months (personal observation), which suggests Bd can survive in the environment without a host as long as nutrients are not limiting. In the aquatic environment, Bd is detected by filtering water samples to capture free-living zoospores and zoosporangia then performing a qPCR analysis. Bd has not been reliably isolated from sediments. The goal of our research was to study free-living Bd in amphibian habitats to better understand its ecology and host-pathogen dynamics

FungicidePoster2.pptx

In the US, significant research is ongoing regarding the interactions of pesticides with species and processes within wetland ecosystems. For example, the effect of such pollutants on amphibian survival and physiology has been an area of focused research. Negative impacts of pesticides to non-target organisms (e.g. amphibians) have been observed. The effects of the fungal disease to amphibians that are also exposed to pesticides is complex, however the effects of pesticides on the free-living fungal pathogen has received little attention. We hypothesized that Tebuconazole would inhibit the growth of the amphibian chytrid fungus in culture. To test this hypothesis, we measured the growth of the fungus exposed to a high concentration of Tebuconazole (0.02mg/ml) over a 12 day period. We observed a rapid increase in fungal density in the first two days of the experiment, followed by a steady decrease in the density. We conclude that a high concentration (0.02 mg/ml) amphibian chytrid fungus growth appears to be inhibited by Tebuconazole, although exposure time appears to be an important factor in the response

FungicidePoster2pdf.pdf

In the US, significant research is ongoing regarding the interactions of pesticides with species and processes within wetland ecosystems. For example, the effect of such pollutants on amphibian survival and physiology has been an area of focused research. Negative impacts of pesticides to non-target organisms (e.g. amphibians) have been observed. The effects of the fungal disease to amphibians that are also exposed to pesticides is complex, however the effects of pesticides on the free-living fungal pathogen has received little attention. We hypothesized that Tebuconazole would inhibit the growth of the amphibian chytrid fungus in culture. To test this hypothesis, we measured the growth of the fungus exposed to a high concentration of Tebuconazole (0.02mg/ml) over a 12 day period. We observed a rapid increase in fungal density in the first two days of the experiment, followed by a steady decrease in the density. We conclude that a high concentration (0.02 mg/ml) amphibian chytrid fungus growth appears to be inhibited by Tebuconazole, although exposure time appears to be an important factor in the response