Kevin B. Lunde

PARASITE (RIBEIROIA ONDATRAE) INFECTION LINKED TO AMPHIBIAN MALFORMATIONS IN THE WESTERN UNITED STATES

Parasites and pathogens can influence the survivorship, behavior, and very structure of their host species. For example, experimental studies have shown that trematode parasites can cause high frequencies of severe limb malformations in amphibians. In a broad-scale field survey covering parts of California, Oregon, Washington, Idaho, and Montana, we examined relationships between the frequency and types of morphological abnormalities in amphibians and the abundance of trematode parasite infection, pH, con- centrations of 61 pesticides, and levels of orthophosphate and total nitrate. We recorded severe malformations at frequencies ranging from 1% to 90% in nine amphibian species from 53 aquatic systems. Infection of larvae by the trematode Ribeiroia ondatrae was associated with, and functionally related to, higher frequencies of amphibian limb malfor- mations than found in uninfected populations (#5%). Parasites were concentrated around the basal tissue of hind limbs in infected anurans, and malformations associated with infection included skin webbings, supernumerary limbs and digits, and missing or mal- formed hind limbs. In the absence of Ribeiroia, amphibian populations exhibited low (0- 5%) frequencies of abnormalities involving missing digits or distal portions of a hind limb. Species were affected differentially by the parasite, and Ambystoma macrodactylum , Hyla regilla, Rana aurora, R. luteiventris, and Taricha torosa typically exhibited the highest frequencies of abnormalities. None of the water-quality variables measured was associated with malformed amphibians, but aquatic snail hosts (Planorbella spp.) were significant predictors of the presence and abundance of Ribeiroia infection. Morphological comparisons of adult specimens of Ribeiroia collected from different sites and raised in experimental definitive hosts suggested that all samples represented the same species— R. ondatrae. These field results, coupled with experimental research on the effects of Ribeiroia on amphibians, demonstrate that Ribeiroia infection is an important and widespread cause of amphibian limb malformations in the western United States. The relevance of trematode infection to declines of amphibian populations and the influence of habitat modification on the pathology and life cycle of Ribeiroia are emphasized as areas requiring further research.

Long-term macroinvertebrate responses to climate change: implications for biological assessment in mediterranean-climate streams

Abstract Climate change is expected to have strong effects on mediterranean-climate regions worldwide. In some areas, these effects will include increases in temperature and decreases in rainfall, which could have important implications for biological assessment programs of aquatic ecosystems. We used a consistently collected, 20-y benthic macroinvertebrate data set from 4 sites along 2 small northern California streams to examine potential climate-change effects on aquatic communities. The sites represented unique combinations of stream order and flow intermittency. The North Coast benthic macroinvertebrate index of biotic integrity (B-IBI) developed for northern California streams was not influenced by temperature extremes (cool and warm) or precipitation extremes (wet and dry). Other common indices and metrics used in biological monitoring studies, such as the ratio of observed to expected taxa (O/E), % Ephemeroptera, Plecoptera, and Trichoptera (EPT) individuals, and total richness were unaffected by temperature and precipitation variability. For future monitoring of climate-change effects on small streams, we developed a local climate-change indicator that is composed of the presence/absence of 9 macroinvertebrate taxa, identified to genus level. This indicator detected significant differences between years that were grouped based on temperature, precipitation, and a combination of temperature and precipitation. It also detected significant differences between groups in an external data set including 40 reference sites throughout the San Francisco Bay area, a result that suggests this indicator could be used at larger spatial scales in this region. Two biological trait categories found in large, long-lived organisms decreased with increasing temperature and decreasing precipitation at the most intermittent site. This result indicates that climate change might selectively affect taxa with certain traits. The robustness of the North Coast B-IBI and other common indices and metrics to temperature and precipitation variability demonstrates their continued applicability for examining water quality under future climate-change scenarios, but suggests that they probably will not be good indicators for detecting climate-change effects. The effects of climate change in mediterranean-climate streams can be monitored effectively within the framework of existing biological assessment programs by using regional indicators based on specific taxa identified to the generic level and information on their species traits.

Development and validation of a macroinvertebrate index of biotic integrity (IBI) for assessing urban impacts to Northern California freshwater wetlands.

Despite California policies requiring assessment of ambient wetland condition and compensatory wetland mitigations, no intensive monitoring tools have been developed to evaluate freshwater wetlands within the state. Therefore, we developed standardized, wadeable field methods to sample macroinvertebrate communities and evaluated 40 wetlands across Northern California to develop a macroinvertebrate index of biotic integrity (IBI). A priori reference sites were selected with minimal urban impacts, representing a best-attainable condition. We screened 56 macroinvertebrate metrics for inclusion in the IBI based on responsiveness to percent urbanization. Eight final metrics were selected for inclusion in the IBI: percent three dominant taxa; scraper richness; percent Ephemeroptera, Odonata, and Trichoptera (EOT); EOT richness; percent Tanypodinae/Chironomidae; Oligochaeta richness; percent Coleoptera; and predator richness. The IBI (potential range 0–100) demonstrated significant discriminatory power between the reference (mean = 69) and impacted wetlands (mean = 28). It also declined with increasing percent urbanization (R2 = 0.53, p < 0.005) among wetlands in an independent validation dataset (n = 14). The IBI was robust in showing no significant bias with environmental gradients. This IBI is a functional tool to determine the ecological condition at urban (stormwater and flood control ponds), as well as rural freshwater wetlands (stockponds, seasonal wetlands, and natural ponds). Biological differences between perennial and non-perennial wetlands suggest that developing separate indicators for these wetland types may improve applicability, although the existing data set was not sufficient for exploring this option.

Parasitic Copepod (Lernaea cyprinacea) Outbreaks in Foothill Yellow-legged Frogs (Rana boylii) Linked to Unusually Warm Summers and Amphibian Malformations in Northern California

Abstract How climate change may affect parasite–host assemblages and emerging infectious diseases is an important question in amphibian decline research. We present data supporting a link between periods of unusually warm summer water temperatures during 2006 and 2008 in a northern California river, outbreaks of the parasitic copepod Lernaea cyprinacea, and malformations in tadpoles and young of the year Foothill Yellow-legged Frogs (Rana boylii). Relative to baseline data gathered since 1989, both 2006 and 2008 had significantly longer periods when daily mean water temperatures exceeded 20°C compared to years without copepod outbreaks. Infestation varied spatially in the watershed, as prevalence increased concomitantly with temperature along a 5.2 km longitudinal transect. At breeding sites of R. boylii with copepods in 2006, infestation ranged from 2.9% of individuals upstream to 58.3% downstream. In 2008, copepods were absent from the most upstream sites and infested up to 28.6% of individuals sampled at downstream locations. Copepods most frequently embedded near a hind limb or the cloaca. Among individuals with parasites in 2006, 26.5% had morphological abnormalities compared to 1.1% of un-infested individuals. In 2008 when the infestation peak occurred late in development (post Gosner stage 39), abnormalities were not associated with copepod infestation. In both years, recently metamorphosed frogs with copepods were, on average, slightly smaller than those not infested. These occurrences represent a sudden increase in local prevalence atypical for this river ecosystem. Previously we had only once seen copepods on amphibians (on non-native Bullfrogs, Rana catesbeiana), six km further downstream. Pacific Chorus Frogs, Pseudacris regilla, which co-occur with R. boylii in shallow near shore habitats were not used as hosts. The data suggest that increasing summer water temperatures, decreased daily discharge, or a combination of both, promote outbreaks of this non-native parasite on an indigenous host, and could present a threat to the long-term conservation of R. boylii under the flow regime scenarios predicted by climate change models.

A Practical Guide for the Study of Malformed Amphibians and Their Causes

Abstract Reports of severely malformed amphibians in the 1990s prompted researchers to examine the causes and extent of the issue. However, disparities in survey methods and a shortage of baseline data have hindered standardization among investigations. Here, we review field-based surveys and experimental approaches used over the last decade to address this complex ecological issue. We offer specific recommendations regarding amphibian-sampling techniques, including methods to capture and examine amphibians, with the aim of enhancing the accessibility of this topic to scientists, students, and educators of diverse backgrounds. Based on established approaches from epidemiology, we provide recommendations regarding methods to identify proximate causes(s) of abnormalities with a focus on using “multiple lines of evidence,” including large-scale field surveys, comparing malformation “signatures” between field and laboratory studies, and using of manipulative experiments at multiple spatial scales. As an example, we describe methods to examine the causal influence of infection by the trematode parasite, Ribeiroia ondatrae, including quantifying presence and abundance within amphibian and snail host populations with adequate power of detection. We conclude by identifying outstanding questions with the goal of stimulating additional research to evaluate the causes and consequences of amphibian malformations.

Evaluating the adequacy of a reference-site pool for ecological assessments in environmentally complex regions

Many advances in the field of bioassessment have focused on approaches for objectively selecting the pool of reference sites used to establish expectations for healthy waterbodies, but little emphasis has been placed on ways to evaluate the suitability of the reference-site pool for its intended applications (e.g., compliance assessment vs ambient monitoring). These evaluations are critical because an inadequately evaluated reference pool may bias assessments in some settings. We present an approach for evaluating the adequacy of a reference-site pool for supporting biotic-index development in environmentally heterogeneous and pervasively altered regions. We followed common approaches for selecting sites with low levels of anthropogenic stress to screen 1985 candidate stream reaches to create a pool of 590 reference sites for assessing the biological integrity of streams in California, USA. We assessed the resulting pool of reference sites against 2 performance criteria. First, we evaluated how well the reference-site pool represented the range of natural gradients present in the entire population of streams as estimated by sites sampled through probabilistic surveys. Second, we evaluated the degree to which we were successful in rejecting sites influenced by anthropogenic stress by comparing biological metric scores at reference sites with the most vs fewest potential sources of stress. Using this approach, we established a reference-site pool with low levels of human-associated stress and broad coverage of environmental heterogeneity. This approach should be widely applicable and customizable to particular regional or programmatic needs.

Using an ecosystem‐level manipulation to understand host‐parasite interactions and how they vary with study venue

We investigated interactions between the virulent trematode Ribeiroia ondatrae, which has been linked to amphibian malformations across the United States, and its amphibian host (Pseudacris regilla) using a hierarchical approach involving multi-year regional field surveys, replicated pond enclosures, and an unreplicated ecosystem manipulation of parasite infection. Results of this multi-tiered approach provide strong evidence of the causal linkage between infection and malformations while offering additional insights about the influence of experimental venue on effect size. Among years and across 18 wetlands in northern California, Ribeiroia infection was a strong predictor of malformation frequency, which ranged from 0 to 77% at all sites (n = 6,511). Correspondingly, the addition of >500 Ribeiroia infected snails to an experimentally divided wetland using a three-year Before After Control Impact (BACI) design caused sharp increases in Ribeiroia infection and severe malformations in P. regilla during the manipulation year (but not pre- or post-manipulation). These results were complemented by the findings from a replicated (n = 16) enclosure/exclosure study conducted on both sides of the divided wetland (Hog Lake), which showed increased infection and malformations only among larvae within cages that allowed parasite entry and only on the manipulated side. No differences in mortality were observed among animals as a function of cage type. A comparison of the slope between observed infection and malformations as a function of venue (previous laboratory work and the two spatial scales of this study (cages and whole-pond)) indicated that small-scale experiments exhibit stronger effects relative to results from larger spatial extents. Multi-year sampling also indicated that malformed frogs were unlikely to return as breeding adults, highlighting the potential for population-level impacts associated with high Ribeiroia infections. Taken together, these results provide support for the causal relationship between Ribeiroia infection and amphibian malformations under realistic conditions while simultaneously emphasizing the influence of study venue on the strength of this relationship.

Seasonal Progression of Aquatic Organisms in a Temporary Wetland in Northern California

Abstract. Seasonal wetlands are important habitats for biodiversity of both invertebrate and vertebrate fauna. Many aquatic species have life history traits adapted to colonizing and developing in temporary aquatic habitats, and these traits influence the annual succession of the macroinvertebrate community. The chronology of taxon appearance and the variation in relative abundances during the hydroperiod are important for understanding population dynamics, trophic interactions, and responses to drought. This study investigated the successional changes in macroinvertebrate abundances in a seasonal wetland in northern California. Water quality parameters were measured regularly, including dissolved oxygen, temperature, pH, surface area, and specific conductance during the wet season ( January–July) in 2007–2009. Macroinvertebrates were collected with net sweeps (mesh > 500 mm), and the presence of life stages of amphibians were visually observed from March to June each year. As the hydroperiod progressed, wetland surface area decreased, while water temperature and specific conductance increased. Macroinvertebrate abundance increased with the progression of the hydroperiod, and the richness of macroinvertebrate predator taxa tripled from 2 families in March to 6 families in June. The earliest part of the hydroperiod in the wetland was dominated by Cyzicus clam shrimp and Linderiella occidentalis fairy shrimp. Limnephilus caddisfly larvae were few in number but were found exclusively in the early season. Sequential changes of dominant invertebrate taxa and relative abundances of macroinvertebrates were evident, particularly among several macroinvertebrate predators. Among these predators, the early-season community was dominated by larval dytiscid beetles, while later-season communities demonstrated increased predator richness (e.g., Notonecta backswimmers) and were dominated by Lestes damselflies. Larvae of the vertebrate predator Taricha torosa, which may act as a top predator, were present during the later stages of the hydroperiod. The phenology of individual aquatic taxa and their specific life history strategies may impact the sensitivity of macroinvertebrate populations to increased annual variation in hydroperiod that may result from climate changes in this region.