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The amphibian disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), has been linked to significant amphibian declines over the past three decades. The most severe effects of the pathogen have been primarily observed in relatively pristine areas that are not affected by many anthropogenic factors.One hypothesis concerning improved amphibian persistence with Bd in disturbed landscapes is that contaminants may abate the effects of Bd on amphibians. Recent laboratory studies have shown that pesticides, specifically the fungicide thiophanate-methyl (TM), can kill Bd outside of hosts and clear Bd infections within hosts. Using aquatic mesocosms, we tested the hypothesis that TM (0.43 mg/L) would alter growth and development of Lithobates sphenocephalus (southern leopard frog) tadpoles and Bd-infection loads in infected individuals. We hypothesized that the scope of such alterations and infection clearing would be affected by aquatic community variables, specifically zooplankton. TM altered zooplankton diversity (reduced cladoceran and increased copepod and ostracod abundances) and caused mortality to all tadpoles in TM-exposed tanks. In TM-free tanks, Bd-exposed tadpoles in high-density treatments metamorphosed smaller than Bd-unexposed, effects that were reversed in low-density treatments. Our study demonstrates the potential adverse effects of a fungicide and Bd on tadpoles and aquatic systems.

Interactions between gut microbes and anthropogenic pollutants have been under study. The authors investigated the effects of larval exposure to polychlorinated biphenyl 126 (PCB-126) on the gut microbial communities of tadpoles and frogs. Frogs treated with PCBs exhibited increased species richness in the gut and harbored communities significantly enriched in Fusobacteria. These results suggest that anthropogenic pollutants alter gut microbial populations, which may have health and fitness consequences for hosts., (© 2015 SETAC.)

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Vertebrates maintain complex symbioses with a diverse community of microbes residing within their guts. The microbial players in these symbioses differ between major taxa of vertebrates, such that fish and amniotes maintain notably different communities. To date, there has not been a culture-independent inventory of an amphibian gut microbial community. Here, we compared gut microbial communities of tadpoles and frogs of the Northern leopard frog (Lithobates pipiens). We utilized Illumina sequencing, which allowed us to inventory more than 450 000 microbial sequences. We found that tadpoles and frogs differ markedly in the composition of their gut microbial communities, with tadpoles maintaining a community more similar to fish, whereas the frog community resembles that of amniotes. Additionally, frogs maintain a community with lower phylogenetic diversity compared with tadpoles. The significant restructuring of the microbiota is likely due to changes in diet as well as the large reorganization of the intestinal organ during metamorphosis. Overall, we propose that amphibians represent an important system in which to study regulation and selection of gut microbial communities., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

One issue of great concern for the scientific community is the continuing loss of diverse amphibian species on a global scale. Amphibian populations around the world are experiencing serious losses due to the chytrid fungus, Batrachochytrium dendrobatidis. This pathogen colonizes the skin, leading to the disruption of ionic balance and eventual cardiac arrest. In many species, antimicrobial peptides secreted into the mucus are thought to contribute to protection against colonization by skin pathogens. Although it is generally thought that antimicrobial peptides are an important component of innate immune defenses against B. dendrobatidis, much of the current evidence relies on correlations between effective antimicrobial peptide defenses and species survival. There have been few studies to directly demonstrate that antimicrobial peptides play a role. Using the northern leopard frog, Rana pipiens, we show here that injection of noradrenaline (norepinephrine) brings about a long-term depletion of skin peptides (initial concentrations do not recover until after day 56). When peptide stores recovered, the renewed peptides were similar in composition to the initial peptides as determined by MALDI-TOF mass spectrometry and in activity against B. dendrobatidis as determined by growth inhibition assays. Newly metamorphosed froglets depleted of their peptide stores and exposed to B. dendrobatidis died more rapidly than B. dendrobatidis-exposed froglets with their peptides intact. Thus, antimicrobial peptides in the skin mucus appear to provide some resistance to B. dendrobatidis infections, and it is important for biologists to recognize that this defense is especially important for newly metamorphosed frogs in which the adaptive immune system is still immature.

The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been linked to amphibian declines and extinctions worldwide. The pathogen has been found on amphibians throughout eastern North America, but has not been associated with mass die-offs in this region. In this study, we conducted laboratory experiments on the effects of Bd infection in a putative carrier species, Lithobates pipiens, using two estimators of fitness: jumping performance and testes morphology. Over the 8-week study period, peak acceleration during jumping was not significantly different between infected and uninfected animals. Peak velocity, however, was significantly lower for infected animals after 8 weeks. Two measures of sperm production, germinal epithelium depth, and maximum spermatic cyst diameter, showed no difference between infected and uninfected animals. The width, but not length, of testes of infected animals was significantly greater than in uninfected animals. This study is the first to show effects on whole-organism performance of Bd infection in post-metamorphic amphibians, and may have important long-term, evolutionary implications for amphibian populations co-existing with Bd infection.

One prediction of optimal digestion theory is that organisms will increase the relative length of their digestive tracts when food resources become limited. We used theory of optimal digestion to test whether tadpoles can adjust the relative length of their intestines when challenged with the fungal pathogen Batrachochytrium dendrobatidis (Bd). The degree of tadpole mouthpart damage, a symptom of Bd infections that reduces food consumption, was associated positively with the length of tadpole intestines relative to their body size, consistent with optimal digestion theory. After controlling for mouthpart damage, tadpoles exposed to Bd had shorter intestines relative to their body size, opposite to the predictions of optimal digestion theory. One explanation of why tadpoles with higher Bd loads have shorter relative intestinal lengths is that they divert energy from maintaining intestinal and overall growth towards anti-parasite defences.

Effects of exposure to contaminants such as pesticides along with exposure to pathogens have been listed as two major contributors to the global crisis of declining amphibian populations. These two factors have also been linked in explanations of the causes of these population declines. We conducted a combined exposure experiment to test the hypothesis that exposure to two agricultural herbicides would increase the susceptibility of post-metamorphic northern leopard frogs (Lithobates pipiens) to the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed the independent and interactive effects of these exposures on the health and survival of the frogs. Wild-caught frogs underwent a 21-day exposure to a nominal concentration of either 2.1 μg/L atrazine (Aatrex(®) Liquid 480) or 100 μg a.e./L glyphosate (Roundup(®) Original), followed by Bd, and then were observed until 94 days post-initial exposure to the herbicides. Actual levels of atrazine were between 4.28 ± 0.04 μg/L and 1.70 ± 0.26 μg/L while glyphosate degraded from 100 μg a.e./L to approximately 7 μg a.e./L within 6 days of initial exposure to the herbicides. Compared to controls, the glyphosate formulation reduced the snout-vent length of frogs during the pesticide exposure (at Day 21), and the atrazine formulation reduced gain in mass up to Day 94. No treatment affected survival, splenosomatic or hepatosomatic indices, the densities and sizes of hepatic and splenic melanomacrophage aggregates, the density and size of hepatic granulomas, proportions of circulating leucocytes, the ratio of neutrophils to lymphocytes, or the ratio of leucocytes to erythrocytes. Histological assessment of samples collected at Day 94 revealed no evidence of Bd infection in any Bd-exposed frogs, while real-time PCR detected only one case of light infection in a single atrazine- and Bd-exposed frog. Frogs exposed to Bd shed their skin significantly more frequently than Bd-unexposed frogs, which may have helped them resist or clear infection, and could explain why no interaction between the herbicides and Bd was detected. The results suggest that these frogs were resistant to Bd infection and that pre-exposure to the herbicides did not alter this resistance. The effects seen on the growth following herbicide exposure is a concern, as reduced growth can lower the reproductive success and survival of the amphibians., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.

Ecological theory predicts that species with restricted geographic ranges will have the highest probability of extinction, but species with extensive distributions and high population densities can also exhibit widespread population losses. In the western United States populations of northern leopard frogs (Lithobates pipiens)-historically one of the most widespread frogs in North America-have declined dramatically in abundance and geographic distribution. To assess the status of leopard frogs in Colorado and evaluate causes of decline, we coupled statewide surveys of 196 historically occupied sites with intensive sampling of 274 wetlands stratified by land use. We used an information-theoretic approach to evaluate the contributions of factors at multiple spatial extents in explaining the contemporary distribution of leopard frogs. Our results indicate leopard frogs have declined in Colorado, but this decline was regionally variable. The lowest proportion of occupied wetlands occurred in eastern Colorado (2-28%), coincident with urban development and colonization by non-native bullfrogs (Lithobates catesbeianus). Variables at several spatial extents explained observed leopard frog distributional patterns. In low-elevation wetlands introduced fishes, bullfrogs, and urbanization or suburbanization associated negatively with leopard frog occurrence, whereas wetland area was positively associated with occurrence. Leopard frogs were more abundant and widespread west of the Continental Divide, where urban development and bullfrog abundance were low. Although the pathogenic chytrid Batrachochytrium dendrobatidis (Bd) was not selected in our best-supported models, the nearly complete extirpation of leopard frogs from montane wetlands could reflect the individual or interactive effects of Bd and climate patterns. Our results highlight the importance of considering multiple, competing hypotheses to explain species declines, particularly when implicated factors operate at different spatial extents., (©2011 Society for Conservation Biology.)

Background: Emerging infectious diseases threaten naïve host populations with extinction. Chytridiomycosis, an emerging infectious disease of amphibians, is caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd) and has been linked to global declines in amphibians., Results: We monitored the prevalence of Bd for four years in the Northern leopard frog, Rana pipiens, which is critically imperiled in British Columbia (BC), Canada. The prevalence of Bd initially increased and then remained constant over the last three years of the study. Young of the year emerging from breeding ponds in summer were rarely infected with Bd. Some individuals cleared their Bd infections and the return rate between infected and uninfected individuals was not significantly different., Conclusions: The BC population of R. pipiens appears to have evolved a level of resistance that allows it to co-exist with Bd. However, this small population of R. pipiens remains vulnerable to extinction.

Since host defenses to infectious disease are often costly, one would expect hosts to use their defenses only when the threat of infection is high. Southern leopard frogs (Rana sphenocephala) at Ellenton Bay in South Carolina (USA) have an extended breeding season and their eggs are exposed to a wide range of temperatures depending on the time of year when they are laid. Adults aggregate their egg masses in cold temperatures, but separate them in warm temperatures. The spatial aggregation of egg masses may insulate eggs from cold temperatures, but may also affect the transmission of pathogens between the eggs. I examined the effects of temperature, pathogens, and spatial distribution on the survival of R. sphenocephala in the egg stage. Field observations found that temperature had little effect on the number of infected eggs within egg masses, but that egg masses in colder water were more likely to be aggregated together. In a controlled laboratory experiment, the presence of aquatic oomycetes led to higher mortality in cold temperatures than they did at warmer temperatures. Infectious disease may be a selective force that favors R. sphenocephala adults that aggregate their offspring when the threat of disease is high. Host aggregation can reduce the risk of infection when pathogens have a slow generation time that hinders them from responding to an abundance of hosts.

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium-host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

During latent infection of humans with Mycobacterium tuberculosis, bacteria persist in the asymptomatic host within granulomas, organized collections of differentiated macrophages, and other immune cells. The mechanisms for persistence remain poorly understood, as is the metabolic and replicative state of the microbes within granulomas. We analyzed the gene expression profile of Mycobacterium marinum, the cause of fish and amphibian tuberculosis, during its persistence in granulomas. We identified genes expressed specifically when M. marinum persists within granulomas. These granuloma-activated genes were not activated in vitro in response to various conditions postulated to be operant in tuberculous granulomas, suggesting that their granuloma-specific activation was caused by complex conditions that could not be mimicked in vitro. In addition to the granuloma-activated genes, the bacteria resident in granulomas expressed a wide range of metabolic and synthetic genes that are expressed during logarithmic growth in laboratory medium. Our results suggest a dynamic host-pathogen interaction in the granuloma, where metabolically active bacteria are kept in check by the host immune system and where the products of granuloma-specific bacterial genes may thwart the host's attempt to completely eradicate the bacteria.

In 222 Rana pipiens frogs and 34 tadpoles captured in and near Minnesota, Aeromonas hydrophila and 29 species of Enterobacteriaceae, including yersinia enterocolitica and Salmonella arizonae, were isolated from intestines. The prevalence of members of the family Enterobacteriaceae was lowest in frogs captured in early spring and highest in frogs captured in late summer.

The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens. Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators. The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species. The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators. Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.

Survival data for over 500 adult frogs (393 Rana pipiens, 35 Rana clamitans , and 97 Rana temporaria) obtained from commercial suppliers were accumulated. Frogs which developed signs of red-leg disease while being kept in 0.15% saline were cured by addition of appropriate antibiotics to the saline. The appropriate antibiotic was determined by sensitivity testing of bacteria isolated from the sick frogs. When many frogs in a shipment were sick, prophylactic treatment of the entire shipment improved the overall survival in the shipment.

A frog pronephric cell line, infected with herpes virus derived from Lucké renal carcinomas of Rana pipiens was examined for the presence of Lucké herpes virus antigens. Non-infected pronephric cells were controls. Antiserum to purified Lucké tumor herpes virus was applied in blind tests to the normal and virus infected cells. Both cytoplasmic and nuclear fluorescence was found in the herpes virus infected cells after indirect immunofluorescence with the antiserum. Infected cells cultivated at the optimum growth temperature of 25 degrees C or maintained at 9 degrees C, a temperature inducive to herpes virus replication, showed equivalent fluorescence reactions. No fluorescence was found in the normal pronephric cell line. Examination of parallel herpes infected cells showed cytopathic effect in monolayers by light microscopy, and nuclear or cytoplasmic immunofluorescence. Electron microscopic examination revealed proviral elements in nuclei and sparsely scattered herpes virus coincident with cytoplasmic fluorescence.

The bacteria in the large intestines of 10 northern leopard frogs (Rana pipiens) were enumerated and partially characterized. Four nonhibernating frogs were collected in the summer, four hibernating frogs were collected in the winter, and two frogs just emerged from hibernation were collected in the spring. All frogs had about 10(10) bacteria per g (wet weight) of intestinal contents and about 10(9) bacteria per g (wet weight) of mucosal scraping, although the counts from the winter frogs were slightly less than those from the other two groups of frogs. Another group of 14 summer frogs, after treatment to induce hibernation, showed a drop in bacterial counts accompanied by a change in the composition of the flora. In most frogs, Bacteroides was the dominant organism. Other bacteria repeatedly isolated at high dilutions were strict anaerobes, including butyrigenic and acetogenic helically coiled bacteria; fusobacteria; and acetogenic, small, gram-positive bacilli. These data indicate that the intestinal flora of frogs is similar to that of mammals and birds and that this flora can be maintained at temperatures close to freezing.

The bacterial flora of the large intestine was examined in 35 laboratory-reared leopard frogs (Rana pipiens) subjected to one of the following four treatments: (i) normal feeding at 21 degrees C (10 frogs); (ii) fasting for 2 weeks at 21 degrees C (8 frogs); (iii) chilling for 1 week at 4 degrees C (9 frogs); and (iv) simulated hibernation for 3 weeks at 4 degrees C (8 frogs). Bacteria from the intestinal contents and mucosa were counted microscopically and by colony counting after strictly anaerobic culturing. The predominant bacteria were isolated and partially characterized. Fasting for 2 weeks produced no significant changes in total counts or in the types of bacteria cultured. Chilling, whether rapid or in the course of simulated hibernation, was associated with a decrease in the numbers and variety of bacteria. Thus it appears that the lowering of temperature rather than the absence of food is the important factor in the reduction of bacterial flora seen in hibernating frogs. However, the bacteria showed some adaptation to the low temperature, as the longer the host had been at 4 degrees C, the higher the proportion of bacteria which could grow when cultured at that temperature.

Mechanisms and factors that normally control the large intestinal flora were investigated to determine whether changes in these parameters could account for the decreased bacterial concentration and facultative nature of the flora found in hibernating frogs. It appeared that low temperatures and limited nutrients were the main factors responsible for the decrease in the bacterial concentration and may also have been responsible for the increase in the proportions of facultative organisms, since no change in the redox potential was seen. The hibernating frogs were extremely sluggish in the removal of India ink particles from the circulatory system by the Kupffer cells of the liver compared with nonhibernating frogs. They were unable to mount an antibody response to bovine serum albumin, but their serum did exhibit killing of Pseudomonas paucimobilis, suggesting opsonization by preformed antibody and complement. The role of these host factors in protecting the hibernating frog against this indigenous flora is discussed.

Leopard frogs (Rana pipiens) were orally inoculated with Listeria monocytogenes. Most listeriae were shed 3-6 days after inoculation. The evidence indicated that the listeriae did not multiply in frogs and were unable to become established in the gastrointestinal tract.

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens, is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4 degrees C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.

Frogs and tadpoles were captured at 14 sites in and near Minnesota during 1978-79 and nearly all appeared healthy. Aeromonas hydrophila was isolated from 94 of 294 (32%) juvenile and adult frogs and from 66 of 104 (63%) tadpoles. Of the isolates from frogs and tadpoles respectively, 68% and 47% were from the intestine only, 12% and 32% were from the intestine and the other sites, and 20% and 21% were from extraintestinal sites only. Isolations were more frequent from frogs collected in March-June than in August-November. Evidence was not found that disease due to Aeromonas hydrophila was a primary cause of declining Rana pipiens populations in Minnesota.