Effects of Temperature on Batrachochytrium dendrobatidis Infection of Amphibians

Alyssa Carroll

3/31/15

Joseph O’Connor

Dr. Christopher Binckley

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Abstract

One third of all amphibian species are listed as endangered, and Batrachochytrium dendrobatidis (Bd), is a chytrid fungus causing mass mortality of amphibians. Perhaps started by the global amphibian trade, it is linked to mass die offs of amphibians on every continent. Temperature is the primary factor regulating its spread, allowing prediction of Bd movement and which specific amphibian populations are at risk. Temperature is correlated to infection prevalence, and is the most consistent factor in the persistence of Bd. Some amphibian species are resistant to infection, possibly helping spread Bd by acting as reservoirs. As explained by the naïve prey hypothesis, populations of amphibians previously unexposed to Bd are dying quickly, as they have little immunity to this novel pathogen. Studies done by Sapsford et al, 2013, Piovia-Scott et al, 2011, and Savage et al, 2011 all indicated an optimal temperature range for Bd growth of between 17 C° and 23 C°. Treatment and conservation tactics should consist of mapping possible areas of outbreaks and prophylactically treating populations with antifungals, or collecting species for preservation in captivity.

Overview

There are over 6,000 species of frogs on the earth. Recently, frog numbers have dwindled

due to a fungus from the Chytrid family called Batrachochytrium dendrobatidis (Bd).

Batrachochytrium dendrobatidis is a fungus that is killing frogs at an alarming rate. Thought to

have been spread by the global amphibian trade, Bd may have originated in Africa and has now

spread so widely it is found everywhere frogs are found. While other factors are attributed to

frog declines like habitat destruction and climate change, infectious diseases like fungal Bd or

viruses like the Ranaviruses which also kills frogs, are coming to the forefront of research. Bd

can spread very fast through an environment causing mass mortalities in frog populations in a

short amount of time. Bd was only recently identified in 1998, and it has been attributed to a

number of amphibian extinctions.

The frog life cycle begins as tadpoles which live in water and are mainly herbivorous. As

tadpoles grow into adults, they go through a process called metamorphosis and grow legs and

losing their tail. As they grow into adults they move onto land, becoming carnivorous, eating

insects and other small organisms. Frogs play a crucial role in our ecosystem by keeping both the

ecosystem and humans healthy. Frogs eat insects like mosquitos that infect humans with deadly

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diseases like Malaria and West Nile Virus. Since adult frogs live on both land and in water, they

can tell us about the health of the environment they live in as they are in such close proximity

with it. Frogs are also ectothermic, meaning they cannot regulate their own body temperature;

they rely on the environment to warm or cool their bodies. They are dependent on their

environments temperature for normal physiological function, and a small imbalance can throw

that homeostatic balance off.

Batrachochytrium dendrobatidis is a fungus that reproduces through spores that travel

from frog to frog causing disease. Bd causes an overproduction of skin cells, resulting in

thickening of the skin. This thickening is detrimental to frogs since they use their skin for

respiration and secretion of fluids. When their skin thickens it disrupts the frog’s ability to

maintain normal body function and eventually leads to death. Some frogs show natural immunity

to the fungus, like the Australian Green Tree frog. Unfortunately that is not true for all frog

species, as Bd has been linked to about 200 species extinctions in recent years. Since Bd has the

ability to kill many frogs in a short amount of time, figuring out how Bd spreads and how to stop

it is crucial to prevent further loss of amphibians. Treatment for sick frogs is being tested and has

been shown to work in captive frogs, but when applied to wild habitats are not as effective. In

captivity, sick frogs can be treated with hot water baths at temperatures high enough to kill off

the fungus, also so does a limited number of antifungal medications.

Recent studies point to temperature as a main factor in the spread of Bd, with optimal

temperature for growth between 17-23 C°. This is useful for planning effective conservation

tactics to prevent spread of Bd. With the knowledge of the fungus’s optimal environment, we can

predict where future outbreaks will occur; allowing researchers to prophylactically treat or

remove frogs from the environment before an outbreak of Bd. Organizations like Amphibian Ark

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collect threatened species of frogs to keep in captivity to reestablish populations decimated by a

Bd infection, when possible. Knowing how the disease spreads, understanding its dependence on

temperature for growth, and developing treatment and preventative methods can help us bring

frog populations back up to normal numbers.

Introduction

Amphibian populations have seen severe declines in recent years. Emerging infectious

disease is one of the leading causes of declines recently, along with habitat destruction, climate

change and introduced predator species. There are many types of pathogens that can harm

amphibians like viruses belonging to the Iridoviridae family, called Ranaviruses which have

caused major mortalities of many amphibian species (Gray, Miller and Hoverman, 2009). A

bacterial pathogen, Aeromonas hydrophila, has been tied to large die offs of the Mountain

yellow-legged frog (Rana muscosa), by causing massive internal hemorrhage and death (Hill et

al, 2010). A recently discovered infectious disease is Batrachochytrium dendrobatidis, identified

only in 1998 by Berger at al, (1998) has been linked to declines in frog populations on every

continent where amphibians are found. Batrachochytrium dendrobatidis may also be tied to

salamander declines (Rovito et al, 2009). Currently one third of all amphibian species are on the

International Union for the Conservation of Natures Red List as threatened (IUCN 2008

Conference). While habitat destruction is the most common reason for amphibian population

declines, protected habitats are becoming infected with Bd at an alarming rate (Skerratt et al,

2007).

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The ecosystem is a complex system of relationships between biotic and abiotic factors.

One very important group of animals that play a major role in keeping the ecosystem in balance

are frogs. There is estimated to be about 6,509 species of frogs, mostly found in tropical regions

but spreading as far north as North America (AmphibiaWeb). Over the last few decades

approximately one third of those species have become threatened or extinct, attributed to the

spread of Batrachochytrium dendrobatidis, known as Chytrid fungus or Bd (Wake &

Vredenburg, 2008). Frogs play a crucial role in keeping the ecosystem healthy and the

conservation of frog species is important for the environment and for humans. Multiple

treatments are in use for infected frogs that range from treating individual frogs to treating whole

populations at once. The spread of Chytrid fungus is a crucial topic of research that needs

attention in order to develop strategies capable of stopping further declines of frog populations.

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Figure 1. A- Histology of a skin secretion from a White’s Treefrog (Litoria caerulea), showing heaving Chytrid fungus infection. I- Immature stage of zoosporangium. D- Mature zoosporangium containing zoospores, where discharge papillae are visible. Arrow- Empty zoosporangia after zoospores have discharged. E- Epidermis. Figure from Berger et al. (1998). B. Scanning electron microscopy of toe skin surface from Lesurer’s Frog (Litoria lesueuri) showing heavy Chytrid fungus infection. One plugged discharge papilla is visible at the surface of each epithelial cell. Figure from Berger et al. (2005). Both figures reproduced from Amphibiaweb.

The frog life cycle includes aquatic development of eggs into tadpoles, and eventually

maturation into terrestrial adults. Over their life cycle, frogs are exposed to a variety of different

environmental factors: they are both aquatic and terrestrial, have different prey as tadpoles and

adults, are thermos-conformers, meaning they are sensitive to temperature changes, and the

permeability of their skin makes them sensitive to changes in water or air quality. Frogs are

important to the ecosystem as they often act as bio-indicators. This means the health of a

population of frogs can be used to gauge the health of the body of water they live in and even

show early signs of pollution. Tadpoles also are vital to the water systems they live in as most

tadpoles are filter feeders that filter microscopic particles out of the water, helping to maintain

water quality. Tadpoles help reduce nitrogen input in the water by feeding on nitrogen fixing

algae, reducing natural eutrophication, which promotes excessive algal growth.  As adults, frogs

prey on many insects including mosquitoes which carry deadly human diseases like West Nile

Virus and Malaria. A decrease in frog populations can lead to a rapid increase of insect

populations which can be harmful to humans and agriculture. Frogs are important parts of the

food chain as prey for other animals in their environments (Mohneke & Rodel, 2009).

Batrachochytrium dendrobatidis itself is an infection found on the skin of frogs that

impairs normal skin function (Carver, et al., 2010). The fungus causes a disruption in gene

expression of the genes that transcribe for collagen, fibrinogen, keratin and elastin, which are all

involved in maintaining skin integrity (Rosenblum et al,. 2012). Disrupting the integrity of a

frogs’ skin can interfere with critical functions like cutaneous respiration and expelling excess

solutes. Laboratory tests revealed infected frogs had problems with fluid and electrolyte balances

and therefore suffered from dehydration (Rosenblum et al, 2012). This is thought to be because

of the disruption of transport of fluid across the skin. The fungus itself has two life stages: an

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immobile reproductive stage, and a motile zoospore stage. The fungus undergoes sporulation

inside the skin of an infected individual and produces mobile zoospores that can go on to infect

new frogs. There is evidence that the amount of zoospores found on the skin of an infected frog

has a correlation with how severe the infection becomes

(Voyles et al., 2012). Eventually, the disruption of

homeostasis causes cardiac arrest and death (Voyles et al.,

2009). Some frogs have shown innate immunity to Bd

(Woodhams et al, 2010). Some species are found to have

natural, built in defenses like skin peptides that protect

them from infection (Roseblum, 2012). Southern Toads

(Anaxyrus terrestris) and Wood frogs (Lithobates

sylvaticus) have had lower mortality rates even with a high

number of zoospores present on the skin (Searle et al.,

2011).  Australian Green Tree frogs have skin peptides

that act as a defense against the fungus by limiting the intensity of the infection (Woodhams et

al., 2010). Unfortunately this is not true for all amphibian species and Chytrid fungus has been

attributed to the extinction of at least 200 species (Skerratt et al., 2007). New treatments

attempting antifungal use and electrolyte and fluid rebalancing are currently being tested.

Researchers are actively trying to find a successful treatment (Harris, 2009).

Aside from habitat destruction, Bd is one of the major causes of amphibian population

declines. The overall effects of any animal going extinct are wide ranging and harmful to the

environment. Extinctions cause a cascade effect, and can cause extinctions of other species by

secondary processes (Sodhi, 2009). Extinctions can cause an overabundance of a prey species

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that can proliferate when relived of predation pressure, and predators of the species can die out

due to lack of food or be forced to switch to another prey population, and cause stress of the new

prey population.

One of the most studied areas of frog populations is in the Sierra Nevada Mountains of

California. Of the seven species of frogs found and well documented from the area, five of them

are currently threatened. Yellow-legged frogs (Rana

mucosa) which were pervasive throughout the Sierra

Nevada when documented in the 1980’s have now

disappeared from more than 90% of their documented

range. They are currently listed as endangered on the

IUCN (International Union for the Conservation of

Nature) Red List of threatened species (Wake &

Vredenburg, 2008).  Fellers et al., (2007) tried to

reestablish populations of yellow-legged frogs that saw

a severe population decline and reestablishment failed.

While Chytrid fungus was not tested for in the dead

frogs, it was later found to be one of the only plausible reasons for the failure due to the patterns

of amphibian die offs at the study sites. There is also an argument that global warming may be a

driver of the infection, as warmer temperatures seem to be conducive to fungal growth and

spread (Wake & Vredenburg, 2008).

It is thought that Chytrid fungus was first spread as a result of the international pet trade.

International trade of animals can be millions a year (Schlaepfer et al., 2005). Previously

unharmed populations were exposed to non-native, infected individuals that spread the fungus

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Figure 3. Dead southern mountain yellow-legged frogs (Rana mucosa) killed by Chytrid Fungus at Sixty Lake Basin, Kings Canyon National Park, Califonia USA. Reproduced from sciencedaily.com. Photo Credit Vance T. Vredenburg.

and caused declines of endemic populations (Fisher et al., 2007). Chytrid fungus is now found in

43 countries, and has been attributed specifically to the decline and extinction of the Panamanian

Gold Frog, the Wyoming Frog, and the Australian gastric-brooding frog, among others (Skerret

et al, 2007). Although most parasitic diseases do not cause total extinction of a host population

because most need the symbiotic relationship with the host to live, it is believed that Bd can

cause total extinctions because it has a reservoir species that can carry, but not be affected by the

fungus (Garmyn et al, 2012). Garmyn et al., (2012) showed that geese could act as reservoirs for

Bd, living on the toes of geese which are heavily keratinized. Since amphibians and waterfowl

normally coexist in their environment, it is plausible that waterfowl are acting as reservoirs for

the fungus, allowing continual infection of a population of amphibians until the population dies

out. Reservoirs are an important aspect of the etiology of Bd infection since they allow the

fungus to persist in an environment even when conditions are unfavorable for the fungus.

There is evidence that a specific temperature range ideal for Bd growth. This could

explain why the fungus thrives in certain regions of the world, and could allow prediction of

populations at risk for infection (Sapsford et al., 2013). Although the spread of Chytrid fungus

internationally is thought to most likely be caused by humans and international trading, the

natural mechanism of how Chytrid fungus spreads in populations is questionable. As Chytrid

fungus is a threat to the ecosystem as a whole, it is vital to study the mechanism of how the

fungus moves and thrives in environments, and leads to declines of frog populations. In this

paper, I suggest temperature is the primary regulating factor of Bd infection in amphibian

populations.  

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Current Investigations

Batrachochytrium Dendrobatidis (Bd), is a Chytrid fungus that alters genes in the skin

causing an overproduction of epidermis, which in turn leads to an imbalance of fluid and

electrolyte levels and ultimately death (Rosenblum, 2012). Infection has been attributed to the

decline and/or extinction of almost 200 species of frogs, secondarily affecting the water quality,

algal levels, and abundance of

mosquitoes, and other pest animals, like

spiders when their natural predator are

gone (Mohneke, 2009). The pet trade

may explain the spread of Bd to

relatively secluded places. Reservoir

hosts, suspected to be tadpoles or other

frog species immune to Bd, may keep

the fungus present in the environment,

even under unfavorable conditions

(Fischer, 2007),.  

Current studies are looking at

how Bd is spread in the environment

and how it persists. Studies have

examined connections between

temperature, site type, and seasonal

dynamics to the spread of Bd, and

possible reservoir species keeping the disease present post amphibian decline. The aim of these

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Figure 4 Proportion of environmental air temperatures in ranges relevant to Batrachochytrium dendrobatidis growth at one high site, one contiguous low site, and one non-contiguous low site. Figure 1 reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Figure 1

current investigation articles are understanding the etiology and pathogenesis of disease in frogs

contracting this fungus both endemically in their own habitats, but also how it spreads to new

novel places.

A study done by Sapsford et al, (2013) sampled adult common mist frogs (Litoria

reocola) over one year from six sites: two at high elevation, two at low elevation contiguous with

different high elevation sites, and two low elevation sites noncontiguous with higher elevations,

all in the Australian wet tropics bioregion in northern Queensland, Australia.  All frogs were

swabbed and tested for Bd, but

only information from adult

male frogs was used since most

data collected was from adult

males. The study also measured

intensity of the infection in

zoospores, which are motile

spores produced by the fungus

and is how Bd is spread. Air

temperatures were measured at

each site. The results of the study

revealed a clear correlation

between the prevalence of Bd

infections and temperature.

High elevation sites had cooler temperatures than both lower sites, contiguous and

noncontiguous. High elevation temperatures were on average less than 17 °C, which slows Bd

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Figure 5 Relationship between prevalence and air temperature. As air temperature increases, the proportion of infected adults declines. Temperatures higher than 23 °C have been seen to kill Bd fungus. Figure 2 reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Figure 2.

growth, or between 17 C° and 23 C° during the summer months which is optimal temperature

range for growth. Temperatures at both low elevation sites were the same and substantially

warmer than the high elevation sites, on average between 23 °C and 28 °C on average,

sometimes reaching 30 °C during the summer season, which kills Bd (Figure 4, reproduced from

Aquatic Connectivity Affects Bd Disease Dynamics, Sapsford et al, 2013). Across all sites and

temperatures there was a negative correlation between mean seasonal temperature and Bd

prevalence in adult male frogs; prevalence of Bd was lower at higher air temperatures (Figure 5,

reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Sapsford et al, 2013). Since

temperatures at high elevations were optimal the mean intensity of infection stayed constant. The

data between season and site type for high elevation sites and contiguous low sites did not differ

significantly, indicating that the effects of temperature or season on Bd prevalence or infection

intensity did not differ between the two site types. Results revealed the mean intensity of

infection stayed constant throughout the year at higher elevation sites. Intensity of infection was

influenced by season, temperature and site. Low sites connected to higher elevation areas by

water flow had a higher prevalence of infection. While prevalence was higher at higher elevation

sites due to optimal temperatures, low elevation sites contiguous with them had higher

prevalence than non-contiguous low elevation sites which sometimes reached temperatures high

enough to kill Bd. The prevalence of infection approached zero at both types of low elevation

sites in the summer months and in autumn when temperatures occasionally reached 30 °C, while

prevalence at high elevation sites remained well above zero in the summer and autumn (Figure 6

reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Sapsford et al, 2013).

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Figure 6 Prevalence of Bd in adult frogs calculated at 2 high sites, 2 contiguous low sites, and 2 non-contiguous low sites. Across all 3 site types prevalence was lower in summer and autumn when temperatures on average were higher. Figure 3 reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Figure 3

Figure 7 shows intensity of Bd in adult frogs at 2 high sites, 2 contiguous low sites, and 2 non-contiguous low sites, measured in zoospores. Intensity of infection was constant at high elevations with optimal temperatures for Bd growth. Figure 4 reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Figure 4.

The seasonal pattern of prevalence varied significantly between high and non-contiguous

low sites, suggesting that zoospore drift, or movement of cooler water from higher elevation sites

attribute to the higher prevalence of infection during summer and autumn months at the

contiguous low sites. Season patterns also affected intensity at the different locations (Figure 7

reproduced from Aquatic Connectivity Affects Bd Disease Dynamics, Sapsford et al, 2013).

Patterns at high elevation sites and contiguous low sites did not differ significantly. This study

suggests that reservoir hosts are most likely a vital part in persistence of Bd at contiguous and

noncontiguous low sites, since prevalence fell near zero in summer and autumn but increased

again in the winter. This research team suggests tadpoles, adults of other frog species, or the

environment such as water or soil, may act as reservoirs (Sapsford et al, 2013).

A second study done by Piovia-Scott et al, (2011) looked at the cascade frogs (Rana

cascadae) in the Klamath Mountains of California. The cascade frogs have recently experienced

large declines in other parts of California, Bd has been found in many of those populations.

Piovia-Scott et al, (2011) wanted to examine the distribution of Bd in the cascade frog

populations in these mountains. In California, the cascade frogs are found in two mountains

ranges: the Cascades and the Klamath mountain ranges. The Cascade mountain frog populations

have gone through severe declines in recent years with populations dwindling down to just a few

remaining populations. In contrast, the Klamath Mountains still have thriving populations.

Surveyors looked at 105 sites between June and September of 2008, and seven in June of 2009.

At each body of water up to 20 frogs at each stage of life were weighed, measured, and tested for

Bd. For tadpoles, the insides of mouths were swabbed as that is the only keratinized part of the

body. Adults were swabbed over the abdomen, inner thighs, and the webbing of each foot. PCR

was run to test swabs for Bd.

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Cascade frogs, Western toads, the

Pacific Chorus frog and the California

Newt were the three most commonly

collected organisms at the sites.

Cascade frogs were found at 88 out of

the 112 sites surveyed. The results

yielded that Bd is commonly found

throughout the Klamath ranges. Bd

was found at 68% of sites and in 16%

of individuals swabbed. Taking all

four commonly founds amphibians

into account; sub adult Cascade frogs

(the life stage between larvae and

adult) had the highest rate of infection

than all other amphibian life stages.

Analysis with cascade frog data alone

revealed sub adult frogs still had a

higher prevalence of infection than

adults, revealing a possible connection between life stage and infection. Temperature and

elevation also played a role in infection. Over the course of a summer season, probability of

infection stayed the same for sub adults but declined over time in adults. Prevalence in both sub

adults and adults were high at higher elevations, with the highest rate at the beginning of the

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Figure 8 shows prevalence of Bd. Each symbol represents a study site, check figure legend on map for corresponding shading and prevalence. Figure 8 reproduced from Factors Related To The Distribution And Prevalence Of The Fungal Pathogen Batrachochytrium Dendrobatidis In Rana Cascadae And Other Amphibians In The Klamath Mountains, Figure 1.

summer. Larval stages had a low prevalence of infection, in both cascade frogs and other frog

species swabbed. This is suggested to be most likely due to the small amount of keratinized areas

larval stages have, leading to a smaller likelihood of being infected or a smaller zoospore load.

This study suggests a mechanism by which adults may be able to clear themselves of

infection over the summer season. As frogs are ectothermic, temperature regulation may be a

part of their immune systems. Piovia-Scott et al, (2011) suggests that adult frogs may be able to

use the warmer temperatures of mid-summer to clear themselves of infection, while sub adult

frogs may be have trade-

offs between immune

function and other

physiological process like

growth. There is also a

suggestion that at higher

elevations when it is cold,

there is lower immune

function, leading to higher

infection intensity (Piovia-

Scott et al, 2011).

A third study done

by Savage et al, (2011)

examined Bd prevalence in

the Lowland Leopard frog (Lithobates yavapaiensis), in the United States. Savage et al, (2011)

sampled frogs from 12 populations in Arizona over the span of five years, and quantified

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Figure 9 shows the prevalence of Bd in each of the four most commonly found amphibians at study sites. L= larvae, M= metamorph, S= sub adult, A=adult. Error bars represents a 95% confidence interval. Figure 9 reproduced from from Factors Related To The Distribution And Prevalence Of The Fungal Pathogen Batrachochytrium Dendrobatidis In Rana Cascadae And Other Amphibians In The Klamath Mountains, Figure 2.

prevalence, intensity, and mortality. Each of the 12 population localities were surveyed in

summer and winter from 2006-2010. Air and water temperatures were logged; two of 12

localities were fed by hot springs which kept the water temperature constant over the study years.

All frogs captured were swabbed and tested for Bd. Frogs that showed signs of Bd infection,

such as increased body mass, lethargy, or failure to seek cover, were collected and swabbed for

intensity of infection: if the frog died within 24 hours of collection and tested positive for Bd

they were considered “chytridiomycosis mortalities.” Frogs that were found dead were also

collected and swabbed, and were considered chytridiomycosis mortalities if they tested positive

for Bd.

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Figure 10 (A) Prevalence of Bd infection (closed circles) and chytridiomycosis mortality (open circles) across all 12 localities over winters of all 5 years sampled. 10 (B) Mean Bd infection prevalence and chytridiomycosis mortality prevalence across all samplings. 10 (C) Average Bd infection intensity measured in zoospore, across sampling winters. Figure 10 reproduced from Disease Dynamics Vary Spatially and temporally in a North American Amphibian, Figure 1.

A total of 692 lowland leopard frogs were sampled. All populations except two (noted as

AS and HS in Figure 10) had infections in the winter. Of the ten infected populations, five of

them suffered from winter mortalities and had varying levels of Bd infection intensity (Figure

10, reproduced from Disease Dynamics Vary Spatially and temporally in a North American

Amphibian, Savage et al, 2011). Overall, Bd infection and mortality were significantly lower in

summer than in winter, indicating that there is a specific temperature range where Bd can

flourish. Winter prevalence and mortality did not change over the five year sampling period, but

intensity of infection decreased over the five year period. Across all localities prevalence was

higher in the winter than in the summer. Over summers there were no mortalities and a very low

prevalence of Bd at all 12 localities. Since two sites were thermal pools and had a constant water

temperature, the interaction between water temperature and chytridiomycosis mortality was also

measured at those sites. As expected, in the winter when air temperatures were more favorable

for Bd, mortality was higher in non-thermal pools due to the lower water temperatures that

fluctuate with the air temperature. Prevalence in thermal pools stayed low and constant due to

above optimal temperatures for Bd. It is also noted that frogs collected with signs of Bd infection

had higher intensities of disease than those found dead, possibly due to how long they laid

outside, which was on average 5.3 days. In general, frogs sampled in the winter that were found

dead and tested positive for Bd, and those collected presenting signs of Bd infection had a larger

body mass than asymptomatic frogs.  

These results of higher intensity and prevalence in winter months in the United States are

consistent with other studies of Bd in natural habitats. The thermal pools averaged 30 C° which

is considered an upper threshold for Bd, and since infection prevalence was lower in these

thermal pools this coincides with Sapsford et al, (2013), Piovia-Scott et al, (2011) which show

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decreased infection prevalence and intensities at warmer than optimal temperatures.  The results

of all three studies reveal a correlation between temperature and Bd prevalence and intensity,

which could in turn help to predict areas of vulnerability and aid in the development of a plan of

action and treatment for Bd outbreaks.

Discussion

The most recent research concludes temperature is the primary factor governing the

spread of Bd. Batrachochytrium dendrobatidis has an optimal temperature for growth and

viability that averages between 17 and 23 °C with temperatures out of this range substantially

slowing the growth or killing the fungus. Although elevation is positively correlated with Bd

prevalence, higher elevations are cooler and more likely to be in optimal temperature range,

compared to warmer temperatures at sea level. The study in Australia by Sapsford et al, (2013), a

continent with high infection rates, showed that aquatic connectivity has an effect on Bd

prevalence, which is also related to temperature. Since higher elevation sites had higher

prevalence of Bd than low elevation counterparts, cool water running between the two produced

higher prevalence of Bd at low elevation connected sites. All of these factors like elevation,

season, and site type are related to temperature, and still need to be researched. The more

information we gain about this new pathogen will aid in planning conservation tactics to save

future populations.

Studies done by Savage et al, (2011) and Piovia-Scott et al, (2011), in the United States

were consistent with the findings of Sapsford et al, (2013), in two different species of amphibian

which have experienced declines due to Bd infection. These studies focused on seasonal

variability in prevalence and intensity of infection causing mortality. Piovia-Scott et al, (2011)

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also showed seasonal and elevation to prevalence of Bd, relating back to temperature. This study

examined what life stage of frog was most likely to be affected by Bd, and revealed that frog life

stage may play a role. Generally, larval stages usually only show sub lethal effects such as

lesions on the insides of their mouth, the only keratinized part of their body, while the infection

is more lethal in sub adult and adult frogs (Berger et al, 1998). Piovia-Scott et al, (2011) showed

that larval stages were less likely to experience mortality from Bd, most likely due to smaller

amounts of keratin, only found in the mouths of larval frogs, disallowing lethal levels of

zoospores to congregate, and suggest adults may have better immune responses to the fungus

than sub adults. Savage et al, (2011) revealed higher Bd mortality in winter when temperatures

were optimal for infection, compared to no mortalities and low Bd prevalence in the summer

when temperatures were above optimal.  Although winter mortality rates did not change across

the sampling years, infection intensities decreased. This suggests that as populations are

continually exposed to Bd, they may be gaining resistance. Frogs may eventually be able to live

in the environment with Bd. The initial onset of Bd in populations kills many individuals as their

immune systems are not prepared to respond to the foreign pathogen.

The naive prey hypothesis explains why initial exposure can be detrimental. Populations

are exposed to a novel pathogen and do not have any defenses. This tends to lead to higher

intensity of infection in individuals of newly exposed populations (DiRenzo et al, 2014).

Intensity of infection, counted by the number of zoospores found on an individual, has been

found to need above 10,000 zoospores to produce lethal effects (Vredenburg et al. 2010). There

have been species of amphibians that are able to carry these zoospores without detrimental

effects, such as the American Bullfrog (Rana catesbeiana) (Rosenblum et al. 2010). Over time,

with constant exposure, frogs may be able to gain an adaptive immunity over the fungus, and

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therefore live and thrive with lower infection intensities. Eventually, populations may be able to

live in the presence of Bd without lethal effects.

It is established that the international trade of amphibians is the explanation for the global

jump of Bd from continent to continent, but amphibian immune systems, along with temperature,

can help explain how Bd persists in individual populations of amphibians (Olson et al, 2013).

Co-habitants of threatened populations, like other frog species that can carry the fungus with no

lethal effects, can keep the fungus steadfast in a population, even when population levels get low.

Briggs, et al. (2010), suggests that there is an external source of zoospores, a reservoir, which

keeps the pathogen from going extinct when frog population density declines. In low host

density conditions, it is possible to come in contact with zoospores from a reservoir and gain low

intensities of infection, without ever reaching the infection intensity needed for death. This

pattern can repeat in a frog many times, yet the frog may never actually succumb to the disease.

Lastly, frogs are ectothermic and maintaining their body temperature is vital to maintaining their

immune system. Studies have showed that cold weather can decrease a frog’s immune system

(Raffel et al, 2006).  Since Bd’s optimal temperature range is 17 C° and 23 C°, this could be

attributed to the degree of infection intensity in populations living in cooler environments rather

than those that live in environments with above optimal temperatures.

There is a debate about how Bd acts as a pathogen, and there are two dueling hypotheses.

Under each hypothesis, the fungus would act differently as it spread, and each hypothesis, if

found to be true, would require different treatment and conservation plans. The novel pathogen

hypothesis suggests that a pathogen is completely novel, introduced to new populations that are

highly susceptible to the pathogen. The second hypothesis is the endemic pathogen hypothesis,

which suggests that a pathogen has been around in the environment, but has just gone unnoticed

Carroll 21

until it became more virulent or entered a new host species. By the theory of novel pathogen, the

spread of Bd would be due to human interaction and introduction of new species. The theory for

endemic pathogen suggests that the change in virulence is due to immunological stress on a

population, or abiotic changes which caused an increased susceptibility in the host population or

increased transmission ability of the pathogen.  Genetically, the pathogen takes on characteristics

of a novel pathogen, with little to no allele variation at multiple sites of infection. As an endemic

pathogen, the pathogen would be expected to have a great deal of genetic variability over time as

it spread from site to site. Conservation tactics differ depending on the hypothesis used. The

novel pathogen hypothesis would require immediate containment of carriers of the pathogen

while the endemic pathogen hypothesis would require further study between biotic and abiotic

relationships in infected environments (Rachowicz et al. 2005). The novel pathogen hypotheses

better fits the description of Bd, since it is genetically not variable across populations and

introduction by humans by the world amphibian trade is an established source of the infection in

new areas. Conservation tactics should be built around this hypothesis.

Future studies should continue to look at the effects of Bd on specific frog species in

laboratory tests so we know which species can be affected. This can help us determine what

species are at risk. Possible reservoir species need to be identified so we know how Bd is persists

in environments and how to treat frogs and reservoirs alike for the infection. Combined, this can

help us map the path of Bd and predict future species that will become infected with the fungus.

Since Bd seems to be acting like a novel pathogen, the appropriate conservation tactics should be

put in place. This means immediate containment of carriers, strict regulation of amphibian trade

intra and inter continentally and treatment before releasing them back into the wild, or collecting

a significant amount of a species before the infection hits a population to release after the wave

Carroll 22

of Bd hits. This process has already begun with the Amphibian Ark, but should be used in

conjunction with field treatment. Antifungal treatments like Itraconazole have shown limited

success in captive populations (Gagliardo et al, 2008, Woodhams et al, 2012). Hot water

treatment of infected frogs has also been successful in laboratory studies. Since temperatures

above 28 C° have been shown to stop growth of Bd, this could be used as possible treatment of

captured sick frogs (Forrest & Schlaepfer, 2011). However, using these treatments in natural

habitats is proving to be difficult. Vredenberg et al, (2010) suggests capturing and treating frogs

with antifungal drugs during an outbreak, and releasing them back into the same area they were

from in hopes of reducing pathogen load and therefore mortality, not necessarily eradicating Bd

as a whole.

Currently, amphibians are under the greatest threat of mass extinction of all species on

the planet. One in four species of amphibians are endangered or vulnerable to extinction in the

United States alone (Adkins Giese, 2013). Since frogs act as bio indicators, their rapid decline

should give us pause to think about the consequences to biodiversity due to a mass extinction

crisis. The Amphibian Ark, started in 2008, is an organization that works to promote awareness

and safety information about dying amphibian populations and Chytrid fungus. Their aim is to

identify which species are at the highest risk and need to be captured for reintroduction if and

when there is a period of time suitable for reestablishment of populations. It is vital that we

prevent the spread of Bd to places it has yet to touch. In a study published as recently as

February 2015, Bletz et al, (2015), Bd was found in Madagascar which was previously

untouched by Bd. Immediate action to prevent mass die offs on this island are crucial to the 300

species of frogs that call Madagascar their home. Chytrid fungus is a still growing, deadly

infectious disease ripping through amphibian species producing extinctions and major die off

Carroll 23

events. Using temperature as the primary regulating factor of Bd spread, we can predict

populations at risk for disease, and take prophylactic measures to treat them. Treatment for those

already infected need to be used to prevent even more deaths, as amphibians are already the most

endangered group of animals on the planet. Helping save amphibians, from Bd and other dangers

including habitat destruction and climate change, may help prevent any further loss of

amphibians.

Carroll 24

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