Continental-extent patterns in amphibian malformations linked to parasites, chemical contaminants, and their interactions.

Widespread observations of malformed amphibians across North America have generated both concern and controversy. Debates over the causes of such malformations-which can affect >50% of animals in a population-have continued, likely due to involvement of multiple causal factors. Here, we used a 13-year dataset encompassing 53,880 frogs and toads from 422 wetlands and 42 states in the conterminous USA to test hypotheses relating abnormalities and four categories of potential drivers: (i) chemical contaminants, (ii) land use practices, (iii) parasite infection, and (iv) targeted interactions between parasites and pesticides. Using a hierarchically nested, competing-model approach, we further examined how these associations varied spatially among geographic regions. Although malformations were rare overall (average = 1.6%), we identified 96 hotspot sites with 5%-25% abnormal individuals. Using the full dataset of 934 collections (without data on parasite infection), malformation frequency was best predicted by the presence of oil and gas wells within the watershed. Among collections also examined for parasite infection (n = 154), average parasite load and its interaction with pesticide application positively predicted malformations: wetlands with a greater abundance of the trematode Ribeiroia ondatrae were more likely to have malformed amphibians, but these effects were strongest when pesticide application was also high, consistent with prior experimental research. Importantly, however, the influence of these factors also varied regionally, helping explain divergent results from previous studies at local scales; parasite infection was more influential in the West and Northeast, whereas pesticide application and oil/gas wells correlated with abnormalities in the Northeast, Southeast, and western regions of the USA. These results, based on the largest systematic sampling of amphibian malformations, suggest that increased observations of abnormal amphibians are associated with both parasite infection and chemical contaminants, but that their relative importance and interaction strength varied with the spatial extent of the analysis.

A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues.

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

Road proximity increases risk of skeletal abnormalities in wood frogs from National Wildlife Refuges in Alaska.

BACKGROUND: Skeletal and eye abnormalities in amphibians are not well understood, and they appear to be increasing while global populations decline. Here, we present the first study of amphibian abnormalities in Alaska. OBJECTIVE: In this study we investigated the relationship between anthropogenic influences and the probability of skeletal and eye abnormalities in Alaskan wood frogs (Rana sylvatica). METHODS: From 2000 to 2006, we examined 9,269 metamorphic wood frogs from 86 breeding sites on five National Wildlife Refuges: Arctic, Innoko, Kenai, Tetlin, and Yukon Delta. Using road proximity as a proxy for human development, we tested relationships between skeletal and eye abnormalities and anthropogenic effects. We also examined a subsample of 458 frogs for the trematode parasite Ribeiroia ondatrae, a known cause of amphibian limb abnormalities. RESULTS: Prevalence of skeletal and eye abnormalities at Alaskan refuges ranged from 1.5% to 7.9% and were as high as 20% at individual breeding sites. Proximity to roads increased the risk of skeletal abnormalities (p = 0.004) but not eye abnormalities. The only significant predictor of eye abnormalities was year sampled (p = 0.006). R. ondatrae was not detected in any Alaskan wood frogs. CONCLUSIONS: Abnormality prevalence at road-accessible sites in the Kenai and Tetlin refuges is among the highest reported in the published literature. Proximity to roads is positively correlated with risk of skeletal abnormalities in Alaskan wood frogs.

Predicting risk of trace element pollution from municipal roads using site-specific soil samples and remotely sensed data.

Studies of environmental processes exhibit spatial variation within data sets. The ability to derive predictions of risk from field data is a critical path forward in understanding the data and applying the information to land and resource management. Thanks to recent advances in predictive modeling, open source software, and computing, the power to do this is within grasp. This article provides an example of how we predicted relative trace element pollution risk from roads across a region by combining site specific trace element data in soils with regional land cover and planning information in a predictive model framework. In the Kenai Peninsula of Alaska, we sampled 36 sites (191 soil samples) adjacent to roads for trace elements. We then combined this site specific data with freely-available land cover and urban planning data to derive a predictive model of landscape scale environmental risk. We used six different model algorithms to analyze the dataset, comparing these in terms of their predictive abilities and the variables identified as important. Based on comparable predictive abilities (mean R2 from 30 to 35% and mean root mean square error from 65 to 68%), we averaged all six model outputs to predict relative levels of trace element deposition in soils-given the road surface, traffic volume, sample distance from the road, land cover category, and impervious surface percentage. Mapped predictions of environmental risk from toxic trace element pollution can show land managers and transportation planners where to prioritize road renewal or maintenance by each road segment's relative environmental and human health risk.

Localized hotspots drive continental geography of abnormal amphibians on U.S. wildlife refuges.

Amphibians with missing, misshapen, and extra limbs have garnered public and scientific attention for two decades, yet the extent of the phenomenon remains poorly understood. Despite progress in identifying the causes of abnormalities in some regions, a lack of knowledge about their broader spatial distribution and temporal dynamics has hindered efforts to understand their implications for amphibian population declines and environmental quality. To address this data gap, we conducted a nationwide, 10-year assessment of 62,947 amphibians on U.S. National Wildlife Refuges. Analysis of a core dataset of 48,081 individuals revealed that consistent with expected background frequencies, an average of 2% were abnormal, but abnormalities exhibited marked spatial variation with a maximum prevalence of 40%. Variance partitioning analysis demonstrated that factors associated with space (rather than species or year sampled) captured 97% of the variation in abnormalities, and the amount of partitioned variance decreased with increasing spatial scale (from site to refuge to region). Consistent with this, abnormalities occurred in local to regional hotspots, clustering at scales of tens to hundreds of kilometers. We detected such hotspot clusters of high-abnormality sites in the Mississippi River Valley, California, and Alaska. Abnormality frequency was more variable within than outside of hotspot clusters. This is consistent with dynamic phenomena such as disturbance or natural enemies (pathogens or predators), whereas similarity of abnormality frequencies at scales of tens to hundreds of kilometers suggests involvement of factors that are spatially consistent at a regional scale. Our characterization of the spatial and temporal variation inherent in continent-wide amphibian abnormalities demonstrates the disproportionate contribution of local factors in predicting hotspots, and the episodic nature of their occurrence.

Petroleum hydrocarbon contamination in Nelson Lagoon, Alaska, sampling three different matrices.

Polycyclic aromatic hydrocarbon (PAH) levels were measured in sediments, bivalves and semi-permeable membrane devices (SPMDs) in the relatively pristine marine environment of Nelson Lagoon, Alaska. Most PAH levels in Nelson Lagoon were low, and similar to global background concentrations. Sampling media type can significantly influence conclusions of PAH contamination in the environment. Concentration of a broad size range of PAHs was observed in the tissues of blue mussels (Mytilus edulis). SPMDs collected some two- to three-ring PAHs from the dissolved water phase, while sediments collected five- to six-ring PAHs that were likely adsorbed onto particulate matter. Benzo(a)pyrene, a potent carcinogen, was found in mussels at levels similar to more industrialized harbors in Alaska.