John M. Romansic

The Fungicide Chlorothalonil Is Nonlinearly Associated with Corticosterone Levels, Immunity, and Mortality in Amphibians

Background: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them. Objective: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States. Methods: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 μg/L). We also conducted two dose–response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone. Results: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 μg/L killed 100% of R. sphenocephala, and 0.0164 μg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species 
showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 μg/L). Conclusions: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

Evaluating the links between climate, disease spread, and amphibian declines

Human alteration of the environment has arguably propelled the Earth into its sixth mass extinction event and amphibians, the most threatened of all vertebrate taxa, are at the forefront. Many of the worldwide amphibian declines have been caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), and two contrasting hypotheses have been proposed to explain these declines. Positive correlations between global warming and Bd-related declines sparked the chytrid-thermal-optimum hypothesis, which proposes that global warming increased cloud cover in warm years that drove the convergence of daytime and nighttime temperatures toward the thermal optimum for Bd growth. In contrast, the spatiotemporal-spread hypothesis states that Bd-related declines are caused by the introduction and spread of Bd, independent of climate change. We provide a rigorous test of these hypotheses by evaluating (i) whether cloud cover, temperature convergence, and predicted temperature-dependent Bd growth are significant positive predictors of amphibian extinctions in the genus Atelopus and (ii) whether spatial structure in the timing of these extinctions can be detected without making assumptions about the location, timing, or number of Bd emergences. We show that there is spatial structure to the timing of Atelopus spp. extinctions but that the cause of this structure remains equivocal, emphasizing the need for further molecular characterization of Bd. We also show that the reported positive multi-decade correlation between Atelopus spp. extinctions and mean tropical air temperature in the previous year is indeed robust, but the evidence that it is causal is weak because numerous other variables, including regional banana and beer production, were better predictors of these extinctions. Finally, almost all of our findings were opposite to the predictions of the chytrid-thermal-optimum hypothesis. Although climate change is likely to play an important role in worldwide amphibian declines, more convincing evidence is needed of a causal link.

Community ecology theory predicts the effects of agrochemical mixtures on aquatic biodiversity and ecosystem properties

Ecosystems are often exposed to mixtures of chemical contaminants, but the scientific community lacks a theoretical framework to predict the effects of mixtures on biodiversity and ecosystem properties. We conducted a freshwater mesocosm experiment to examine the effects of pairwise agrochemical mixtures [fertiliser, herbicide (atrazine), insecticide (malathion) and fungicide (chlorothalonil)] on 24 species- and seven ecosystem-level responses. As postulated, the responses of biodiversity and ecosystem properties to agrochemicals alone and in mixtures was predictable by integrating information on each functional groups (1) sensitivity to the chemicals (direct effects), (2) reproductive rates (recovery rates), (3) interaction strength with other functional groups (indirect effects) and (4) links to ecosystem properties. These results show that community ecology theory holds promise for predicting the effects of contaminant mixtures on biodiversity and ecosystem services and yields recommendations on which types of agrochemicals to apply together and separately to reduce their impacts on aquatic ecosystems.

ADDING INFECTION TO INJURY: SYNERGISTIC EFFECTS OF PREDATION AND PARASITISM ON AMPHIBIAN MALFORMATIONS

We explored the importance of interactions between parasite infection and predation in driving an emerging phenomenon of conservation importance: amphibian limb malformations. We suggest that injury resulting from intraspecific predation in combination with trematode infection contributes to the frequency and severity of malformations in salamanders. By integrating field surveys and experiments, we evaluated the individual and combined effects of conspecific attack and parasite (Ribeiroia ondatrae) infection on limb development of long-toed salamanders (Ambystoma macrodactylum). In the absence of Ribeiroia, abnormalities involved missing digits, feet, or limbs and were similar to those produced by cannibalistic attack in experimental trials. At field sites that supported Ribeiroia, malformations were dominated by extra limbs and digits. Correspondingly, laboratory exposure of larval salamanders to Ribeiroia cercariae over a 30-day period induced high frequencies of malformations, including extra digits, extra limbs, cutaneous fusion, and micromelia. However, salamander limbs exposed to both injury and infection exhibited 3-5 times more abnormalities than those exposed to either factor alone. Infection also caused significant delays in limb regeneration and time-to-metamorphosis. Taken together, these results help to explain malformation patterns observed in natural salamander populations while emphasizing the importance of interactions between parasitism and predation in driving disease.

Nonmonotonic and Monotonic Effects of Pesticides on the Pathogenic Fungus Batrachochytrium dendrobatidis in Culture and on Tadpoles

Pesticides and the pathogenic fungus Batrachochytrium dendrobatidis (Bd) co-occur and are implicated in the global decline of amphibians, a highly threatened taxon. Here, we investigated the effects of ecologically relevant concentrations of chlorothalonil and atrazine, two of the most commonly used, immunomodulatory pesticides in the United States, on tadpole (Osteopilus septentrionalis) survival and Bd growth. Tadpole survival was unaffected by the pesticides but was reduced by Bd. Atrazine monotonically (i.e., consistently increasing or decreasing) reduced Bd in culture and on tadpoles, and every concentration tested (0.0106-106 μg/L) significantly reduced Bd growth compared to controls. Chlorothalonil had a nonmonotonic (i.e., nonlinear) effect on Bd growth both in culture and on tadpoles, where low (0.0176-1.76 μg/L) and high (32-176 μg/L) concentrations inhibited Bd growth significantly more than did intermediate concentrations (8.2-17.6 μg/L) and controls. To our knowledge, this is one of only a handful of studies to document a nonmonotonic dose response of a nonvertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on frogs, neither cleared the infection entirely, and because we know little about the long-term effects of the pesticides on hosts, we do not recommend using these chemicals to control Bd.

Individual and combined effects of multiple pathogens on Pacific treefrogs

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens.

Ambient Levels of Ultraviolet-B Radiation Cause Mortality in Juvenile Western Toads, Bufo boreas

Abstract Numerous anthropogenic and natural factors affect living organisms in nature. Anthropogenic factors include a wide array of contaminants and processes that alter the habitat on both local and global scales. For example, chlorofluorocarbons (CFCs) and other industrial gases contribute to the depletion of the earths protective ozone layer, resulting in increased amounts of cell damaging ultraviolet-B (UV-B) radiation reaching the surface of the earth. Recent experiments provide evidence that increasing ambient levels of UV-B radiation harm many amphibian species. UV-B radiation can kill amphibians and can cause sublethal damage to them. However, most studies that have examined the effects of UV-B radiation on amphibians have focused on developing embryos. There is little information on how UV-B radiation affects amphibians at later stages of development. In experimental laboratory tests, we exposed one group of juvenile western toads (Bufo boreas) to full spectrum lighting with ambient levels of UV-B radiation and control toads to full spectrum lighting excluding most UV-B. Juvenile toads exposed to ambient levels of UV-B radiation showed significantly greater mortality rates compared with controls. These results add to a growing body of literature demonstrating that UV-B is harmful to amphibians. Furthermore, our results suggest that investigators should look at the effects of UV-B radiation on different life stages before making conclusions about the overall impact of UV-B on amphibians.

VARIABLE BREEDING PHENOLOGY AFFECTS THE EXPOSURE OF AMPHIBIAN EMBRYOS TO ULTRAVIOLET RADIATION and OPTICAL CHARACTERISTICS OF NATURAL WATERS PROTECT AMPHIBIANS FROM UV‐B IN THE U.S. PACIFIC NORTHWEST: COMMENT

1Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, Oregon 97331 USA 2United States Geological Survey, Oregon Cooperative Fish and Wildlife Research Unit, Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331 USA 3Department of Evolutionary Biology, Donana Biological Station, CSIC, Apartado 1056, Sevilla 41013, Spain 4Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, Saskatchewan S7N5E2 5Department of Biology, 2125 Derring Hall, Virginia Polytechnic Institute and State University Blacksburg, VA 24061 USA 6Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802 USA 7Departamento de Biologia Animal, Ecologia, Parasitologia y Edafologia, Universidad de Salamanca, 37071 Salamanca, Spain 8Natural Science Division, Pepperdine University, Malibu, CA 90263 USA

Does UV-B Radiation Affect Embryos of Three High Elevation Amphibian Species in California?

Abstract Declines and extinctions of amphibians in well-protected habitats suggest that global atmospheric factors may be responsible. We tested effects of field exposures of ultraviolet radiation (UV-B) on embryo hatching success and time to hatching in three anurans that inhabit high elevation areas of the Sierra Nevada in California, USA. While few obvious environmental impacts have occurred in the high elevation area of the Sierra Nevada, two of the three most common anurans, the Yosemite Toad (Bufo canorus) and the Southern Mountain Yellow-legged Frog (Rana muscosa), have suffered severe population declines while the sympatric Pacific Treefrog (Pseudacris regilla) has remained relatively abundant. Previous studies have shown that hatching of P. regilla embryos at lower elevations are not affected negatively by UV-B radiation. We hypothesized that differences in UV sensitivity may help explain why P. regilla remain abundant while B. canorus and R. muscosa have declined sharply. We conducted field experiments at two remote sites above 3030 m elevation over two years. No effect of UV-B was found on hatching success or rate of development in embryos of B. canorus, P. regilla, or R. muscosa, except for a small, context-dependent increase in time to hatching in R. muscosa. We recommend that research efforts on these species in the Sierra Nevada concentrate on post-hatching effects of UV-B, or on other decline hypotheses.

Complex interactive effects of water mold, herbicide, and the fungus Batrachochytrium dendrobatidis on Pacific treefrog Hyliola regilla hosts

Infectious diseases pose a serious threat to global biodiversity. However, their ecological impacts are not independent of environmental conditions. For example, the pathogenic fungus Batrachochytrium dendrobatidis (Bd), which has contributed to population declines and extinctions in many amphibian species, interacts with several environmental factors to influence its hosts, but potential interactions with other pathogens and environmental contaminants are understudied. We examined the combined effects of Bd, a water mold (Achlya sp.), and the herbicide Roundup® Regular (hereafter, Roundup®) on larval Pacific treefrog Hyliola regilla hosts. We employed a 2 wk, fully factorial laboratory experiment with 3 ecologically realistic levels (0, 1, and 2 mg l-1 of active ingredient) of field-formulated Roundup®, 2 Achlya treatments (present and absent), and 2 Bd treatments (present and absent). Our results were consistent with sublethal interactive effects involving all 3 experimental factors. When Roundup® was absent, the proportion of Bd-exposed larvae infected with Bd was elevated in the presence of Achlya, consistent with Achlya acting as a synergistic cofactor that facilitated the establishment of Bd infection. However, this Achlya effect became nonsignificant at 1 mg l-1 of the active ingredient of Roundup® and disappeared at the highest Roundup® concentration. In addition, Roundup® decreased Bd loads among Bd-exposed larvae. Our study suggests complex interactive effects of a water mold and a contaminant on Bd infection in amphibian hosts. Achlya and Roundup® were both correlated with altered patterns of Bd infection, but in different ways, and Roundup® appeared to remove the influence of Achlya on Bd.