Effects of Infection of the Protist Parasite, Dermomycoides Sp., in Dusky Gopher Frog Tadpoles

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Master's Theses

Fall 12-1-2020

Effects of Infection of the Protist Parasite, Dermomycoides sp., in Dusky Gopher Frog Tadpoles

Jaime Smith

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Recommended Citation Smith, Jaime, "Effects of Infection of the Protist Parasite, Dermomycoides sp., in Dusky Gopher Frog Tadpoles" (2020). Master's Theses. 775. https://aquila.usm.edu/masters_theses/775

This Masters Thesis is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Master's Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. EFFECTS OF INFECTION OF THE PROTIST PARASITE, DERMOMYCOIDES SP. IN DUSKY GOPHER FROG TADPOLES

Jaime E Smith

A Thesis Submitted to the Graduate School, the College of Arts and Sciences and the School of Ocean Science and Engineering at The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Master of Science

Approved by:

Robert J Griffitt, Committee Chair Robin Overstreet, Thesis Director Zachary Darnell Joseph H.K. Pechmann, Western Carolina University

December 2020

Jaime E Smith

2020

Published by the Graduate School

ABSTRACT

Infections of the protist parasite, Dermomycoides sp. are thought to have caused several years of low recruitment in the dusky gopher frog (Rana sevosa) populations. I evaluated the effects of density of the infective zoospores, host developmental stage, and tadpoles' ability to acquire resistance to Dermomycoides sp. on dusky gopher frog tadpoles.

Tadpoles were exposed to zoospore densities of 0, 250, 500, and 750 zoospores/µL at

Gosner stage 25, and we found no significant differences among treatments in tadpole mortality. In evaluating susceptibility by development stage, I exposed R. sevosa to 50 zoospores/µL as eggs, embryos, hatchlings, and 2-weeks post hatching tadpoles.

Hatchlings (17 days, SE = 2.09 days) and tadpoles exposed 2-weeks post-hatching (17.65 days, SE = 1.48 days) had a significantly lower days to mortality than tadpoles exposed as eggs(27.56 days, SE = 2.09 days) or embryos ( 25.81 days, SE = 2.09 days). To assess if gopher frog tadpoles can acquire resistance to Dermomycoides sp. I exposed tadpoles around Gosner stage 25 to an initial zero (0 zoospores/µL), low (50 zoospores/µL), or high (250 zoospores/µL) dose of Dermomycoides sp. Two-weeks later each treatment was exposed to a second zero, low, or high dose for a total of 9 treatments. Survival was lowest in tadpoles that received a low initial dose followed by a zero or high challenge dose (0 and 3.03% respectively). I found that gopher frog tadpoles can acquire resistance to Dermomycoides sp., and that age at initial exposure plays an important role in tadpole survival.

ACKNOWLEDGMENTS

I would like to thank Dr. Joseph Pechmann and Dr. Robin Overstreet for taking me on as a student; and Dr. Joe Griffitt and Dr. Zack Darnell for supporting me as committee members. I would also like to thank Jean Jovonovich Allivar, Janet Wright,

Matt Atkinson, Jim Lee, and John Tupy for their advice and guidance throughout this project. A special thanks to Linda LaClaire and the U.S. Fish and Wildlife Service, and the Gulf Coast Research Laboratory, for funding and support.

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TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES ...... vi

LIST OF ILLUSTRATIONS ...... vii

LIST OF ABBREVIATIONS ...... ix

CHAPTER I – INTRODUCTION TO DERMOMYCOIDES SP. AND THE DUSKY

GOPHER FROG ...... 1

CHAPTER II – EFFECTS OF TADPOLE DEVELOPMENTAL STAGE AND

ZOOSPORE EXPOSURE ON MORTALITY OF RANID TADPOLES ...... 8

Methods...... 11

Collection of animal and infectious material ...... 11

Experiment 1. Investigation of effects of gopher frog developmental stage at the

time of infection on morbidity ...... 12

Experiment 2. An investigation into the relationship between zoospore exposure

concentration and infection of gopher frog and leopard frog tadpoles...... 15

Molecular analysis ...... 16

Statistical analysis ...... 16

Results ...... 17

Average days to mortality across developmental stages ...... 17

Average infection level across developmental stage groups detected by qPCR ...... 20

Mortality across water baths containing different numbers of zoospores ...... 20

Discussion ...... 23

Host developmental stage ...... 23

Zoospore exposure ...... 26

Future work ...... 27

CHAPTER III - ACQUIRED RESISTANCE TO DERMOMYCOIDES SP. IN

PREVIOUSLY INFECTED DUSKY GOPHER FROG TADPOLES ...... 29

Methods...... 33

Results ...... 36

Days to mortality...... 36

Survival ...... 36

Molecular results ...... 38

Discussion ...... 38

CHAPTER IV – SUMMARY ...... 45

APPENDIX A – Preliminary study on infection threshold tolerance of dusky gopher frog tadpoles ...... 48

REFERENCES ...... 50

LIST OF TABLES

Table 1 Average days to mortality in dusky gopher frog tadpoles exposed at different developmental stages...... 18

Table 2 Average days to mortality in individual tadpoles exposed to zoospores of

Dermomycoides sp...... 21

Table 3 ANOVA summary for survival to experimental endpoint in tadpoles that received an initial and challenge dose treatment...... 37

Table 4 Average infection load of tadpoles in each replicate egg clutch at experimental mid-point and endpoint...... 42

LIST OF ILLUSTRATIONS

Figure 1. Range map of the dusky gopher frog (Rana sevosa) by county in Mississippi. .. 2

Counties with established gopher frog populations are dark green, and counties with emerging populations aided by human assistance are in light green...... 2

Figure 2. Life history interaction between the dusky gopher frogs and the protist parasite

Dermomycoides sp...... 3

Gopher frogs migrate into ponds to breed. Drawing by (Cook, 2008)...... 3

Figure 3. Gosner stages for amphibian development (Gosner, 1960) used in this experiment...... 13

Figure 4. Tank map for experiment 1...... 14

Figure 5. Cumulative mortality of tadpoles at different days...... 19

Figure 6. Average days to the mortality of dusky gopher frog tadpoles exposed to 50 zoospores/µL at different developmental stages...... 19

Figure 7. Average infection quantity across developmental stages at the time of collection as detected using qPCR...... 20

Figure 8. Cumulative mortality of individual gopher frog tadpoles exposed to baths with different numbers of zoospores of Dermomycoides sp. (0, 250, 500, 750)...... 22

Figure 9. Cumulative mortality of individual leopard frog tadpoles exposed to baths with different numbers zoospores of Dermomycoides sp. (0, 250, 500, and 750)...... 22

Figure 10. Average days to the mortality of gopher frog (R. sevosa) and leopard frog tadpoles (R. sphenocephala) exposed to zoospore water baths...... 23

Figure 11. The average survival of gopher frog tadpoles at different developmental stages exposed to 50 zoospores/µL in experiment 1 of this thesis compared to different

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developmental stages of leopard frog tadpoles exposed 100 zoospores/µL by Atkinson

(2016) and Cook (2008) ...... 25

Figure 12. Gopher frog tadpoles were exposed to an initial and challenge dose of zoospores...... 34

Figure 13. Tank map of tadpole groups exposed to a challenge dose of zoospores...... 35

Figure 14. Survival to the endpoint of all initial and challenge dose treatment combinations...... 37

Figure 15. Average days to the mortality of tadpoles exposed to an initial and challenge dose of zoospores of Dermomycoides sp., post-exposure to the challenge dose...... 39

Figure 16. Survival to the experimental endpoint of tadpoles that received a high (H), low

(L), or zero (O) exposure dose of zoospores of Dermomycoides sp...... 41

Figure 17. Average infection level among initial treatment groups at experimental midpoint and endpoint when outliers were removed from the analysis...... 43

Average days to mortality in gopher frog tadpoles exposed between 0-250 zoospores/µL. There were no significant differences in days to mortality across treatment groups...... 49

Average survival to end-point of tadpoles exposed between 0-250 zoospores/µL. Survival was only significantly different between tadpole exposed to 0 zoospores/µL and 250 zoospores/µL (p= 0.04)...... 49

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LIST OF ABBREVIATIONS

USM The University of Southern Mississippi

WCU Western Carolina University

HEF Harrison Experimental Forest

GCRL Gulf Coast Research Laboratory

USFWS United States Fish and Wildlife Service

TNC The Nature Conservancy

CHAPTER I – INTRODUCTION TO DERMOMYCOIDES SP. AND THE DUSKY

GOPHER FROG

The protist parasite Dermomycoides sp. can utilize a wide range of amphibian hosts and has been documented across the United States. Documented hosts include members of the genus Rana (R. sylvatica, R. catesbeiana, R. sphenocephala, R. clamitans, R. captio, R. septentrionalis, R. pipens, and R. sevosa) the species Pseudacris crucifer, Acris gryllus, Gyrinophilus porphyriticus, and Bufo terrestris (Aurelie

Chambouvet et al., 2020; Green, Converse, & Schrader, 2002; Isidoro-Ayza et al., 2017;

Isidoro-Ayza, Lorch, Ballmann, & Businga, 2019). The majority of infections and mortality associated with Dermomycoides reported have been in primarily late developmental stage tadpoles around Gosner stages 31-46 (Isidoro-Ayza et al., 2017).

Infection is characterized by hemorrhaging of the liver and spleen with red or black discoloration of the tissues (Isidoro-Ayza, Grear, & Chambouvet, 2019). While zoospores can be found in nearly all tissues of an infected host, the liver is typically the most heavily infected (Landsberg et al., 2013). Mortality events associated with infection of zoospores have been documented in New Hampshire, Minnesota, Virginia, Tennessee,

Alaska, Maryland, Louisiana, Florida, Maine, Mississippi, New York, and Georgia

(Green et al., 2002; Isidoro-Ayza et al., 2017). External gross characteristics of infected individuals include abdominal swelling, skin discoloration, and redness, and puffiness caused by fluid retention (Isidoro-Ayza, Grear, et al., 2019). As infectious zoospores reproduce asexually in the host, extracellular spores invade and replace the liver, spleen, kidneys, and pancreas. Because of the ability of Dermomycoides sp. to utilize a wide range of hosts and environments, if one host species were to go extinct, the parasite could

still survive and cause declines among other hosts (Gleason, Chambouvet, Sullivan, Lilje,

& Rowley, 2014). For example, bullfrogs are a common and widespread species throughout the United States, and they use some of the same territory and habitat as the endangered dusky gopher frog (Fig. 1.1). Both species have had mortality events associated with infection of Dermomycoides sp. across their respective ranges (Green et al., 2002). Because of this overlap in the territorial range of the host, even if

Dermomycoides sp. were to drive the dusky gopher frog to extinction, the parasite would persist in alternative hosts. This is extremely problematic for endangered species reintroduction efforts, as it can dramatically decrease uninfected habitat for release.

Figure 1. Range map of the dusky gopher frog (Rana sevosa) by county in Mississippi.

Counties with established gopher frog populations are dark green, and counties with emerging populations aided by human assistance are in light green.

Amphibian tadpoles function as the definitive host for Dermomycoides sp. reproduction and spread (Cook, 2008). Tadpoles are exposed to both infectious zoospores and non-infectious spores of Dermomycoides sp. in the water column until they emerge from the pond as juvenile metamorphs and migrate to the upland habitat. Infectious zoospores can penetrate and directly infect amphibian eggs and tadpoles (Fig. 1.2). Once in the amphibian host, zoospores reproduce asexually in the lumen and intestine, replacing the host tissue with spores. These spores are shed by the host through the digestive system and re-enter the pond basin. Within the pond, non-infectious spores can hatch into infective zoospores when the pH is between 5.5-7.0 (Cook, 2008). Non- infective spores can be ingested indirectly by tadpoles or transport hosts such as birds and invertebrates that also use that pond basin.

Figure 2. Life history interaction between the dusky gopher frogs and the protist parasite Dermomycoides sp.

Gopher frogs migrate into ponds to breed. Drawing by (Cook, 2008). 3

The eggs develop into tadpoles that undergo metamorphosis from early May to late August. As tadpoles are developing in the pond basin, they are exposed to infectious zoospores that can directly penetrate the tadpoles and non-infectious spores which the tadpoles ingest while swimming. Mortality occurs when the tadpole is unable to shed spores faster than the spores can replicate asexually within the tadpole. While the frog is the only terminal host of Dermomycoides sp., the parasite can use additional transport hosts such as birds and invertebrates, that carry spores in their feces.

Endangered and threatened species are at high risk from extinction due to disease because of their small population sizes. Decreased survival and reproductive success of hosts caused by disease can cause the mortality rate to surpass the host recruitment rate, causing extinction (De Castro & Bolker, 2004). This enhanced extinction risk is especially true of hosts that experience increased stochasticity of natural recruitment due to habitat constraints. Many amphibians require ephemeral ponds for breeding, and early drying of the pond basin can lead to years of zero natural recruitment in the population(Joseph H K Pechmann et al., 1991). Several years of low or zero natural recruitment coupled mass mortality events caused by infection can greatly increase the risk of extinction.

One buffer against low recruitment caused by a shortened hydroperiod is the presence of a metapopulation habitat structure. A metapopulation is defined as a collection of populations of the same species, within the same general geographic area, that may exchange individuals through migration, dispersal, or human interference

(Hanski & Simberloff, 1997; Marsh & Trenham, 2001; McCullough, 1996). Juvenile frogs originating from one pond within the metapopulation may disperse to the other 4

ponds for breeding. While the metapopulation structure can buffer against low juvenile recruitment due to early pond drying, it does not necessarily prevent low recruitment due to disease. Hosts that utilize metapopulation structures are susceptible to extinction at local and meta-population levels when the rate of the pathogen to infect habitat exceeds the rate of host colonization of uninfected habitat (De Castro & Bolker, 2004). If hosts are unable to find uninfected habitat, the populations at each infected pond will continually decrease over time, with no long-term recovery due to a lack of uninfected habitat for the population to continuously experience recruitment.

At the time of their listing, the dusky gopher frog only occupied a single breeding pond known as Glen's Pond (LaClaire, 2001). Since then the Glen's Pond population expanded into two additional ponds which were altered or created by humans, Pony

Ranch Pond and New Pond, respectively, that make up the Glen's Pond metapopulation.

Pony Ranch Pond and New Pond are within 2 km of Glens Pond and have had gopher frog breeding documented via egg mass surveys as early as 2016 at New Pond and 2013 at Pony Ranch Pond (Joseph H.K. Pechmann, 2016).

Both Pony Ranch Pond and New Pond have been documented as having over

95% tadpole mortality associated with infection of Dermomycoides sp. (M. Atkinson,

2016). Low recruitment in these ponds during several years with a sustained hydroperiod is thought to have been caused by exposure to Dermomycoides sp. One management tactic used to mitigate declines caused by disease is expanding the range of the host and creating more metapopulations at uninfected sites (Hanski & Simberloff, 1997; Heard et al., 2017; Marsh & Trenham, 2001; McCullough, 1996). However, endangered species are at higher risk of disease driven extinction if the pathogen can use a more common

and widespread reservoir host that can colonize new habitat at a faster rate than the endangered host (De Castro & Bolker, 2004). The southern leopard frog, for example, is more likely to colonize and infect surrounding ponds before the gopher frog can be introduced into these ponds. Once the gopher frog starts using these ponds, their offspring would have a lower probability of survival because of infection introduced by leopard frogs. Because of the ability of Dermomycoides sp. to use more common species to expand its range, avoidance of this pathogen by the dusky gopher frog is nearly impossible.

Head-starting programs that collect a portion of each wild-hatched gopher frog egg mass and rear the animals through metamorphosis before release at the origin site or translocation to a new site allow for supplementary recruitment of juveniles to the wild population. These head-starting programs have prevented extinction over multiple consecutive years of low or zero recruitment of gopher frogs at Glens' Pond (Joseph H.K.

Pechmann, 2019). However, the small population size of the dusky gopher frog at the time of listing resulted in a low genetic diversity in the species (Hinkson & Richter,

2016). Additional years of zero juvenile recruitment reduced allele richness and contributed to more loss of heterozygosity (Hinkson & Richter, 2016).

Low genetic variability in a population can cause a decrease in the population's immune response making the species more susceptible to disease (De Castro & Bolker, 2004).This occurs when infection causes a positive feedback loop in populations with reduced genetic diversity and allele richness. The low genetic variability can result in a decrease in the population immune response, causing higher mortality in infected individuals, driving down the population size and genetic variability further (De Castro & Bolker,

2004; Soul, 1998). In populations that experience multiple stressors such as disease, early pond drying, temperature fluctuations, or habitat alteration population decline, and extinction are more likely. This type of disease-driven extinction model, or "extinction vortex", could apply to the gopher frog population, with gopher frogs serving as the declining host population and Dermomycoides sp. as the parasitic pathogen driving the population to extinction.

CHAPTER II – EFFECTS OF TADPOLE DEVELOPMENTAL STAGE AND

ZOOSPORE EXPOSURE ON MORTALITY OF RANID TADPOLES

Despite conjecture that Dermomycoides sp. may be the third most common infectious disease behind wild frog mortalities reported (Isidoro-Ayza, 2017), very little is known about what causes mortality in infected hosts. Parasite-host interactions are influenced by a balance of environmental factors, parasite characteristics, and the host response (Blaustein et al., 2012). Prior work by Cook (2008) and Atkinson (2016) showed that Dermomycoides sp. hatches into infective zoospores between a pH of 5.5 and 7.0 and has the highest rate of host mortality in ponds that dry and fill rapidly. Both researchers also investigated the effect of developmental stage on southern leopard frog tadpoles, and as of this project no further investigations have been conducted on the interaction of the parasite-host relationship between tadpoles and Dermomycoides sp.

While the taxonomy of Dermomycoides sp. and a full species description is still unknown, the primary pathology and description have begun to be understood (Isidoro-

Ayza, Grear, et al., 2019). Understanding host response to infection using different amphibian species is imperative to furthering our understanding of this emerging pathogen.

Host developmental stage can influence the likelihood that the host becomes infected after exposure and the level of pathology after that infection (Johnson,

Kellermanns, & Bowerman, 2011). The sensitivity and susceptibility of amphibian hosts to infection is known to vary as hosts develop. For example, these factors vary in wood frog (R. sylvaticus) and bullfrog (R. catesbianus) tadpoles infected with

Batrachochytrium dendrobatidis (Bd), ranavirus, and trematode infections based on the

host's developmental stage (Blaustein et al., 2012; De Jesus Andino, Chen, Li, Grayfer, &

Robert, 2013). Earlier developmental stage green frog (R. clamitans) tadpoles exposed to three different types of parasites (Echinostoma trivovis, Ribeiroia ondatrae, or plagiochid trematode cercariae) had a lower tolerance to infection than older developmental stage tadpoles (Rohr, Raffel, & Hall, 2010). African clawed frog (Xenopis laevis) larvae possess both B and T cells but have no consistent expression of MHC cells until metamorphosis (De Jesús Andino, Chen, Li, Grayfer, & Robert, 2012). When infected,

African clawed frog tadpoles can mount an adaptive immune response using B and T cells, and tolerate the infection, but are unable to express innate immunity pre- metamorphosis.

Understanding the developmental stages at which gopher frog tadpoles are most vulnerable to infection and mortality is a critical first step in a greater understanding of the parasite-host interaction of Dermomycoides sp. Either from observational bias or real association, infection and mortality from Dermomycoides sp. has most commonly been observed in tadpoles during the later stages of development, just prior to metamorphosis.

In an examination of 225 tadpoles sampled from 21 different mortality events, the majority of infected individuals were between Gosner stages 31-46 (Isidoro-Ayza et al.,

2017). Gosner stages 31-46 represent the last developmental stages before metamorphosis (Gosner, 1960). While knowing that these late developmental stages are at risk is important for monitoring efforts, it is also possible that earlier mortality events may have been missed and thus not included. Tadpole mortalities occurring at earlier developmental stages are likely underreported due to sinking and rapid decomposition

(Landsberg et al., 2013). A combination of field observations coupled with laboratory

experiments is ideal so as not to underestimate the disease effects on the host population due to observational bias in either the field or laboratory setting (McCallum & Dobson,

1995).

Previous laboratory work examining the effects of host developmental stage on mortality associated with infection of Dermomycoides sp. used only southern leopard frogs (R. sphenocephala). Cook (2008) exposed leopard frog tadpoles within the first 30-

150 hours of deposition into a pond basin to 100 zoospores/µL and collected all surviving tadpoles ten days after the initial exposure to understand infection during the earliest developmental stages. Atkinson (2016) exposed leopard frogs between Gosner stages 25-

40 following the same protocol as Cook (2008), to understand infection during the later developmental stages of tadpoles. Both researchers found that, excluding eggs, mortality decreased with increased developmental stage at exposure. However, each experiment used a different reference for tadpole development at the time of exposure, and these experiments did not sufficiently overlap in the developmental stages observed, presenting us with a knowledge gap in host susceptibility.

There is no known tolerance threshold of host susceptibility to infection of

Dermomycoides sp. and we have an incomplete understanding of the effects of gopher frog developmental stage on mortality associated with infection of Dermomycoides sp. A common assumption in parasite-host relationships is that the rate of infection is proportionate to the quantity of the parasite (Ben-Ami, Regoes, & Ebert, 2008). I designed this experiment to better understand if infection quantity of zoospores of

Dermomycoides sp. and mortality of tadpole hosts follow this infection model. This experiment was replicated using both gopher frogs and leopard frogs in order to

determine if there was a species-specific response. I focused on eggs, hatchlings, and tadpoles 2 weeks post-hatching in order to encompass the stages used by Cook (2008) and Atkinson (2016). I hypothesized tadpoles exposed at earlier developmental stages would have higher mortality than tadpoles exposed at later developmental stages. I also examined how the quantity of zoospores that tadpoles were initially exposed to affected average days to mortality. I hypothesized that tadpole mortality would increase with increasing zoospore quantity, and that gopher frog tadpoles would experience higher mortality than leopard frog tadpoles. I based my hypothesis on the assumption that because of their small population size gopher frogs have a lower genetic diversity than leopard frogs and should be more susceptible to infection. The overall purpose of this study was to understand susceptibility and sensitivity of dusky gopher frog tadpoles to infection of zoospores of Dermomycoides sp. in a laboratory setting.

Methods

Collection of animal and infectious material

I collected gopher frog eggs from a portion of three egg-clutches laid in the same pond within 48 hours of deposition. Eggs were placed in small plastic containers with approximately 300 mL filtered water used to cover the eggs while maximizing oxygen availability and taken to a laboratory at the Harrison Experimental Forest (HEF), where they were washed in filtered water to remove large contaminants. After being rinsed, the eggs were kept in 42.5 cm x 30.2 cm x 17.8 cm plastic boxes (henceforth referred to as frog boxes) and monitored for growth and mortality. Once at the developmental stage needed for each experiment, the animals were placed in small, lidded, plastic containers

(approximately 12 cm x 8 cm deep) filled with enough water to cover the eggs and

maximize oxygen availability and transported to the Gulf Coast Research Laboratory

(GCRL). Before the tadpole groups were placed into the experiment, a sample of 1-2 eggs or tadpoles from each clutch was analyzed for contamination with Dermomycoides sp. using qPCR.

Infectious material of Dermomycoides sp. was extracted from the liver and intestine of dead infected dusky gopher frog tadpoles, homogenized into a slurry, added to 500 mL of 6.5 ppt NaCl, and then re-refrigerated at 4 o C until needed for the experimental exposure. Spores of Dermomycoides sp. were processed into infectious zoospores from the refrigerated material. Before exposure, the slurry was filtered through a 0.2 µL filter using a vacuum pump, dried for 1 Hr. in a desiccation chamber, placed in

50 mL of clean 6.5 ppt NaCl, and rehydrated for several hours to maximize the number of spores hatching into infectious zoospores. The number of zoospores in each dose was determined by counting zoospores from a known concentration. Five cells were randomly selected and counted within the center square of a hemocytometer and averaged. This number was then multiplied by 25 for the number of total cells in the center square and finally multiplied by 10 for the final concentration (Equation 1). This process was repeated four times, and the average of the counts was used for the final zoospore/µL of the solution.

n zoospores 25 cells x zoospores (1) ( ) ∗ ( ) ∗ 10 = 5 cells 0.1µL square µL

Experiment 1. Investigation of effects of gopher frog developmental stage at the time of infection on morbidity

Experimental treatments consisted of gopher frogs exposed as eggs (between

Gosner stages 1-10), embryos (between stages 11-19), hatchlings (between stages 20-22), 12

and 2-weeks post-hatching (at stage 25; Fig. 2.1), and unexposed controls. Ten eggs from each of three different clutches were placed in 2.5 L aquaria in a separate, quarantined room to serve as additional control groups. Both in-room and out-of-room control groups were randomly selected at the same time as tadpoles being experimental exposed and were separated when eggs were collected for the first experimental exposure. The control groups were exposed to filtered water while the exposed eggs were placed in infected water.

Figure 3. Gosner stages for amphibian development (Gosner, 1960) used in this experiment.

Once the larvae grew to the developmental stage needed for their treatment group,

10 randomly selected individuals from each of the three egg clutches were removed from their frog box and placed in a 1000 mL plastic bowl containing 500 ml of 40 zoospore/µl stock solution. After 1 hour, the animals were removed from the bowl with a sterile

pipette and placed into a 10-L aquarium (Fig 2.2). This was replicated three times, with each replicate group representing a different egg mass. Two- weeks after initial exposure, one individual was collected from each replicate for a mid-point sample, and the remaining animals were collected 4- weeks after their initial exposure for the end-point sample. Each replicate group was kept in a different 26 o C water bath, and tadpoles were monitored daily for morbidity and mortality.

Figure 4. Tank map for experiment 1.

Replicates were separated by egg mass (E.M.), and each treatment consisted of 10 tadpoles randomly assigned to and maintained in

10-L aquaria. Aquaria (represented by brown boxes) were maintained in water baths (blue boxes) to keep at a temperature of 26 o C. A second control group was kept in a separate quarantined control room to monitor the level of baseline infection in the egg masses. Dead or morbid animals (morbidity was defined as bloated and non-responsive to touch) were removed and preserved in either 90 % EtOH or 10 % non-buffered formalin for molecular or histological examination, respectively, with a minimum of a 9:1 ratio of the respective preservation models based on tissue condition at the time of collection.

Tadpoles used for molecular examination were stored in a -20 o C freezer before DNA extraction of the liver and intestine. A portion of the liver and intestine was extracted and 14

analyzed for the quantity of Dermomycoides sp. present in the tadpole at the time of collection using qPCR, following the protocol described for molecular analysis below.

The average days to mortality were analyzed for significance, as described for statistical analysis below.

Experiment 2. An investigation into the relationship between zoospore exposure concentration and infection of gopher frog and leopard frog tadpoles.

As soon as the tadpoles maintained at the HEF developed to Gosner stage 25

(spiracle formed to the left and obvious mouthparts), each of the three egg-clutches used in each experiment was randomly assigned to a replicate group. Five tadpoles, from each replicate egg-clutch, were randomly assigned to one of five exposure treatments (out-of- room control, in-room control, 250, 500, or 750 zoospores/µL). Each group of five tadpoles was placed into a small plastic container (12 x 12 x 8 cm) with 350 mL filtered water and transported to GCRL.

The infectious zoospore solution was diluted with filtered water from the HEF to create the experimental exposure treatment (250, 500, or 750 zoospores/µL). A homogenous aliquot of 500 mL of each exposure solution was pipetted into a 600 mL plastic bowl, and five tadpoles were placed into the exposure solution for their treatment.

Tadpoles were left in the bowls for 30 minutes before they were removed to their plastic containers and taken to the Maturation Laboratory at GCRL, where each group of tadpoles was placed into a 1.5 L plastic container with approximately 1 L of water. In- room and out-of-room control groups were exposed to filtered water while the other treatments were exposed to infected water. Placement of individual aquaria on shelving after exposure was randomly assigned within each replicate.

Tadpoles were kept at 70o F air temperature, with UVB lights for 8 hours a day, fed one Hakkari algal wafer once a week, and checked twice a day. Morbid and dead tadpoles were removed, placed in a 2 mL microcentrifuge tube, and preserved in 90%

EtOH. One tadpole was removed from each treatment group 2 weeks after the initial exposure for the midpoint sample, and the remaining tadpoles were removed 4weeks after the initial exposure for the end-point sample. The liver and intestine of preserved tadpoles were extracted and analyzed for infection of Dermomycoides sp. using qPCR.

Molecular analysis

Tadpoles livers and intestines were analyzed using qPCR to test the prevalence and intensity of infection of individuals across treatments. The qPCR sequence used was probe 6-FAM-5'- ACC GTG TTG ATC GAG GCA TT-3' denatured for 2 min at 95 o C, followed 36-cycles of 30 s at 95 o C, 30 s at 55 o C, 2 min at 72 o C, and an extension period of 10 min at 72 o C (Karwacki, Atkinson, Ossiboff, & Savage, 2018). The amount of Dermomycoides sp. present per 1 µL of each sample was calculated by dividing the mean quantity measured by the total amount of DNA/µg observed via nanodrop. This quantity was compared with the qPCR results from the representative samples taken initially.

Statistical analysis

I used a linear mixed model with package "lme4" version 1.1-23 (Bates, Machler,

Bolker, & Walker, 2015) in R studio version 3.6.1 to investigate the association between the developmental stage and zoospore exposure at the time of initial exposure and days to mortality. P values were calculated using the Satterthwaite's method, where the mean of each individual treatment is compared individually to the model mean (Kuznetsova,

Brockhoff, & Christensen, 2017). This analysis provides us individual p-values for each treatment in the model. Shapiro-Wilk normality test and Levene's test for homogeny of variance were analyzed to examine if the use of parametric statistics were appropriate.

Because each block represented a different egg clutch, analysis of block effect was used to investigate for differences in mortality due to clutch differences. Tukey's HSD was used as a post hoc analysis of the differences between average days to mortality among treatment groups.

For the first experiment, the dependent variables analyzed were: days to mortality and infection level at the experimental endpoint. Days to mortality was determined as the number of days after experimental exposure until the tadpole was observed as morbid, dead, or the experiment ended for that treatment. Infection level at experimental endpoint was calculated using the results of my molecular analysis. The fixed effect for these models was developmental stage exposed and the random effect was the clutch that represented each replicate.

For the second experiment the dependent variable was average days to mortality.

The fixed effects for the model were treatment and replicate number, and the random effect was clutch. I also analyzed for any interaction between treatment, replicate number, and egg clutch.

Results

Average days to mortality across developmental stages

The average days to mortality was significantly different among developmental stage exposure groups (see Table 2.1). Tadpoles exposed and hatchlings and 2-weeks post-hatching had the lowest days to mortality (Table 2.1 and Fig. 5). Tukey HSD

showed no significant difference between hatchlings and tadpoles exposed 2-weeks post- hatching (p= 0.99). The average days to mortality in eggs and embryos were both significantly different from hatchlings (Fig. 2.4). There were also significant differences in tadpoles exposed 2 weeks post-hatching and all other treatment groups, including both control groups (Fig. 2.4).

Table 1 Average days to mortality in dusky gopher frog tadpoles exposed at different developmental stages.

Tadpoles were tracked for 4 weeks after exposure to zoospores of Dermomycoides sp. P-values represent significance for each individual treatment using the Satterthwaite's method in lmerTest.

Figure 5. Cumulative mortality of tadpoles at different days.

Individuals across all three replicates were combined and used to calculate percent mortality across the experimental period. Each line started on the day that the treatment group was experimentally exposed and ended 28 days after the initial exposure.

Days to mortality to Days 5

0 0 Out Control1 Control2 Eggs3 Embryo4 Hatchling5 6 Post-Hatching

Figure 6. Average days to the mortality of dusky gopher frog tadpoles exposed to 50 zoospores/µL at different developmental stages.

Each developmental stage group consisted of 10 tadpoles and was replicated three times. An additional out of room control group was kept in a quarantined room to assure the lack or level of contamination that the tadpoles already had. Tadpoles were monitored for morbidity and mortality twice daily for 28 days after the initial exposure of their developmental stage group.

Average infection level across developmental stage groups detected by qPCR

The average number of zoospores at the end of the experiment was not significantly different among developmental stage groups (Fig. 2.5). There was also no significant difference in the number of zoospores detected by qPCR in subjects at the end of the experiment across developmental stages exposed.

Figure 7. Average infection quantity across developmental stages at the time of collection as detected using qPCR.

The average infection level was determined by the number of copies of Dermomycoides sp. in each sample, divided by the amount of

DNA in the sample, and averaged across the treatment group. Mortality across water baths containing different numbers of zoospores

All water baths containing zoospores of Dermomycoides sp. had lower days to mortality compared with the control groups across both gopher frog and leopard frog 20

tadpoles. Mortality of gopher frogs was significantly lower in the in-room control group among gopher frogs, and only the additional out-of-room control group had significantly lower mortality among leopard frog treatment groups (Table 2.2). Cumulative mortality of individual gopher frog and leopard frog tadpoles showed higher mortality in tadpoles exposed in water baths containing 750 zoospores/µL than all other treatments (see Fig.

2.6 and Fig. 2.7).

Table 2 Average days to mortality in individual tadpoles exposed to zoospores of

Dermomycoides sp.

P-values represent significance for each individual treatment using the Satterthwaite's method in lmerTest.

Figure 8. Cumulative mortality of individual gopher frog tadpoles exposed to baths with different numbers of zoospores of Dermomycoides sp. (0, 250, 500, 750).

Tadpoles that died within the first and second days after the initial exposure were omitted from analysis because mortality may have been caused by electrical exposure of misfiring wiring in the laboratory. It is also unlikely that the mortality event was caused by handling stress or stress associated with the experimental exposure as tadpoles in the out-of-room control group did not experience mortality, whereas tadpoles in the in-room control group did. If there had been mortality in the out-of-room control group as well, this mortality would be attributed to handling stress. And because tadpoles in the in-room control group experienced mortality I was able to rule out stress associated with the infection process. Tadpoles were monitored twice daily, and all tadpoles were collected 28 days after the initial exposure.

Figure 9. Cumulative mortality of individual leopard frog tadpoles exposed to baths with different numbers zoospores of Dermomycoides sp. (0, 250, 500, and 750).

Their initial zoospore exposure organized individuals, and all surviving tadpoles were collected 28 days after the initial exposure. 22

There was no significant species or clutch effect on average days to mortality across water bath treatments. Overall mortality was higher in exposed gopher frog versus exposed leopard frog tadpoles (mean of 25 days and 27 days, respectively). However, there were no statistically significant differences between the two species and average days to mortality (t value = -0.09). Average days to mortality varied across treatments among the species in tadpoles exposed to 500 or 750 zoospores/µL (Fig. 2.8).

Figure 10. Average days to the mortality of gopher frog (R. sevosa) and leopard frog tadpoles (R. sphenocephala) exposed to zoospore water baths.

All gopher frogs that died within the first two days after initial exposure were omitted from analysis because electrical failure instead of infection might have caused their mortality. Tadpoles were monitored twice daily, and morbid tadpoles were removed; all surviving tadpoles were collected 28 days after the initial infection. Discussion

Host developmental stage

Contrary to our hypothesis, gopher frogs exposed at earlier developmental stages were less susceptible to zoospores of Dermomycoides sp. This result may be due to the 23

jelly capsule layer surrounding the eggs serving as a protective barrier against infection

(Robert & Ohta, 2009). While mortality was high in eggs that are directly injected with ranavirus, animals exposed in water baths had the lowest mortality of all developmental stage groups (Haislip, Gray, Hoverman, & Miller, 2011). Similarly, eggs of R. temporaria exposed to Polycyclic Aromatic Hydrocarbons had higher mortality when exposed while the jelly capsule surrounding the eggs was removed (Marquis,

Guittonneau, Millery, & Miaud, 2006). Even though embryos are still enveloped in the jelly capsule, the structure of this protective barrier changes as early as the elongation from eggs to embryo state (Salthe, 1963). We hypothesize that the structural reshaping of the jelly capsule during elongation into the embryo state is what caused higher mortality in the gopher frogs exposed as embryos vs. eggs.

Gopher frogs appear to have a similar mortality response to the infection of zoospores as leopard frog tadpoles used in prior work. Despite using half of the zoospore dosage used both Atkinson (2018) and Cook (2006), 40 zoospores/µL vs 100 zoospores/µL gopher frogs exposed around the same developmental stages had lower survival than leopard frogs (Fig. 2.9). We observed an exception in the survival of gopher frog eggs vs. leopard frog eggs. This difference is either due to the lower exposure dose, or the structural make-up of gopher frog jelly capsules, which are more compact than leopard frog egg masses in the wild population. The difference in survival between leopard frog tadpoles and gopher frog tadpoles exposed at Gosner stage 25 is likely due to a lack of genetic variation in the gopher frog. Low genetic variation of the gopher frog may predispose their tadpoles to lower MHC allele expression, which is already limited during metamorphosis (De Jesus Andino et al., 2013; Robert & Ohta, 2009). MHC alleles

are responsible for immune system functions and regulation. Until metamorphosis, there is no consistent expression of these genes, causing susceptibility to infection to fluctuate.

In populations that experience loss of genetic diversity and allele richness, a reduction of

MHC alleles can also occur (Belasen, Bletz, Leite, Toledo, & James, 2019). This reduction in MHC expression increases susceptibility to infection and associated mortality. Future research into the gene expression of infected gopher frog and leopard frog tadpoles between Gosner stages 25-30 would likely show dramatic differences in alleles expression between the two species.

Figure 11. The average survival of gopher frog tadpoles at different developmental stages exposed to 50 zoospores/µL in experiment 1 of this thesis compared to different developmental stages of leopard frog tadpoles exposed 100 zoospores/µL by Atkinson (2016) and Cook (2008)

Average survival is grouped by the developmental stage at which the tadpoles were exposed, and each color corresponds to a different author. The developmental stage at initial exposure was decided by hours after collection in Cook (2008), and Gosner staging was used by Atkinson (2016). The average mortality in all three experiments was lowest among hatchlings and tadpoles just after hatching.

Fluctuations in the survival of tadpoles at different developmental stages may reflect immune system fluctuations associated with metamorphosis. Tadpole's response to the infection of Dermomycoides sp. does not appear to be a linear relationship and is likely influenced by fluctuations in the immune system structure during metamorphosis. Immune system studies of Xenopus laecis show fluctuations in

the adaptive immune system throughout metamorphosis (Lau, Igawa, Komaki, & Satta,

2020; Rollins-Smith, 1998). β-lymphocyte populations were found to vary throughout metamorphosis and decreased during the later stages of metamorphosis. This decrease is coupled with the organogenesis of the tadpole, which culminates in the restructuring of the immune system from the tadpole stage to the adult stage (Robert & Ohta, 2009).

Because the immune response is being restructured while the tadpole host is infected, the host's ability to tolerate the parasite will decrease during these periods of low immunity.

Zoospore exposure

There does not appear to be a tolerance threshold to tadpoles exposed to different doses of zoospores of Dermomycoides sp., in either this study or our preliminary study

(Appendix A). Gopher frog tadpoles exposed to water baths containing between 0 and

250 zoospore/µL in a preliminary study also showed no significant difference in days to mortality. Three groups of 10 tadpoles were exposed to water baths containing 0, 49, 75,

100, and 250 zoospores/µL, following the same protocol as described above. These tadpoles were left to develop for 3 months, fed Hakkari algae wafer once a week, and monitored daily for mortality. There was no significant difference in average days to mortality across any of the developmental treatment groups, including 250 zoospores/µL

(Appendix A).

I believe that the conditions of the maturation laboratory may have influenced the results of my second experiment exposing tadpoles to a range of zoospores of

Dermomycoides sp. Faulty electrical wiring in the exposure room observed through investigator shock at the beginning of the experiment may have also caused mortality in tadpoles across all treatment groups. Because mortality of tadpoles within the first 2 days

of the experiment may have been caused by exposure to faulty electrical wiring and not zoospore exposures, we chose to remove the animals that died within the first 48 hours of the experiment across all treatments.

Quantitative PCR of Dermomycoides sp. detected in hosts does not appear to be related to exposure quantity. Across all zoospore treatment groups in both this experiment and our preliminary experiment, the quantity of Dermomycoides sp. detected was not significantly different at the end-point collection period. Other studies using

Osteopilus septentrionalis exposed to water baths containing between 0 and 100 zoospores/µL also found that exposure had no direct relationship with infection level at the experimental end-point (M. S. Atkinson & Savage, 2020; Blow, Atkinson, & Savage,

2020). These results are contrary to our hypothesis that there would be a negative linear relationship between exposure and mortality. We believe that while exposure does lead to infection, the host response to this infection is most likely proportional to the initial exposure quantity. Because the immune function is metabolically taxing, tadpole hosts may only be responding to the parasite threat level present at the initial exposure and then tapering off once they are at equilibrium with the parasite (Kirschman, Quade, Zera, &

Warne, 2017).

Future work

Future studies analyzing the other divers of mortality associated with

Dermomycoides sp. in amphibian hosts should focus on susceptibility at later developmental stages as well as post-metamorphic tolerance. After metamorphosis, the gopher frogs migrate from the pond basin to find their permanent terrestrial burrow where they will spend approximately 90% of their adult lives. Understanding if these

adults retain a low level of infection like tadpoles that are exposed in water baths can help broaden our understanding of carrier vectors and the persistence of this pathogen in breeding ponds. The only amphibian whose immune system has been studied thoroughly throughout metamorphosis is Xenopus laecis, and as such, should be considered as a host species for understanding the effects of Dermomycoides sp. at different infection levels.

CHAPTER III - ACQUIRED RESISTANCE TO DERMOMYCOIDES SP. IN

PREVIOUSLY INFECTED DUSKY GOPHER FROG TADPOLES

Several years of near-zero recruitment of juvenile gopher frogs in the wild population have been attributed to infection of Dermomycoides sp. in the pond. Unlike years of low recruitment caused by premature pond drying, these events are marked by high but not total mortality of all tadpoles (Joseph H K Pechmann et al., 2008).

Commonly, not all hosts that are exposed to a parasite or pathogen will become infected; furthermore, not all hosts that are infected will experience mortality caused by the infection. The processes by which hosts avoid or mitigate infection is described as resistance and tolerance. Resistance is the ability of a host to avoid or decrease the pathogen burden, whereas tolerance is the ability of the host to mitigate the detrimental effects of the infection (Miller, White, & Boots, 2006; Vredenburg, Knapp, Tunstall, &

Briggs, 2010). Typical resistance strategies are avoidance of the pathogen, shortened recovery after infection, or remaining immunity post-infection (Boots, 2008; Miller et al.,

2006). Resistance targets the fitness of the pathogen so that when the pathogen load is constant, the number of parasites that can infect the host is decreased (Boots, 2008; Rohr et al., 2010). If the host is unable to resist the pathogen, tolerance strategies work to mitigate the physical damage caused by the infection. These strategies include tissue repair and a more extended period of survival at lethal infection loads than for non- tolerant hosts (Blaustein et al., 2005; Wilber, Knapp, Toothman, & Briggs, 2017). Both resistance and tolerance can affect the host-pathogen relationship because of the trade- offs involved in each strategy (D A Zelmer, 1998). Understanding what strategies used by the surviving tadpoles after a mortality event caused by Dermomycoides sp. can help

improve understanding of the host species if, for example, the surviving individuals were able to acquire resistance.

Acquired resistance is the ability of a host to change behavior, activate an immune response, or reduce infection loads upon multiple exposures within the individual's lifetime (McMahon et al., 2014). Multiple periods of rapid pond drying and refilling within a tadpole’s lifetime can result in multiple hatching events of zoospores of

Dermomycoides sp. The multiple hatching events that tadpoles are likely subjected to throughout their metamorphosis validates the requirement of multiple exposures throughout the individual's lifetime(M. Atkinson, 2016; Cook, 2008). However, acquired resistance can only be achieved when the pathogen infects the host to a certain level

(Richmond, Savage, Zamudio, & Rosenblum, 2009). This infection threshold is activated when the pathogen overwhelms the host innate immune system, and thus triggers the adaptive immune system. Because the infection has to exceed the host tolerance threshold, there are associated costs to the hosts' health, and mortality may occur on an individual level (Kirschman et al., 2017; Lochmiller & Deerenberg, 2000). These trade- offs can include a nutritional cost, metabolic burden, or increased predation risk of the host (Kirschman et al., 2017; Lochmiller & Deerenberg, 2000). Prolonged infection of the disease can increase the hosts' risk of predation or starvation, such as adult frogs increasing time outside of the burrow basking in response to Bd infection (Richmond et al., 2009). The energy required of the immune system of metamorphosing tadpoles can also limit the energy required for foraging resulting in a metabolic cost. In tadpoles, metabolic cost results in smaller body size, an extended larval period, or disruption to the gut microbiome (Knutie, Wilkinson, Kohl, & Rohr, 2017; Mesquita et al., 2017).

Morbidity and mortality are avoided through acquired resistance in the individual, but the population response to the pathogen can still result in low survival.

To understand how acquired resistance affects an individual tadpole infected with

Dermomycoides sp., we must understand the intersection of incidence, mortality, and survival (Ellis et al., 2014). Incidence is the occurrence of a specific disease, mortality is death caused by that specific disease, and survival is death from any cause after the incidence. The incidence of Dermomycoides sp. infection could be a zoospore hatching event caused by the pond rapidly filling. Mortality is the death of an individual caused by only Dermomycoides sp., and survival is the death of any tadpoles after the initial infection. When a pathogen is rapidly lethal, mortality can serve as a proxy for incidence, as is the case for mass mortality events attributed to infection of

Dermomycoides sp. observed in field surveys (Green et al., 2002; Isidoro-Ayza, Lorch, et al., 2019). While an individual is either alive or dead (mortality), survival aims to reflect the course of the disease after a known incidence. A mass mortality event of several individuals can increase the overall population survival for that year because of the increase in resource availability (Ainley & DeMaster, 1980). Mortality affects the individual and can be influenced by age, sex, and life-history traits (Ainley & DeMaster,

1980; McDowell et al., 2008; Pike, Pizzatto, Pike, & Shine, 2008). Understanding how mortality and survival are affected by a disease incidence can allow for better management decisions (Haines, Tewes, & Laack, 2005). If gopher frog tadpoles that acquire resistance to Dermomycoides sp. have higher survival in subsequent years than adults not exposed as tadpole’s population reinforcement efforts through head-started individuals may be curbed to allow for long term benefits.

The intersection of incidence, mortality, and survival of Dermomycoides sp. needs to be conducted in an experimental laboratory setting. Laboratory experiments can measure the progress of a disease at a given time because they can follow all individuals exposed at the same time (Ellis et al., 2014). To date, there have been no experiments examining if tadpoles infected with Dermomycoides sp. can acquire resistance to later exposure if they survive the initial exposure. Also, very few studies have used wild- caught animals in the examination of resistance research (Raffel, Lloyd-Smith, Sessions,

Hudson, & Rohr, 2011; Rohr et al., 2010). I used wild bred gopher frog tadpoles to investigate if tadpoles exposed to an initial high, low, or zero zoospore dose can acquire resistance to a second high or low dose. I also wanted to investigate the relationship between survival and mortality of infected individuals. I hypothesized that tadpoles exposed to an initial dose of zoospores would acquire resistance to a challenge dose and that tadpoles exposed to an initial high dose would be more resistant than those exposed to an initial low dose. I also hypothesized that mortality and survival would be highly correlated, given prior studies and field observations (M. Atkinson, 2016; Aurélie

Chambouvet et al., 2015; Cook, 2008; Gleason et al., 2014; Green et al., 2002; Isidoro-

Ayza, Lorch, et al., 2019). The purpose of this study was to inform future management models for years with near-zero natural juvenile recruitment in the wild dusky gopher frog population. If tadpoles can acquire resistance and survive multiple infections, this will help managers better model the future population under different scenarios (Haines et al., 2005).

Methods

Tadpoles were selected from three single egg clutches head-started at the HEF and taken to the GCRL for experimental exposure. Each egg clutch originated from the same pond and was maintained in 15 L plastic containers filled with filtered water. Water quality, tadpole development, and mortality were monitored every other day. When tadpoles developed to Gosner stage 25, they were separated into 27 groups of ten tadpoles and transferred to 500 mL square plastic boxes filled with approximately 350 mL filtered water to prevent mortality during transport to GCRL. Then 250 mL of water was removed from each container of tadpoles, and the initial exposure dose was randomly assigned to the containers. Tadpoles were exposed to 150 mL of water containing the initial zoospore dose exposure quantity.

Experimental doses were created from a stock solution of zoospores collected from dead, infected gopher frogs and approximated using a hemocytometer. The liver and intestine of dead and infected tadpoles were extracted, ground into a homologous slurry, desiccated in a vacuum chamber for 1-hour, and then resuspended in 6.5 ppt NaCl. The slurry was shaken, and four different representative samples were examined by counting the number of zoospores present in the center square of the hemocytometer. This concentration was then used to calculate the number of zoospores/ µL in the stock solution. The concentration of zoospores/µL was approximated once a day until it exceeded the highest experimental exposure quantity needed. The amount of stock solution that needed to be added to each container for the experimental exposure was calculated, and exposure groups were randomly assigned.

The experimental doses approximated 0 zoospores/µL, 50 zoospores/µL, and 250 zoospores/µL. Tadpole groups were exposed for 30 minutes and then placed into five 2.5

L aquaria specific to their initial treatment group (for experimental design see Fig. 3.1).

Tadpoles were monitored daily for morbidity and mortality and fed one Hakari algae wafer once a week. Two weeks after the initial exposure, tadpoles were removed from their aquaria divided into three groups of ten tadpoles and exposed to a second challenge dose of zoospores (Fig. 3.1 and 3.2).

Figure 12. Gopher frog tadpoles were exposed to an initial and challenge dose of zoospores

Exposure doses approximated 0 zoospores/µL (zero), 50 zoospores/µL (low), and 250 zoospores/µL (high), with a 2-week separation between the initial and challenge doses. The experimental exposure followed the same protocol as the initial exposure, and one tadpole from each the initial dose group was collected for the midpoint sample just

before the challenge dose of approximately 0 zoospores/µL, 50 zoospores/µL, and 250 zoospores/µL. After the exposure to the challenge dose, each group of ten tadpoles was placed in a new 5-L aquarium specific to their treatment (Fig. 3.2). Each treatment was replicated three times, and each replicate came from a different egg clutch with all tadpoles in that replicate representing the same egg clutch. Tadpoles were monitored daily for morbidity and mortality and fed once a week, and any morbid tadpoles were collected and preserved in either 95% EtOH or 10% non-buffered formalin.

Figure 13. Tank map of tadpole groups exposed to a challenge dose of zoospores.

Treatments are read as Initial-Challenge, (H) is high, (L) is low, and (0) for no exposure. Each replicate represented a different egg mass, and additional control groups for each egg mass were placed in a separate, quarantined room. Two weeks after the challenge exposure, all remaining tadpoles were killed and analyzed for infection of zoospores for our end-point sample. The liver and intestine of each tadpole was extracted and either sectioned for histology or analyzed using qPCR.

Average days to mortality were analyzed statistically in RStudio version 3.6.1 using the

'survival,' 'Rmisc,' 'tidyverse,' and 'GGgally' packages. Significance was tested using a

linear mixed model under package "lmer4". The fixed effects were the initial treatment, challenge treatment, and interaction of the initial and challenge treatment, and the random effects were block and egg mass. Tukey HSD was then used as a post hoc test to determine differences among individual treatments.

Results

Days to mortality

Analysis of average days to mortality across all treatments showed no significant differences among or between initial and challenge treatment groups (Table 3). Across all treatments, days to mortality was shortest in tadpoles that received a low initial dose of zoospores (Fig. 14). Average days to mortality in tadpoles that received a low initial zoospore exposure were approximately 2.85 days shorter than tadpoles that received a high initial dose, and 2.08 days shorter than tadpoles received no initial exposure.

Treatment groups that received a high initial zoospore exposure had the second-longest days to mortality, and no mortality was observed in the out of room control group.

Survival

Analysis across treatment groups was significantly different across treatments only after the challenge dose exposure. When initial and challenge treatments were for survival to the endpoint, initial, and challenge dose treatments were significantly different

(Table 3.1). Tukey HSD showed that only tadpoles that received a low initial and high challenge (L-H) dose were significantly different from tadpoles that received a zero initial and low challenge (L-O) dose (p = 0.02), see Figure 3.3.

Table 3 ANOVA summary for survival to experimental endpoint in tadpoles that received an initial and challenge dose treatment.

Figure 14. Survival to the endpoint of all initial and challenge dose treatment combinations.

Treatments are labeled H (high), L (low), and (O) for zero dose exposures, with the initial treatment listed first and the challenge treatment second. Tukey HSD showed significant differences in average survival between L.H. and O.L. (p = 0.02). A midpoint sample of one tadpole was taken from each replicate treatment two weeks after the initial exposure dose and directly before the challenge exposure dose. All surviving tadpoles were collected 28 days after the initial exposure.

Molecular results

There were no statistically significant differences between treatment groups and the intensity of infection observed by qPCR. Infection intensity was also not statistically significant when analyzing survival (p = 0.643, SE = 7.731e-13) or days to mortality (p =

0.426, SE = 1.331e-11). Neither days to survival nor days to mortality became statistically significant when outliers were removed (p = 0.122, SE = 0.0001; and p =

0.356, SE = 0.0018 respectively).

Discussion

Tadpoles that received a low initial dose appeared to be less resistant to a challenge dose, and tadpoles that received a high initial dose appeared to be more resistant to a challenge dose exposure. Average days to mortality after the challenge dose was lowest across all treatments with an initial low exposure dose (Fig. 3.4). Tadpoles that received no initial zoospore dose and a low challenge dose had the shortest days to the mortality of that initial treatment group. Inversely, tadpoles that received a high initial dose experienced longer days to mortality when exposed to a low or high challenge dose.

The high initial dose of zoospores may have triggered the immune system's use, which is typically shut off during metamorphosis to allow for growth. If the higher initial dose activated the immune system, this could trigger an adaptive resistance response in the tadpoles to the second challenge dose exposure. This would also explain why tadpoles that received a high initial dose and no challenge dose had the highest days to mortality across that treatment. This is because an additional metabolic strain of an adaptive response in the tadpoles received the challenge dose (Lochmiller & Deerenberg, 2000).

While not statistically significant, this trend of heightened susceptibility in low exposure

treatment groups is notable for wildlife managers hoping to catch a mortality event when it occurs.

Figure 15. Average days to the mortality of tadpoles exposed to an initial and challenge dose of zoospores of Dermomycoides sp., post-exposure to the challenge dose.

Tadpoles were initially exposed to either a high, low, or zero doses of zoospores (approximating 250 zoospores/µL, 50 zoospores/µL, or 0 zoospores/µL respectively), allowed to develop for two-weeks then exposed to a second high, low, or zero doses. A second control group was kept in a separate room separated from experimentally exposed tadpoles (green line).

Survival to endpoint was influenced by the initial and challenge dose groups used, but there was no interaction effect between initial and challenge dose treatments. Across all challenge dose treatments, tadpoles that received a low initial dose treatment had the lowest survival to end-point (Fig. 5). Conversely, across all initial dose treatments, tadpoles that received a high challenge treatment had the lowest survival to end-point

(Fig. 16). The low survival in tadpoles that received a low initial dose treatment reflects the lower days to mortality across all treatments, but there is less of a trend in tadpoles that received a high challenge dose. The lack of significance in the survival of tadpoles that received a zero-initial dose across all challenge dose groups warrants future exploration. However, by comparing the survival of tadpoles that received a high challenge dose across all initial treatment groups to tadpoles that received both a high initial and challenge dose, we see that the survival curves are similar (Fig. 3.5).

Figure 16. Survival to the experimental endpoint of tadpoles that received a high (H), low (L), or zero (O) exposure dose of zoospores of Dermomycoides sp.

a) shows survival by initial dose groups. b) shows survival by challenge dose groups. Exposure did not equate infection levels at endpoint, and egg clutches vary in their ability to respond to infection. The infection level varied among both clutches infected and individuals within each clutch (Table 3.2). Overall average infection level observed by qPCR increased from midpoint to end-point when all individuals were included but decreased when outlier individuals, defined as individuals infected above

1x106, were removed (Fig. 3.6). This difference displays the variability in infection and associated resistance among individuals. Tadpoles that can tolerate or resist the infection of Dermomycoides sp. showed an overall decrease in infection between the two-week mid-point and four-week endpoint. Outliers were re-analyzed using qPCR in triplicate and verified before statistical analysis. The verification of these values demonstrates the variability among individuals' susceptibility and ability to resist initial infection. The decline in infection levels when these highly susceptible individuals are removed,

coupled with the increase in both in-room and out-of-room control groups, indicates an adaptive response in infected individuals.

Table 4 Average infection load of tadpoles in each replicate egg clutch at experimental mid-point and endpoint.

Figure 17. Average infection level among initial treatment groups at experimental midpoint and endpoint when outliers were removed from the analysis.

Resistance and tolerance strategies appeared to be used by all tadpole groups, including both control groups. In both the in-room and out-of-room control groups, the low infection levels increased from the midpoint and end-point sample periods. The dramatic difference between the in-room versus the out-of-room control group's infection level may be explained by the difference in the associated stress of being in the same room as infected individuals. Another possibility is that tadpoles kept as in-room controls were exposed to more zoospores via air exchange. This unintentional exposure may have caused an adaptive immune response explaining both the mortality observed in our in- room control groups coupled with low infection and high infection loads in the out-of- room control groups with no mortality. In this way, our in-room control groups could resist infection, whereas the out-of-room control groups tolerated the infection.

Dusky gopher frog tadpoles appear to acquire resistance to a later challenge dose, but initial exposure level is crucial in overall survival to acquire resistance. Treatment groups with a low initial exposure level had the shortest days to mortality. The lowest 43

survival had higher infection levels than the control groups and had lower infection levels than the high initial treatment groups. This difference in infection, survival, and mortality is likely due to the metabolic cost of mounting an immune response. While the control groups can tolerate or resist the infection, and tadpole exposed to a high initial dose can acquire resistance to a later high dose, tadpoles infected with a low dose appear to be unable to resist or tolerate infection. The low dose of Dermomycoides sp. may not be enough to activate an immune response, but also be too much for that tadpole's natural ability to tolerate infection, thus causing high mortality and low survival of infected individuals.

Conservation biologists should focus on understanding baseline levels of infection in breeding ponds and seasonal fluctuations in infection levels at these sites. Years of prolonged water retention in ponds could contribute to a slow increase in infection levels at these sites. Years of low recruitment could be caused if these levels exceed what tadpoles are naturally able to tolerate. Gopher frog ponds are also subject to prescribed burning on a 2-3-year basis, years where the pond basin can be burnt could reduce the infection load to a level that tadpoles can tolerate. Multiple drying and filling events within the pond, due to unstable hydrology may cause higher mortality due to repeated infections mimicking a low-high, or high-high exposure. This type of hydroperiod is known to occur at New Pond and may explain the high mortality rates observed by

Atkinson (2016). Future work should evaluate other environmental stressors on individuals infected with a baseline level from the natal pond to explore what factors mitigate the tadpoles' ability to tolerate infection.

CHAPTER IV – SUMMARY

Sensitivity and susceptibility to infection of Dermomycoides sp. vary between species and developmental stage. Prior studies using leopard frogs confirm that there are differences in gopher frogs' susceptibility and leopard frogs when exposed to

Dermomycoides sp. (M. Atkinson, 2016; Cook, 2008). Leopard frogs exposed to 100 zoospores/µL experienced fluctuations in mortality associated with developmental time, but gopher frogs exposed to 50 zoospores/µL only experienced similar mortality. I used a lower exposure dosage if gopher frogs were more susceptible than leopard frog tadpoles due to their lowered genetic diversity (Hinkson, Henry, Hensley, & Richter, 2016;

Hinkson & Richter, 2016; Richter, Crother, & Broughton, 2009). Because mortality levels of gopher frogs infected with half the exposure dose used by Atkinson (2016) and

Cook (2008) with leopard frogs showed similar results, gopher frogs are likely more susceptible as a species than leopard frogs.

When exposure dosage is high (> 250 zoospores/µL), there was no significant difference between exposed gopher frogs and leopard frog tadpoles. High exposure dosages likely exceed natural tolerance levels of both species, causing similar mortality patterns. Because gopher frogs and leopard frogs share similar life-history traits such as breeding season, larval period, and breeding ponds within a metapopulation, each species' generational exposures to Dermomycoides sp. should be indistinguishable. The heightened susceptibility of gopher frogs versus leopard frogs would explain years of low recruitment in the gopher frog population coupled with lowered recruitment in leopard frogs at the same time. Dipnet surveys after a mortality event in gopher frog breeding ponds typically yield almost no gopher frogs and a drastically reduced number of leopard

frog tadpoles. Further work should focus on interacting with the two species cohabitating within infected mesocosms to understand the ecological drivers behind these field observations.

The infection of Dermomycoides sp. can be tolerated by the host and help establish resistance to future infections. A parasite's adult form has to live within the host and uses the host as a habitat for growth and reproduction (Derek A Zelmer, 1998).

Tadpoles exposed to a low initial dose are least able to resist a later exposure to

Dermomycoides sp. This result implies that contrary to the assumption that single large- exposure events cause mass mortality events, they are likely the result of a prolonged low-level exposure that increases over time. Gopher frogs use ephemeral ponds that fill and retain water due to rain events (Thurgate & Pechmann, 2007). During periods of heavy rainfall, the pond's pH can fluctuate dramatically, causing an increase in zoospore hatching (Cook, 2008). While these hatching events expose tadpoles to a large dose of zoospores, years where the ponds retain water and the ponds' pH stay relatively stable, allowing for slow and continuous hatching of zoospores may be more lethal to the overall host survival. In 2018, Glen's Pond remained full for 11 consecutive months; during this time, the pond pH slowly fluctuated between 4.42 and 6. Of the 86 gopher frog egg masses observed in the pond that breeding season, only two tadpoles survived to metamorphosis. While this is only a single event, it may be an indicator of a larger pattern that we are only beginning to understand. Future work should examine the abiotic factors of these ponds, such as water and soil temperature, to analyze fluctuations in zoospore levels compared to rain events.

This thesis is the first study of Dermomycoides sp. using gopher frog tadpoles.

While I believe it is worthwhile to replicate these experiments with zoo-hatched gopher frogs, as they should not have any baseline infection, I do not believe studies need to use wild-hatched gopher frogs. Many of my results were non-significant due to baseline infection levels of wild-caught tadpoles, and I do not believe that a lack of baseline infection is possible. This recommendation does not negate the need to understand how

Dermomycoides sp. affects gopher frogs directly, only that use of wild-hatched animals should continue to be limited. All animals used in these experiments had to be killed and cannot contribute to future breeding events. With approximately 200 breeding adults in

Glen's Pond metapopulation, our work should focus on understanding the disease ecology within the breeding ponds. Head-starting programs should be maintained as they supplement the population in years of low recruitment. As the mechanisms, this pathogen becomes more understood; these programs may allow us to breed tadpoles selected to be less susceptible to infection (Rohr et al., 2010). Further study of this pathogen is essential to the gopher frog and global amphibian conservation efforts' long-term recovery.

APPENDIX A – Preliminary study on infection threshold tolerance of dusky gopher frog

tadpoles

Three different gopher frog egg clutches were collected from Glen's Pond and reared at the Harrison Experimental Forest until they reached Gosner stage 25 (obvious mouthparts and spiracle formed on the left). The tadpoles were then transported to the

Gulf Coast Research Laboratory Wet Laboratory in small frog boxes, where they were separated into five different exposure groups. The exposure groups approximated 0, 25,

50, 75, 100, 150, and 200 zoospores/µL. Once randomly assigned to an exposure group,

10 tadpoles were placed in 300 mL of water contaminated with the exposure dose.

Tadpoles were kept in exposure containers for 1 hour before they were removed from the exposure water and placed into 2.5 L aquaria.

The tadpoles were kept in a water bath maintained at 26 o C and monitored once a day for morbidity and mortality. Morbid tadpoles were removed and preserved in 70%

EtOH or formalin. The experiment was run for 103 days, at which point all remaining tadpoles were killed. All individuals were analyzed for infection level at the experimental endpoint using qPCR.

The average days to mortality were not significantly different among treatment groups, and survival was only significantly different from the lowest and highest treatment groups. There was no significant difference in infection levels of groups at the experimental endpoint. Only tadpoles exposed at 250 or 0 zoospores/µL were significantly different for survival to endpoint (p = 0.04).

Average days to mortality in gopher frog tadpoles exposed between 0-250 zoospores/µL. There were no significant differences in days to mortality across treatment groups.

Average survival to end-point of tadpoles exposed between 0-250 zoospores/µL. Survival was only significantly different between tadpole exposed to 0 zoospores/µL and 250 zoospores/µL (p= 0.04).

REFERENCES

Ainley, D. G., & DeMaster, D. P. (1980). Survival and mortality in a population of adelie penguins. Ecology, 61(3), 522–530. doi:10.2307/1937418

Atkinson, M. (2016). The effects of the protist parasite Dermomycoides sp. on the dusky

gopher frog (Rana sevosa) and the southern leopard frog (Rana sphenocephala)

(Master thesis). Western Carolina University.

Atkinson, M. S., & Savage, A. (2020). Understanding the implications of Perkinsea

infection in Cuban treefrogs (Osteopilus septentrionalis). Presented at the

Southeast Partners of Reptiles and Amphibians.

Bates, D., Machler, M., Bolker, B., & Walker, S. (2015). lme4 (1.1-23). Computer

software, Journal of Statistical Software.

Belasen, A. M., Bletz, M. C., Leite, D. da S., Toledo, L. F., & James, T. Y. (2019). Long-

Term Habitat Fragmentation Is Associated With Reduced MHC IIB Diversity and

Increased Infections in Amphibian Hosts. Frontiers in ecology and evolution, 6.

doi:10.3389/fevo.2018.00236

Blaustein, A. R., Gervasi, S. S., Johnson, P. T. J., Hoverman, J. T., Belden, L. K.,

Bradley, P. W., & Xie, G. Y. (2012). Ecophysiology meets conservation:

understanding the role of disease in amphibian population declines. Philosophical

Transactions of the Royal Society of London. Series B, Biological Sciences,

367(1596), 1688–1707. doi:10.1098/rstb.2012.0011

Blaustein, A. R., Romansic, J. M., Scheessele, E. A., Han, B. A., Pessier, A. P., &

LONGCORE, J. E. (2005). Interspecific Variation in Susceptibility of Frog

Tadpoles to the Pathogenic Fungus Batrachochytrium dendrobatidis.

Conservation Biology, 19(5), 1460–1468. doi:10.1111/j.1523-1739.2005.00195.x

Blow, M., Atkinson, M. S., & Savage, A. (2020). Using eDNA to test whether Cuban tree

frogs (Osteopilus septentrionalis) can amplify the amphibian pathogen Perkinsea.

Presented at the Southeast Partners of Reptiles and Amphibians.

Boots, M. (2008). Fight or learn to live with the consequences? Trends in Ecology &

Evolution, 23(5), 248–250. doi:10.1016/j.tree.2008.01.006

Chambouvet, Aurélie, Gower, D. J., Jirků, M., Yabsley, M. J., Davis, A. K., Leonard, G.,

… Richards, T. A. (2015). Cryptic infection of a broad taxonomic and geographic

diversity of tadpoles by Perkinsea protists. Proceedings of the National Academy

of Sciences of the United States of America, 112(34), E4743–51.

doi:10.1073/pnas.1500163112

Chambouvet, Aurelie, Smilansky, V., Jirků, M., Isidoro-Ayza, M., Itoïz, S., Derelle, E.,

… Richards, T. A. (2020). Diverse alveolate infections of tadpoles, a new threat

to frogs? PLoS Pathogens, 16(2), e1008107. doi:10.1371/journal.ppat.1008107

Cook, J. O. (2008). Transmission and Occurrence of Dermomycoides sp. in Rana sevosa

and other ranids in the north central gulf of mexico states (Master thesis).

De Castro, F., & Bolker, B. (2004). Mechanisms of disease-induced extinction. Ecology

Letters, 8(1), 117–126. doi:10.1111/j.1461-0248.2004.00693.x

De Jesus Andino, F., Chen, G., Li, Z., Grayfer, L., & Robert, J. (2013). Susceptibility of

Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with

a reduced and delayed innate immune response in comparison with adult frogs,

432(2), 435–443.

De Jesús Andino, F., Chen, G., Li, Z., Grayfer, L., & Robert, J. (2012). Susceptibility of

Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with

a reduced and delayed innate immune response in comparison with adult frogs.

Virology, 432(2), 435–443. doi:10.1016/j.virol.2012.07.001

Ellis, L., Woods, L. M., Estève, J., Eloranta, S., Coleman, M. P., & Rachet, B. (2014).

Cancer incidence, survival and mortality: explaining the concepts. International

Journal of Cancer, 135(8), 1774–1782. doi:10.1002/ijc.28990

Gleason, F. H., Chambouvet, A., Sullivan, B. K., Lilje, O., & Rowley, J. J. L. (2014).

Multiple zoosporic parasites pose a significant threat to amphibian populations.

Fungal Ecology, 11, 181–192. doi:10.1016/j.funeco.2014.04.001

Gosner, K. (1960). A Simplified Table for Staging Anuran Embryos and Larvae with

Notes on Identification. Herpetologica, 16(3), 183–190.

Green, D. E., Converse, K. A., & Schrader, A. K. (2002). Epizootiology of sixty-four

amphibian morbidity and mortality events in the USA, 1996-2001. Annals of the

New York Academy of Sciences, 969, 323–339. doi:10.1111/j.1749-

6632.2002.tb04400.x

Haines, A. M., Tewes, M. E., & Laack, L. L. (2005). Survival and sources of mortality in

ocelots. Journal of Wildlife Management, 69(1), 255–263. doi:10.2193/0022-

541X(2005)069<0255:SASOMI>2.0.CO;2

Haislip, N. A., Gray, M. J., Hoverman, J. T., & Miller, D. L. (2011). Development and

disease: how susceptibility to an emerging pathogen changes through anuran

development. Plos One, 6(7), e22307. doi:10.1371/journal.pone.0022307

Hanski, I., & Simberloff, D. (1997). The metapopulation approach, its history, conceptual

domain, and application to conservation. In Metapopulation Biology (pp. 5–26).

Elsevier. doi:10.1016/B978-012323445-2/50003-1

Heard, G. W., Scroggie, M. P., Ramsey, D. S. L., Clemann, N., Hodgson, J. A., &

Thomas, C. D. (2017). Can habitat management mitigate disease impacts on

threatened amphibians? Conservation letters, 11(2). doi:10.1111/conl.12375

Hinkson, K. M., Henry, N. L., Hensley, N. M., & Richter, S. C. (2016). Initial founders

of captive populations are genetically representative of natural populations in

critically endangered dusky gopher frogs, Lithobates sevosus. Zoo Biology, 35(5),

378–384. doi:10.1002/zoo.21309

Hinkson, K. M., & Richter, S. C. (2016). Temporal trends in genetic data and effective

population size support efficacy of management practices in critically endangered

dusky gopher frogs (Lithobates sevosus). Ecology and evolution, 6(9), 2667–

2678. doi:10.1002/ece3.2084

Isidoro-Ayza, M., Grear, D. A., & Chambouvet, A. (2019). Pathology and case definition

of severe perkinsea infection of frogs. Veterinary Pathology, 56(1), 133–142.

doi:10.1177/0300985818798132

Isidoro-Ayza, M., Lorch, J. M., Ballmann, A. E., & Businga, N. K. (2019). Mass

Mortality of Green Frog ( Rana clamitans) Tadpoles in Wisconsin, USA,

Associated with Severe Infection with the Pathogenic Perkinsea Clade. Journal of

Wildlife Diseases, 55(1), 262–265. doi:10.7589/2018-02-046

Isidoro-Ayza, M., Lorch, J. M., Grear, D. A., Winzeler, M., Calhoun, D. L., &

Barichivich, W. J. (2017). Pathogenic lineage of Perkinsea associated with mass

mortality of frogs across the United States. Scientific Reports, 7(1), 10288.

doi:10.1038/s41598-017-10456-1

Johnson, P. T. J., Kellermanns, E., & Bowerman, J. (2011). Critical windows of disease

risk: amphibian pathology driven by developmental changes in host resistance and

tolerance. Functional ecology, 25(3), 726–734. doi:10.1111/j.1365-

2435.2010.01830.x

Karwacki, E. E., Atkinson, M. S., Ossiboff, R. J., & Savage, A. E. (2018). Novel

quantitative PCR assay specific for the emerging Perkinsea amphibian pathogen

reveals seasonal infection dynamics. Diseases of aquatic organisms, 129(2), 85–

98. doi:10.3354/dao03239

Kirschman, L. J., Quade, A. H., Zera, A. J., & Warne, R. W. (2017). Immune function

trade-offs in response to parasite threats. Journal of Insect Physiology, 98, 199–

204. doi:10.1016/j.jinsphys.2017.01.009

Knutie, S. A., Wilkinson, C. L., Kohl, K. D., & Rohr, J. R. (2017). Early-life disruption

of amphibian microbiota decreases later-life resistance to parasites. Nature

Communications, 8(1), 86. doi:10.1038/s41467-017-00119-0

Kuznetsova, A., Brockhoff, P. B., & Christensen, R. H. B. (2017). lmertest package: tests

in linear mixed effects models. Journal of statistical software, 82(13).

doi:10.18637/jss.v082.i13

LaClaire, L. V. Endangered and Threatened Wildlife and Plants; Final Rule To List the

Mississippi Gopher Frog Distinct Population Segment of Dusky Gopher Frog as

Endangered, RIN 1018-AF90 50 CFR § 17 62993–63001 (2001).

Landsberg, J. H., Kiryu, Y., Tabuchi, M., Waltzek, T. B., Enge, K. M., Reintjes-Tolen,

S., … Pessier, A. P. (2013). Co-infection by alveolate parasites and frog virus 3-

like ranavirus during an amphibian larval mortality event in Florida, USA.

Diseases of aquatic organisms, 105(2), 89–99. doi:10.3354/dao02625

Lau, Q., Igawa, T., Komaki, S., & Satta, Y. (2020). Expression Changes of MHC and

Other Immune Genes in Frog Skin during Ontogeny. Animals : an open access

journal from MDPI, 10(1). doi:10.3390/ani10010091

Lochmiller, R. L., & Deerenberg, C. (2000). Trade-offs in evolutionary immunology: just

what is the cost of immunity? Oikos (Copenhagen, Denmark), 88(1), 87–98.

doi:10.1034/j.1600-0706.2000.880110.x

Marquis, O., Guittonneau, S., Millery, A., & Miaud, C. (2006). Toxicity of PAHs and

jelly protection of eggs in the Common frog Rana temporaria. Amphibia-Reptilia,

27(3), 472–475. doi:10.1163/156853806778189945

Marsh, D. M., & Trenham, P. C. (2001). Metapopulation dynamics and amphibian

conservation. Conservation Biology, 15(1), 40–49. doi:10.1111/j.1523-

1739.2001.00129.x

McCallum, H., & Dobson, A. (1995). Detecting disease and parasite threats to

endangered species and ecosystems. Trends in Ecology & Evolution, 10(5), 190–

194. doi:10.1016/s0169-5347(00)89050-3

McCullough, D. R. (1996). Metapopulations and wildlife conservation.

books.google.com.

McDowell, N., Pockman, W. T., Allen, C. D., Breshears, D. D., Cobb, N., Kolb, T., …

Yepez, E. A. (2008). Mechanisms of plant survival and mortality during drought:

why do some plants survive while others succumb to drought? The New

Phytologist, 178(4), 719–739. doi:10.1111/j.1469-8137.2008.02436.x

McMahon, T. A., Sears, B. F., Venesky, M. D., Bessler, S. M., Brown, J. M., Deutsch,

K., … Rohr, J. R. (2014). Amphibians acquire resistance to live and dead fungus

overcoming fungal immunosuppression. Nature, 511(7508), 224–227.

doi:10.1038/nature13491

Mesquita, A. F. C., Lambertini, C., Lyra, M., Malagoli, L. R., James, T. Y., Toledo, L. F.,

… Becker, C. G. (2017). Low resistance to chytridiomycosis in direct-developing

amphibians. Scientific Reports, 7(1), 16605. doi:10.1038/s41598-017-16425-y

Miller, M. R., White, A., & Boots, M. (2006). The evolution of parasites in response to

tolerance in their hosts: the good, the bad, and apparent commensalism.

Evolution, 60(5), 945–956. doi:10.1111/j.0014-3820.2006.tb01173.x

Pechmann, Joseph H K, Scott, D. E., Semlitsch, R. D., Caldwell, J. P., Vitt, L. J., &

Gibbons, J. W. (2008). Declining Amphibian Populations : The Problem of

Separating Human Impacts from Natural Fluctuations Published by : American

Association for the Advancement of Science Stable URL :

http://www.jstor.org/stable/2878941. Advancement Of Science, 253(5022), 892–

895.

Pechmann, Joseph H.K. (2016). Research on conservation and recovery of the

endangered dusky gopher frog (Federal Report). U.S. Fish and Wildlife.

Pechmann, Joseph H.K. (2019). How research has contributed to proactive, adaptive, and

responsive management of the endangered dusky gopher frog. In Staying on PAR:

Using Proactive, Adaptive, and Responsive Management. SEPARC.

Pike, D. A., Pizzatto, L., Pike, B. A., & Shine, R. (2008). Estimating survival rates of

uncatchable animals: the myth of high juvenile mortality in reptiles. Ecology,

89(3), 607–611. doi:10.1890/06-2162.1

Raffel, T. R., Lloyd-Smith, J. O., Sessions, S. K., Hudson, P. J., & Rohr, J. R. (2011).

Does the early frog catch the worm? Disentangling potential drivers of a parasite

age-intensity relationship in tadpoles. Oecologia, 165(4), 1031–1042.

doi:10.1007/s00442-010-1776-0

Richmond, J. Q., Savage, A. E., Zamudio, K. R., & Rosenblum, E. B. (2009). Toward

immunogenetic studies of amphibian chytridiomycosis: linking innate and

acquired immunity. Bioscience, 59(4), 311–320. doi:10.1525/bio.2009.59.4.9

Richter, S. C., Crother, B. I., & Broughton, R. E. (2009). Genetic Consequences of

Population Reduction and Geographic Isolation in the Critically Endangered Frog,

Rana sevosa. Copeia, 2009(4), 799–806. doi:10.1643/CH-09-070

Robert, J., & Ohta, Y. (2009). Comparative and developmental study of the immune

system in Xenopus. Developmental Dynamics, 238(6), 1249–1270.

doi:10.1002/dvdy.21891

Rohr, J. R., Raffel, T. R., & Hall, C. A. (2010). Developmental variation in resistance and

tolerance in a multi-host-parasite system. Functional ecology, 24(5), 1110–1121.

doi:10.1111/j.1365-2435.2010.01709.x

Rollins-Smith, L. A. (1998). Metamorphosis and the amphibian immune system.

Immunological Reviews, 166, 221–230. doi:10.1111/j.1600-065x.1998.tb01265.x

Salthe, S. N. (1963). The egg capsules in the amphibia. Journal of Morphology, 113,

161–171. doi:10.1002/jmor.1051130204

Soul, M. E. (1998). POPULATION genetics:enhanced: no need to isolate genetics.

Science, 282(5394), 1658–1659. doi:10.1126/science.282.5394.1658

Thurgate, N. Y., & Pechmann, J. H. K. (2007). Canopy closure, competition, and the

endangered dusky gopher frog. Journal of Wildlife Management, 71(6), 1845–

1852. doi:10.2193/2005-586

Vredenburg, V. T., Knapp, R. A., Tunstall, T. S., & Briggs, C. J. (2010). Dynamics of an

emerging disease drive large-scale amphibian population extinctions. Proceedings

of the National Academy of Sciences of the United States of America, 107(21),

9689–9694. doi:10.1073/pnas.0914111107

Wilber, M. Q., Knapp, R. A., Toothman, M., & Briggs, C. J. (2017). Resistance,

tolerance and environmental transmission dynamics determine host extinction risk

in a load-dependent amphibian disease. Ecology Letters, 20(9), 1169–1181.

doi:10.1111/ele.12814

Zelmer, D A. (1998). An evolutionary definition of parasitism. International Journal for

Parasitology, 28(3), 531–533. doi:10.1016/s0020-7519(97)00199-9

Zelmer, Derek A. (1998). Definition of Parasitism. Journal of Parasitology, The, 28.