Tawnya L. Cary

Fipronil effects on estuarine copepod (Amphiascus tenuiremis) development, fertility, and reproduction: a rapid life-cycle assay in 96-well microplate format.

Fipronil is a novel gamma-aminobutyric acid receptor-specific phenylpyrazole insecticide commonly used near estuarine environments for rice production, turf-grass management, and residential insect control. In this study, we evaluated the acute, developmental, and reproductive toxicity of fipronil to the estuarine harpacticoid copepod Amphiascus tenuiremis. Fipronil was highly toxic to A. tenuiremis (adult 96-h median lethal concentration [LC50] = 6.8 microg/L) and was more toxic to male copepods (96-h LC50 = 3.5 microg/L) than to nongravid female copepods (96-h LC50 = 13.0 microg/L). By using a newly developed 96-well microplate-based life-cycle toxicity test, we successfully reared single individuals of A. tenuiremis to adulthood in 200-microl microwells and concurrently assessed developmental and reproductive effects (after paired virginal matings) of environmentally relevant aqueous fipronil concentrations (0.16, 0.22, and 0.42 microg/L measured). Throughout the entire life cycle, copepod survival in all treatments was >90%. However, fipronil at 0.22 microg/L and higher significantly delayed male and female development from stage 1 copepodite to adult by approximately 2 d. More importantly, fipronil significantly halted female egg extrusion by 71% in the 0.22-microg/L fipronil treatment, and nearly eliminated reproduction (94% failure) in the 0.42-microg/L fipronil treatment. A three-generation Leslie matrix-based population growth model of fipronil reproductive and life-cycle impacts predicted a 62% decline in population size of A. tenuiremis relative to controls at only 0.16 microg/L.

Restructuring of the amphibian gut microbiota through metamorphosis

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.

Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis

SUMMARY 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.

Larval exposure to polychlorinated biphenyl 126 (PCB-126) causes persistent alteration of the amphibian gut microbiota.

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.

Immunomodulation in Post-metamorphic Northern Leopard Frogs, Lithobates pipiens, Following Larval Exposure to Polybrominated Diphenyl Ether

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.

Interspecific and Postmetamorphic Variation in Susceptibility of Three North American Anurans to Batrachochytrium dendrobatidis

Abstract Batrachochytrium dendrobatidis(Bd), the fungus responsible for chytridiomycosis, grows on the keratinized parts of the body (i.e., the entire skin of juveniles and adults, and the oral disk of tadpoles). The fungus affects epidermal structures, generating an imbalance of the osmotic equilibrium through the skin that ultimately leads to death. Therefore, larvae are likely to be much more resistant compared with juveniles and adults, which may suffer high mortality. However, nothing is known about how susceptibility varies during the juvenile life stages (from the end of metamorphosis to sexual maturity). The coexistence of tolerant hosts (either tolerant species or unaffected developmental stages) with susceptible hosts is a major reason why chytridiomycosis has become an epidemic disease. The aim of this study was to analyze the interspecific and developmental variation in susceptibility to Bd in juveniles of three North American anurans. Leopard Frog (Lithobates pipiens) incurred no lethal effects at 104 Bd zoospores ml−1, regardless of the age at which they were exposed to the fungus. On the contrary, Bd infection was more severe for newly metamorphosed juveniles of American toads (Anaxyrus americanus) than for 4-week-old juveniles. When exposed to 104 Bd zoospores ml−1, newly metamorphosed juveniles of this species experienced mortality rates above 70%, whereas 4-week-old juveniles had mortality rates below 30%. Variations in structural characteristics of the skin or the antifungal efficiency of skin peptides are proposed as potential reasons to explain the observed developmental differences in susceptibility to Bd. Resumen Batrachochytrium dendrobatidis (Bd), el hongo responsable de la quitridiomicosis, crece sobre las partes queratinizadas del cuerpo (i.e. la piel de juveniles y adultos y el disco oral de las larvas). El hongo afecta a las estructuras epidérmicas generando una alteración del equilibrio osmótico en la piel que termina produciendo la muerte. De este modo, las larvas suelen ser mucho más resistentes que los juveniles o los adultos, los cuales pueden sufrir elevadas mortalidades. Sin embargo, se desconoce cómo varía la susceptibilidad a lo largo de la etapa juvenil (desde el final de la metamorfosis hasta la adquisición de la madurez sexual). La coexistencia de hospedadores tolerantes (ya sean especies tolerantes o estadios de desarrollo que no se vean afectados) con hospedadores susceptibles es una de las principales razones por las que la quitridiomicosis se ha convertido en una epidemia. El objeto de este estudio fue analizar las variaciones interespecíficas y a lo largo del desarrollo en la susceptibilidad de tres especies de anuros de Norteamérica a Bd. Los juveniles de rana leopardo (Lithobates pipiens) no sufrieron efectos letales a 104 zoosporas de Bd mL−1, independientemente de la edad a la que fueron expuestos al hongo. Por el contrario, la infección por Bd resultó más severa para individuos recién metamorfoseados de sapo americano (Anaxyrus americanus) que para juveniles con cuatro semanas de vida. Al exponerlos a 104 zoosporas de Bd mL−1, los juveniles recién metamorfoseados experimentaron tasas de mortalidad por encima del 70%, mientras que los juveniles con cuatro semanas de edad sufrieron tasas de mortalidad inferiores al 30%. Las variaciones en las características estructurales de la piel y/o en la eficacia antifúngica de los péptidos cutáneos se proponen como razones potenciales para explicar las diferencias en la susceptibilidad a Bd observadas a lo largo del desarrollo.

Risk assessment of the National Institute of Standards and Technology petroleum crude oil standard water accommodated fraction: Further application of a copepod‐based, full life‐cycle bioassay

The U.S. National Institute of Standards and Technology (NIST) petroleum crude oil was used to generate NIST water-accommodated hydrocarbon fractions (WAFs) for standardized assessment of crude oil effects on the copepod Amphiascus tenuiremis. Effects were assessed using a 96-well microplate, full life-cycle test. Briefly, nauplii (age, 24 h) were reared individually to adults (n > or =120 nauplii/treatment) in microplate wells containing 200 microl of treatment solution (seawater control [0%] or 10, 30, 50, or 100% NIST-WAF). Nauplii were monitored through development to adulthood, and mature virgin male:female pairs mated in wells containing original treatments (<30 d). A second bioassay using 0, 10, 30, and 50% WAFs (n > or =60 nauplii/treatment) was conducted to assess the effects of ultraviolet (UV) light on naupliar endpoints (<16 d). In the first experiment, nauplius-to-copepodite survival in exposures to 100% WAF was 27% +/- 6% lower than in controls (92% +/- 1%), but copepodite-to-adult survival was greater than 90% across all treatments. Analysis of development curves showed that nauplii in the 10% WAF developed into copepodites 25% faster, whereas nauplii in the 50 and 100% WAFs developed 17% slower, than controls. Copepodite development into male and female copepods was significantly delayed (2 and 4 d, respectively) in the 100% WAF compared to controls. Although none of the WAF exposures had significant effects on fertilization success or total viable production (p > 0.05), embryo hatching in the 100% WAF was significantly less (70.0% +/- 21.2%) than that in controls (87.0% +/- 19.4%). Results from the UV bioassay showed that relatively short exposures (<14 d) to 30 and 50% WAFs in the presence of UV light caused negative effects on copepod survival and development. Naupliar-stage survival and developmental endpoints were the most sensitive indicators of exposure to the NIST crude oil WAF

Effects of chronic polybrominated diphenyl ether exposure on gonadal development in the northern leopard frog, Rana pipiens

Polybrominated diphenyl ethers (PBDEs) are bioaccumulative, persistent organic pollutants used as flame retardants in consumer goods. Concentrations of PBDEs in North American wildlife have been increasing for decades and been shown to have estrogenic effects on sexual development. No studies, however, have examined the effects of PBDEs on the sexual development of North American frogs at ecologically relevant concentrations. This study examined the effects of five dietary concentrations of DE-71 (0, 1.1, 6.1, 71.4, and 634 ng ΣPBDEs/g diet), a technical PBDE mixture, on the gonadal development of the northern leopard frog, Rana pipiens. Tadpoles were exposed chronically from the time they became free-swimming until metamorphosis. Frogs were killed either at metamorphic climax or 10 weeks after completing metamorphosis, processed for histology, and examined for alterations in sexual development. The experimental group exposed to PBDEs at 1.1 ng/g had a significantly larger proportion of females compared with the expected 50:50 sex ratio. At 10 weeks post-metamorphosis, male frogs exposed to 6.1 and 71.4 ng/g had significantly smaller testes, but all other measure of gonadal development tested showed no effects. No intersex or increased incidence of gonadal abnormality were detected. These findings indicate that PBDEs may disrupt sexual differentiation in frogs at low, environmentally relevant concentrations.

Toxicokinetics of polybrominated diphenyl ethers across life stages in the northern leopard frog (Lithobates pipiens)

Polybrominated diphenyl ethers (PBDEs), a class of flame retardants, are bioaccumulative toxins that can biomagnify in food webs. However, little is known about the toxicokinetics of total and congener-specific BDEs in lower vertebrates. The authors exposed northern leopard frog (Lithobates (Rana) pipiens) tadpoles to diets containing DE-71 (a pentabromodiphenyl ether mixture (0 ng/g as control, 71.4 ng/g, and 634 DE-71 ng/g wet mass)) for 50 d, followed by a period of depuration during which they were fed only undosed (control) food. After 28 d, tadpoles eliminated over 94% of the ΣPBDEs from their tissues (t½  = 5.9 ± 1.9 d) with no significant differences in elimination rates for the predominant congeners. Elimination of BDE-99 was independent of dose, indicating first-order kinetics. It did not fit a biexponential model significantly better than a monoexponential model, indicating single-compartment elimination. To compare developmental life-stage kinetics following larval exposure, the authors collected individuals at the beginning and end of metamorphosis and at 70 d postmetamorphosis. During metamorphosis, total-body residues per individual did not significantly change, implying little to no elimination. After 70 d, juvenile frogs eliminated 89.7% of the ΣPBDEs from their tissues, and BDE-47 was eliminated at a faster rate (t½ = 17.3 d) than BDE-99 and BDE-100 (t½  = 63.0 d and 69.3 d, respectively). Because the kinetics of PBDEs in L. pipiens differed among life stages, developmental life stage-especially for species that undergo metamorphosis-should be considered when determining the toxicity of persistent organic pollutants.

Conceptual Elements: A Detailed Framework to Support and Assess Student Learning of Biology Core Concepts.

The authors break down the Vision and Change core concepts into elements that transcend biological subdisciplines and scales in a new learning framework. The Conceptual Elements Framework can be used to guide the development of core concept learning outcomes, instructional materials, and assessments of student learning.