A. Title: The Ecology of Disease and Anthropogenic Stressors in Amphibians
B. Authors: Kellie French, Julia Buck, Trang Nguyen, Jenny Urbina Gonzalez, Andrew
Blaustein
C. Abstract:
We proposed to test the hypothesis that ecologically relevant concentrations of pesticide
mixtures will increase susceptibility of metamorphic amphibians to a fungal pathogen known as
Batrachochytrium dendrobatidis, or Bd. Bd has received considerable attention as one of the
causes of declining amphibian populations. We hypothesized that exposure to the contaminants
in the larval stage would cause increased pathogen load, decreased growth, and increased
mortality of amphibians. Our amphibians included 2 species of tree frogs (Spring Peepers and
Pacific tree frogs), 1 toad species (Western Toads), and 2 true frog species (Leopard frogs and
Cascades frogs). Each species of frog was split into five different pesticide treatments: high
herbicide, low herbicide, high insecticide, low insecticide, and a control treatment. Half of the
frogs in each pesticide treatment were exposed as tadpoles and half were exposed as metamorphs.
In the end, three of the five species showed significant mortality rates. We saw similarities
between the tree frogs. They both showed a Bd effect with a significant p-value. The toads
showed an increased susceptibility compared to the other species. Our true frogs showed very
little mortality and had no significant effects.
D. Introduction:
As part of an overall “biodiversity crisis”, amphibian populations are undergoing
unprecedented population declines and extinctions (Stuart et al 2004). Numerous factors may be
contributing to these declines including disease and contaminants (Blaustein et al. 2011).
Amphibians offer an ideal system for studying the interaction of contaminants (e.g., pesticides)
and pathogens (Blaustein et al. 2001). They live in and out of water and are therefore more
susceptible to terrestrial and water-borne pesticides. They also have permeable skin and
unshelled eggs which make them hypersensitive to the environment (Blaustein 1994). Disease
caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has received considerable
attention as a cause of declining populations. However, there are amphibian populations with the
presence of this pathogen without amphibians showing symptoms of disease or experiencing
population declines. Therefore, co-factors such as pesticides and pathogens may be involved. By
considering each factor in isolation, we may be missing a critical explanatory connection.
Therefore, we studied the effects of exposure to both pesticides and pathogens on frogs during
the larval and metamorphic stages of life.
The benefits to society include the potential to offer new insights into the spread of
infectious disease. All organisms, including humans are exposed to pesticides and pathogens.
What we learn about amphibians can tell us generally about how simultaneous exposure to both
pathogens and pesticides may affect organisms, including humans. Moreover, we will be
focusing on amphibians, which are animals that are integral parts of ecosystems. Disappearing
amphibians may affect whole ecosystems because they are food for a variety of animals and they
consume plants and other animals, including many insect pests.
E. Materials and Methods
All of the exposure to the pesticides took place at the University of Pittsburg. The animals
were then shipped to us as metamorphs and we exposed half of them to Bd. We had 2015
animals total in our experiment. Each of the five species of frogs was split into five different
pesticide treatments: high herbicide, low herbicide, high insecticide, low insecticide, and a
control treatment. Half of the frogs in each pesticide treatment were exposed as tadpoles and half
were exposed as metamorphs. When we received the animals each week, we weighed and
measured them and then placed them in a petri dish labeled with their treatment group, life stage
exposure group and Bd exposure group. Half of the petri dishes contained water that had been
infected with Bd. Each animal was kept in the lab for two weeks while we monitored them for
mortality, feed them twice a week, and changed their water once.
F. Results
The tadpole exposed Spring Peepers had a Bd effect p-value of <0.01 and a hazard ratio
of 38.84. The metamorph exposed frogs of this species showed the same Bd effect p-value and a
hazard ratio of 18.737. The tadpole exposed Pacific Tree Frogs displayed a Bd effect p-value of
<0.01 and a hazard ratio of 38.105. The metamorph exposed frogs of the same species showed
the same Bd effect p-value and a hazard ratio of 58.415. The high insecticide group was
statistically shown to be safer than the control group of the metamorph exposed Pacific Tree
Frogs, with a p-value of 0.0023 and a hazard ratio of 0.239. The Western Toads that were
exposed to Bd as tadpoles, had a Bd effect p-value of <0.01 and a hazard ratio of 279.9. The
metamorph exposed toads had the same p-value and a hazard ratio of 27.97. All of the Leopard
Frogs and Cascades Frogs showed very little mortality and no significant effects. There was an
SVL effect in the metamorph exposed Leopard Frogs. This effect had a p=value of 0.000048 and
a hazard ratio of 0.44.
G. Discussion
In the end, three of the five species showed significant mortality rates. We saw similarities
between the tree frogs. They both showed a Bd effect with a significant p-value. The toads
showed an increased susceptibility compared to the other species. We’re not sure why toads are
more susceptible. It could have something to do with the amount of keratin in the skin, or how
they regulate water uptake since Bd infection disrupts the osmotic balance of amphibians,
causing cardiac arrest. Our true frogs showed very little mortality and had no significant effects.
We saw a couple of interesting effects in our data analysis. The pesticide effect in the
metamorph exposed Pacific Tree Frogs shows that the high insecticide group was statistically
shown to be safer than the control group. It’s possible that these frogs may have had some
insecticide remaining on their skin which could have actually reduced the number of Bd
zoospores in the water that they were living in, therefore reducing their risk of infection by Bd.
The SVL effect in the metamorph exposed Leopard Frogs showed that the smaller animals were
actually more at risk than those that were larger. Therefore, although we didn’t see any
significant interactions between the pesticides and pathogens, we did have some interesting
findings.
H. References:
Blaustein, A.R. 1994. Chicken Little or Nero’s fiddle? A perspective on amphibian population
declines. Herpetologica. 50:85-97.
Blaustein, A.R., B.A. Han, R.A. Relyea, P.T.J. Johnson, J.C. Buck, S. Gervasi and L.B. Kats.
2011. The complexity of amphibian population declines: Understanding the role of cofactors in driving amphibian losses. Annals of the New York Academy of Sciences.
The Year in Ecology and Conservation Biology 1223: 108-119.
Stuart, S.N., J.S. Chanson, N.A. Cox, B.E. Young, A.S.L. Rodrigues, D.L. Fishman, and R.W.
Waller 2004. Status and trends of amphibian declines and extinctions worldwide. Science
306:1783-1786.