Immunomodulation in Post-metamorphic Northern Leopard Frogs

Article pubs.acs.org/est

Immunomodulation in Post-metamorphic Northern Leopard Frogs, Lithobates pipiens, Following Larval Exposure to Polybrominated Diphenyl Ether Tawnya L. Cary,*,† Manuel E. Ortiz-Santaliestra,‡ and William H. Karasov†,‡ †

Department of Zoology and ‡Department of Forest & Wildlife Ecology, University of Wisconsin, Madison, Wisconsin 53706, United States ABSTRACT: 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.

■

INTRODUCTION Global amphibian populations have been declining at an alarming rate, and currently almost one-third of amphibian species are endangered.1,2 Pollution is a key factor contributing to the loss of amphibian biodiversity,2 and one class of flame retardants, polybrominated diphenyl ethers (PBDEs), has garnered increased attention over the past two decades. These compounds have been used extensively in plastics and textiles and are ubiquitous in the environment in both biotic and abiotic matrices.3,4 Although production of the penta- and octa-BDE mixtures has been banned in the European Union and voluntarily phased out in the United States, lowerbrominated PBDE concentrations continue to persist at levels reported to cause toxicity to humans and wildlife.5−7 This is likely due to environmental persistence of PBDEs and the debromination of the deca-brominated congener BDE-209,8−10 which has remained in production in the United States (although production, sale, and import of decaBDE will be phased out by the end of 201311). Concentrations of PBDEs in Great Lakes food webs range from 1.4 ng/g in zooplankton to >1000 ng/g tissue in top-predator fishes.3,12 Water concentrations of PBDEs are relatively low due to the hydrophobic nature of PBDEs (54.9 pg BDE-47/L in San Francisco Bay watershed (no Great Lakes data available)13), while organic © 2014 American Chemical Society

matter contains greater concentrations and creates the basis for potential biomagnification (tri- through hepta-BDEs in surficial sediments are 2.8 ng/g dry wt).14 Toxicity of PBDE exposure includes impaired neurological function (decreased spatial memory and learning15,16), thyroid hormone disruption,17−19 and developmental20−23 and reproductive toxicity.24,25 Additionally, alterations in immune function have been reported in humans and mice.26,27 Mice subchronically exposed to DE-71, a commercial pentaBDE mixture, had suppressed plaque-forming cell response against sheep red blood cells and decreased thymus weight.28 Exposure to BDE-47, a major congener component of DE-71, reduced splenocyte numbers in mice.29 Ranch mink, Mustela vison, exposed to DE-71 had altered secondary antibody production to keyhole limpet hemocyanin as well as a higher percentage of neutrophils compared to nonexposed individuals.30 In contrast, these same DE-71-exposed mink had lowered hematocrits and lowered percent lymphocytes compared to control mink.30 Amphibians have many of the same immune components as Received: Revised: Accepted: Published: 5910

December 27, 2013 March 30, 2014 April 15, 2014 April 15, 2014 dx.doi.org/10.1021/es405776m | Environ. Sci. Technol. 2014, 48, 5910−5919

Environmental Science & Technology

Article

mammals,31,32 and therefore, PBDEs may have similar impacts on anuran immune function. Also, thyroid hormone homeostasis is necessary for normal immunological development in frogs,33 and toxicants that disturb neuroendocrine control of metamorphosis may also alter immune function. It is hypothesized that direct or indirect suppression of the immune system by contaminants could prevent adequate immune responses in amphibians leading to increased susceptibility to pathogens.34 Because disease is another major factor in amphibian population declines,2 it is of interest to assess immunotoxicity in amphibians, and recently, increased disease susceptibility following tadpole exposure to an herbicide was shown in juvenile tree frogs.35 Although there has been extensive research on the immune system of Xenopus laevis (reviewed in refs 32 and 36), only a few studies have tested the effects of contaminants on amphibian immune function (e.g., refs 37 and 38). Our goal was to assess whether PBDE exposure of larval Lithobates (Rana) pipiens resulted in long lasting, carryover effects on the immune function of post-metamorphic frogs. The present study tested for changes in measures of both innate and adaptive immunity in northern leopard frogs in response to dietary exposure of environmentally relevant levels of PBDEs and, to our knowledge, is the first study that investigated immunotoxic effects of PBDEs in amphibians.

and zooplankton from the Laurentian Great Lakes’ waters.3,12 Specifically, tadpoles fed concurrently in our facility on two of the same diets described above (71.4 and 634 ng PBDE/g) had body burden residues of 46.3 and 632.4 ng PBDE/g tissue, respectively, after feeding for 50 days.43 The fertilized embryos obtained from Nasco are offspring of field-collected adult males and females from southeastern Minnesota and Wisconsin, such that we expect the tadpoles in our study to be to representative of natural populations. However, information regarding genetic relatedness within and/or across batches was not provided by the supplier, so as a precaution batches of tadpoles were not mixed and each batch was replicated twice at each treatment level. Therefore, each dietary treatment was replicated in 10 different glass aquaria (5 batches × 2 aquaria/batch) containing 20 tadpoles each in 12 L of dechlorinated, filtered tap water. Tadpoles were housed communally at this density (1.7 tadpoles per 1 L water) in order to balance healthy living conditions and resources necessary to accommodate this number of tadpoles. Environmental conditions were 14:10 h light:dark light cycle, 23 ± 1 °C water temperature, and humidity ≥30%. Static renewal of the aquaria water (≥80% water change) was performed four times per week to provide satisfactory water quality: pH = 7.8− 8.3, nitrite < 1.0 mg/L, total NH3 < 1 mg/L). Additionally, air stones in each aquarium provided sufficient aeration throughout the experiment (dissolved oxygen ≥75% saturation). Tadpoles were fed ad libitum each day by placing three blocks of the appropriate diet into the aquaria (the size of blocks increased with tadpole growth but averaged 0.5 g wet mass; range of 0.25−0.95 g). Prior to water changing and the addition of fresh food, any remaining food from the previous day as well as feces were removed. The present immune toxicity study was part of a broader experiment designed to test for both immune and reproductive toxicity of PBDE exposure. The findings of reproductive toxicity have been reported previously.41 Tadpoles were assigned to each component of the broader study by using a lottery system within each replicated aquarium to ensure random placement. Therefore, only a portion of the frogs described above was assigned to the present study to analyze immunotoxic end points [at metamorphic climax (GS 42), 218 frogs were assigned to immunotoxic end points; however only 102 frogs survived to 10 weeks post-metamorphosis for sample collection]. Dietary exposure continued until metamorphic climax. When larvae reached metamorphic climax (emergence of forelimbs at GS 42), they were removed from the aquaria and placed individually into a 500 mL polypropylene jar. Because metamorphosis and early post-metamorphosis are sensitive periods for frogs, we housed frogs individually to minimize mortality risks (e.g., competition, pathogen transmission, stress). Twenty-five milliliters of dechlorinated, filtered tap water was added to the jars, and the jars were tilted to provide both a wet and dry surface for the metamorphosing frogs. Frogs were not fed during metamorphosis as tail resorption and fat reserves provide adequate energy during this time period. Newly metamorphosed juveniles (GS 46) were fed uncontaminated crickets and mealworms six times per week until collection at 10 weeks post-metamorphosis. In order to provide proper nutrition, crickets were dusted with calcium and vitamins prior to being fed to the froglets. The depuration of contaminant levels during this period (e.g., ref 43) is not a major concern because the primary research question focuses

■

METHODS Study Organism. Five batches of Lithobates pipiens embryos (∼200 embryos/batch) were purchased from Nasco (Ft. Atkinson, WI, USA), transferred to the laboratory, and immediately placed in 0.5 L Nalgene containers filled with 300 mL of dechlorinated, filtered tap water. Each container housed 40 embryos, and water was changed twice daily until hatch. Nonviable embryos were removed daily to minimize bacterial growth. Following hatch, the number of tadpoles was culled to 20 healthy tadpoles per container, and water was changed daily until the tadpoles became free swimming, developed operculum, and began foraging (developmental Gosner Stage 25, GS 2539). All experimental procedures involving tadpoles and/or recent metamorphs were approved by the University of Wisconsin’s Institutional Animal Care and Use Committee. Dietary PBDE Exposure of Tadpoles. Polybrominated diphenyl ethers are lipophilic compounds with high octanol− water partition coefficients (e.g., log KOW pentaBDE mixture = 6.5840). Because PBDEs are readily detected in sediments and biota and less so in dissolved and/or particulate aquatic phases,4 we used dietary exposure to better mimic the primary ecological exposure route. Beginning on February 22, 2009, free-swimming tadpoles (GS 25) were transferred to 18.9 L aquaria and fed diets without (control) and with a technical pentaBDE mixture, DE-71, at four doses. The technical DE-71 mixture consisted primarily of pentaBDE [56%], tetraBDE [35%], and hexaBDE [9%] and was purchased from Wellington Laboratories (product TBDE-71; purity undetermined; percentages as determined by Wellington Laboratories). Control and PBDE-spiked tadpole food were made using powderized rabbit chow23 (Harlan Rabbit Chow catalog no. 2030, 3.3% lipids). Measured concentrations of DE-71 adsorbed to the diets were 0 (nondetect), 1.1, 6.1, 71.4, and 634 ng DE-71/g diet wet weight.41 These exposure levels have been previously reported to yield ecologically relevant tissue concentrations in tadpoles and froglets (≤660 ng ΣPBDEs/g wet mass42) and were chosen based on a previous PBDE exposure study with L. pipiens23 and reported PBDE body burdens in fish, crustaceans, 5911

dx.doi.org/10.1021/es405776m | Environ. Sci. Technol. 2014, 48, 5910−5919


Article

anti-Xenopus IgY (11D5) or mouse anti-Xenopus IgM (10A9)] was added to each well. Following incubation, the plates were washed and the secondary antibody was added. Antibody detection was determined as above. The negative control for total antibody response was plasma from a nonreactive species (Zebra finch collected by Tess Killpack, University of WIMadison), and the positive control was Xenopus tropicalis plasma. Innate Immune Assay: Leukocyte Recruitment and Phagocytic Activity. A lavage assay designed to collect peritoneal leukocytes was adapted from Vatnick et al.46 Ten weeks post-metamorphosis, frogs were given an i.p. injection of thioglycollate medium (0.067 mL/g body mass) to stimulate leukocyte extravasation. This same inoculum contained fluorescently labeled 1 μm microbeads (Fluoresbrite Carboxy BB microspheres, 2.5 × 107 beads/mL) at a concentration of 0.55 μL/mL thioglycollate. After 24 h, individuals were anesthetized in 0.1% MS-222 (tricaine methanesulfonate), and phosphate buffered saline (PBS) was i.p. injected into the lower abdomen at a volume of 0.33 mL/g frog using a 27G needle. The abdomen was then gently massaged in order to flush the intraperitoneal cavity and suspend peritoneal leukocytes. A 23G needle was used to aspirate the PBS solution containing suspended cells. The cell suspension was diluted and stained with trypan blue for determination of cell viability, and total leukocyte counts (cells/mL) were obtained using a hemacytometer. Neutrophils comprised >95% of the leukocytes collected from the peritoneal cavity, and thus, leukocyte counts were characterized as the number of neutrophils. Phagocytic activity of the neutrophils was determined by counting 100 neutrophils and determining the percentage of cells that contained fluorescently labeled microbeads. Fluorescently labeled microbeads within neutrophils were visualized using a Nikon fluorescent microscope with a UV filter under 600× magnification. The assay was optimized for the proper microbead to neutrophil ratio such that excess microbeads would allow cells amble opportunity to engulf microbeads, but not so high to create aggregates of microbeads. Skin Peptide Collection and Quantification. A total of 45 7-week-old frogs from all treatments were injected in the dorsal lymph sac with 5 μL per g body mass of 1 mM norepinephrine (i.e., 5 nmol/g) as norepinephrine-bitartrate salt in amphibian phosphate buffered saline (APBS), which stimulates antimicrobial peptide (AMP) secretion.47 Frogs were then placed in 50 mL of collection buffer (2.92 g NaCl and 2.05 g sodium acetate in 1 L of HPLC-grade water) for 15 min as previously described.47−49 Following collection, the frogs were removed from the buffer, and the buffer was acidified by adding 1 mL of 50% HCl. Peptides were partially enriched over C18 Sep-Paks (Waters Corp. WAT020515, Milford, MA, USA) as described by Rollins-Smith et al.47 and quantified using the MicroBCA assay (Pierce, Rockford, IL, USA) according to manufacturer’s instructions except that the peptide bradykinin (RPPGFSPFR) (Sigma Chemical, St. Louis, MO, USA) was used as a standard.47−49 Serially diluted bradykinin solutions were measured at 562 nm to establish a standard curve, and unknown samples were referenced against the standard curve. The mass of each frog was determined at the time of the injection, and total secreted peptides were quantified. To estimate the total amount of peptides recovered, the surface area was calculated according to the method of McClanahan and Baldwin50 [surface area (cm2) = 9.9 (mass in grams)0.56].

on how PBDE exposure during larval development affects immune function post metamorphosis. Adaptive Immune Assay: Enzyme-Linked Immunosorbent Assay (ELISA). Vaccination and Blood Collection. Seven weeks post-metamorphosis, frogs were intraperitoneal (i.p.) injected with 25 μL of the antigen [keyhole limpet hemocyanin (2 mg KLH/25 mL PBS)] and Titer Max Gold (Sigma), an adjuvant previously used in L. pipiens and known to stimulate antibody production.44 Keyhole limpet hemocyanin is a large protein with many epitopes45 and is able to elicit antibody responses in frogs.44 Two weeks later, frogs were boosted with a second injection of KLH (2 mg KLH/25 mL PBS; without adjuvant) to initiate a secondary adaptive immune response to the antigen. One week following the booster injection, frogs were anesthetized in 0.2% MS-222, and blood was collected directly from the heart using a 28G needle. The blood was immediately transferred to heparin-coated microhematocrit capillary tubes and centrifuged to separate the plasma from the red blood cells. The plasma was stored at −20 °C until ELISA analysis. Specific Antibody Response to Keyhole Limpet Hemocyanin. Antibody response in 10-week-old L. pipiens was determined using a modification of enzyme-linked immunosorbent assays (ELISA) previously reported using frog plasma.42 High binding, flat-bottomed microtiter plates (NUNC, cat. no. 442404) were coated with 100 μL of KLH (1 mg/mL) diluted in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6) and incubated at 4 °C overnight. Each plate was washed with wash buffer (PBS with 0.05% Tween) using a Biotek ELx405 microplate washer and blocked with blocking buffer (5% nonfat dry milk with 0.5% Tween). Plates were incubated for 1 h at 37 °C and washed with wash buffer. Diluted plasma samples (1:100 in blocking buffer) were then added to the appropriate wells and incubated at 37 °C for 1 h. Specific anti-KLH antibodies in the frog plasma were detected by adding 100 μL of monoclonal mouse anti-Xenopus IgY (11D5, Xenopus laevis Research Resource for Immunology, University of Rochester Medical Center) to the wells. Plates were incubated at 37 °C for 1 h. After washing, 100 μL of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Sigma Chemicals, cat. no. A-9044) diluted 1:5000 (in blocking buffer) was added to each well and again incubated for 1 h at 37 °C. The plates were washed, and 150 μL of horseradish peroxidase substrate (1-Step ABTS, Pierce) was added to the wells. The reaction was allowed to develop for 45 min at room temperature and then stopped by adding 100 μL of 1% sodium dodecyl sulfate to each well. Absorbance values were read at 405 nm on a Wallac Victor2 microplate reader, and adjusted absorbance values (mean sample absorbance minus the mean negative control absorbance value) were recorded. Plasma from non-immunized frogs was used as a negative control. Positive controls consisted of plasma from frogs that were previously determined to produce anti-KLH IgY ≥ 2-fold that of the negative control plasma. Both negative and positive control samples were included on each plate to validate and normalize plate-to-plate differences. Total Antibody Response. Total IgY and IgM were evaluated for the same juvenile frogs that were analyzed for specific anti-KLH IgY. The ELISA method used was the same as above for specific anti-KLH, except microtiter plates were coated with 100 μL of diluted frog plasma in coating buffer and incubated at 4 °C overnight. Each plate was washed and blocked as above and the appropriate primary antibody [mouse 5912



Article

The thickness of the mucus was assumed to be 50 μm,51 and therefore the volume of mucus covering 1 cm2 of skin would be 5 μL. As a result, the concentration of peptides in mucus (μg/ mL) = total peptides (μg) per cm2 × 200 (because 1 mL = 5 μL × 20048,49). Determination of Antifungal Activity of Skin Peptides. Collected skin secretions were freeze-dried and stored at −20 °C until analysis. Skin secretions are known to be a major immune mechanism of amphibians against the deadly fungus Batrachochytrium dendrobatidis (Bd).52 The antifungal efficacy of collected skin peptides and the potential effect of PBDE exposure on potency were estimated through the calculation of the minimum inhibitory concentration (MIC), i.e., the lowest peptide concentration capable to significantly reduce fungal growth. Because of the low peptide amount in some secretion samples, we pooled together the samples corresponding to several individuals (n = 3 or 4) from the same DE-71 treatment. Dried secretion samples were resuspended in PBS to an estimated concentration of 4000 μg equivalents of bradykinin/ mL. The concentration in the resulting dilution was checked again using the MicroBCA assay, and serial dilutions of skin peptides (2000−15.625 μg eq/mL) were prepared for each sample. We added 25 μL of each dilution in triplicate to wells of a 96-well microplate; in addition, we prepared three extra wells of the highest peptide concentration to be used for the negative control. Batrachochytrium dendrobatidis culture (JEL197) was received from stocks maintained by Joyce Longcore at the University of Maine. This culture corresponds to the type isolate of Bd and was isolated from a Blue Poison Dart Frog (Dendrobates azureus) from the National Zoological Park (Washington, DC, USA) in 1997.53 Pieces of thalli taken directly from the culture received were immersed in vials containing 1% tryptone broth and incubated at 16 °C. Zoospore harvesting was performed as described in previous studies (e.g., ref 54). Briefly, we added ∼500 μL of this broth culture to 1% tryptone agar in 9 cm culture dishes and incubated them at 16 °C for 7 days. Dishes with growing thalli were then flooded with 5 mL of water that was previously disinfected by passing through a 0.45 μm filter. After 30 min, we decanted the plates to collect the zoospore solution. Zoospore concentration was measured by counting a measured aliquot on a hemocytometer, and sterile distilled water was then added to adjust for a final working dilution of 5 × 104 zoospores/mL. We added 25 μL of the working solution of Bd into each well with the exception of those corresponding to negative controls, where we added 25 μL of the working solution that was previously autoclaved for 20 min at 120 °C to kill the zoospores. Microplates were then incubated at 16 °C for 7 days. Fungal growth was estimated directly by measuring the optical density at 492 nm in a Wallac Victor2 microplate reader. A linear regression between the skin peptide concentration and the optical density (both log transformed to attain linearity) was calculated for each pooled sample. MIC was calculated as the concentration corresponding to the value where this regression line crossed the upper limit of the 95% confidence interval of the optical density readings obtained from the negative control (adapted from ref 55). Statistical Analyses. Statistical analyses were performed in SAS (Version 8.01) or R (Version 2.10.21, R Foundation for Statistical Computing). Proportional data for survival and phagocytosis were adjusted with arcsine square-root trans-

formation. Gosner Stage data were rank transformed. Leukocyte counts and absorbance data were adjusted with log10 transformation to ensure normality. A mixed model oneway analysis of variance (ANOVA) was performed to determine differences in tadpole parameters among treatments (fixed factor was treatment, random factor was replicate tank nested in embryo batch, and response variables were percent survival, total length, and Gosner Stage). A one-way ANOVA was performed to determine differences in innate immune parameters among treatments (independent variable was treatment, and response variables were leukocyte number, percent phagocytosis, extracted skin peptide mass per cm2 and MIC). An analysis of covariance was performed to determine differences in adaptive immune parameters among treatments (independent variable was treatment, covariate was snout−vent length or mass of the juvenile frog, and response variable was the adjusted absorbance values for specific anti-KLH IgY, total IgY, or total IgM). Akaike information criterion (AIC) was used for model selection. Post hoc multiple comparisons were made simultaneously using Tukey’s HSD. Statistical significance was accepted for p < 0.05.

■

RESULTS Growth and Development of Tadpoles. To determine whether PBDE exposure impacted survival, growth, and development of the tadpoles, we assessed percent survival, total length, and Gosner Stage (GS) on day 53 of the experiment. This time point corresponded to the longest exposure window before tadpoles began undergoing metamorphosis, which would confound measurements and the ability to make statistical comparisons among treatments. Seven tadpoles per replicated aquaria were measured for total length and visually analyzed for GS development (N = 70 for 0, 1.1, and 634 ng/g; N = 56 for 6.1 and 71.4 ng/g because two aquaria per treatment had to be removed from the sample due to experimenter error). On day 53, percent tadpole survival was not significantly different across treatments (F4,26 = 0.52, p = 0.721) and was ≥94.5% for all treatment groups. At this same time point, total length of tadpoles differed significantly among treatments (F4,308 = 2.81, p = 0.026). The 71.4 ng PBDE/g treated tadpoles were on average 10% shorter than the control tadpoles (p = 0.013; Figure 1a). Tadpole GS also differed significantly among treatments (F4,308 = 4.11, p = 0.003). The 71.4 ng PBDE/g treatment had significantly slower development compared to the control tadpoles (p = 0.003; Figure 1b). Adaptive Immune Function. Specific IgY antibody response to KLH was significantly different among treatment groups (F4,44 = 2.73, p = 0.041; n = 6−13). Specifically, dietary exposure of 71.4 ng PBDE/g significantly decreased anti-KLH IgY response compared to the control-treated frogs (p = 0.018; Figure 2a). We further tested the amount of total IgY and IgM levels in these frogs to measure overall antibody response to all antigens (Figure 2b). Due to insufficient sample volume, we were unable to test some individuals for total immunoglobulin levels. This is reflected in the sample size difference between the total IgY and IgM compared to the specific-KLH response. The ability of juvenile frogs to generate total immunoglobulins of both isotypes did not differ significantly with PBDE treatment [IgY: F4,39 = 0.66, p = 0.624 (n = 6−12); IgM: F4,39 = 0.70, p = 0.597 (n = 6−12)]. Innate Immune Function. The recruitment of neutrophils, which ranged from 7.0 × 104 to 5.6 × 105 cells/mL, differed significantly among treatments (F4,30 = 3.36, p = 0.022; n = 5− 5913



■

Article

DISCUSSION

Growth and Development. Tadpoles were fed environmentally realistic concentrations of DE-71 in order to assess potential immunotoxic effects of PBDEs; however, we also measured survival, growth, and developmental end points to compare with other studies. Survivorship of tadpoles until exposure day 53 was greater than 94.5% for all treatment groups and was not significantly affected by exposure to PBDEs, although our earlier study did find a significant decline due to PBDE exposure.23 As in our earlier study,23 both growth and development rate were significantly depressed, compared with controls, in some of the dose groups (Figure 1). The nonlinear dose response in tadpole growth and development is similar to previous findings with PBDE exposure23 and is consistent with exposure to endocrine disrupting compounds in which low doses have large impacts at the hormone level.56 Tadpole growth and development is driven by circulating thyroid hormone levels, and PBDEs have been shown to lower circulating thyroid hormones,17,57 which may explain the growth and developmental delays demonstrated in this study. Adaptive Immune Response. Amphibians possess many components of adaptive immunity that have been characterized in mammals including B and T lymphocytes, immunoglobulins (Ig), T-cell receptors, major-histocompatibility complex, and recombination-activating genes.32 We tested the ability of juvenile frogs to mount a specific antibody response following antigenic challenge to keyhole limpet hemocyanin (KLH). Larval PBDE exposure significantly lowered the ability of postmetamorphic frogs to mount an adaptive humoral response. Juvenile frogs exposed to 71.4 ng PBDE/g as tadpoles had 92.4% lower levels of antibodies produced that were specific to KLH compared to control frogs. The other PBDE-exposed frogs (1.1, 6.1, and 634 ng/g) had, on average, 89.2%, 48.8%, and 66.8% lower secondary antibody responses, respectively, compared to the controls. Exposure to PBDEs did not affect total production of either IgY or IgM isotypes in 10-week-old frogs. As total IgY and KLH-specific IgY both measure the ability of frogs to mount a secondary antibody response, we might expect both measures to follow a similar pattern. Because we found a significant decrease in specific-KLH IgY antibody production with PBDE exposure, which was not apparent with total IgY measures, immunological memory against KLH was a more sensitive immunotoxic measure than total IgY. Measuring antibody responses to a specific antigen, such as KLH, controls for the timing of antibody class-switching and provides a more refined measure of the organism’s adaptive antibody response. Total IgY includes all antibodies produced against all possible antigens regardless of the timing of when the organism was exposed, such that the response may be muddled, therefore not providing as clear a response as specific-KLH IgY. Total IgM levels were similar among treatments; this provides evidence that PBDE exposure did not suppress the ability of juvenile frogs to produce antibodies in general, but that perhaps the process of generating memory B cells with antibodies specific to KLH was impaired. In mammals, alteration of lymphocyte numbers, lymphoid organs, and antibody responses following PBDE exposure have been reported (e.g., refs 27−30 and 58). For example, mice exposed subchronically to 1000 μg DE-71/g body mass had suppressed primary antibody response to sheep red blood cells (sRBC), which was associated with decreased thymus weight at the same concentration,28 and in harbor porpoises, both thymic

Figure 1. (A) Tadpole length and (B) Gosner Stage (i.e., developmental stage) on day 53 of the DE-71 exposure experiment. Data were analyzed using a mixed model analysis of variance. Treatment groups that share the same letter are not statistically significant from each other (p > 0.05; Tukey post-hoc comparisons). Gosner Stage data were rank transformed for statistical analysis but are presented here with observed Gosner Stage values for biological clarity. (B) N = 70 for 0, 1.1, and 634 ng/g; N = 56 for 6.1 and 71.4 ng/g. Data is presented as the mean ± standard error of the mean.

10 per treatment). Recruitment means were higher in the three higher concentration PBDE treatments, and post-hoc Tukey’s comparisons determined that the 1.1 and 6.1 ng PBDE/g treatments differed significantly from each other in number of neutrophils (p = 0.046), but the mean number of neutrophils in the control treatment was not statistically different from the mean number of neutrophils in any of the PBDE treatments (p ≥ 0.15; Figure 3a). Phagocytic activity was broken down into four levels to discriminate between cells that engulfed no beads (none), 1−2 beads (low), 3−9 beads (medium), and 10 or more beads (high). The percentage of neutrophils that phagocytosed microbeads did not differ significantly among treatment groups for all four levels: none (F4,25 = 2.13, p = 0.107), low (F4,25 = 1.77, p = 0.167), medium (F4,25 = 1.32, p = 0.289), and high (F4,25 = 1.31, p = 0.294) (Figure 3b). In all samples, excess microbeads were present in the interstitial fluid, and no aggregates of microbeads were observed outside of the cells. Neither the amount of antimicrobial skin peptides extracted per cm2 (F4,10 = 0.832, p = 0.535) nor their potency to inhibit Bd growth, estimated from the MIC (F4,8 = 0.507, p = 0.732) were significantly affected by larval exposure to PBDE (Figure 4). 5914



Article

Figure 2. (A) Box-whisker plot of the level of specific anti-KLH IgY in plasma of frogs following a second (booster) injection of KLH. The horizontal line in each box is the median, the top and bottom of the box represent the 75th and the 25th percentile, respectively, and the whiskers define the 5th and 95th percentile observations. Specific anti-KLH IgY levels differed significantly across treatments (analysis of covariance, p = 0.041). Treatments sharing the same letter (above box-whisker plot) are not significantly different (Tukey post-hoc comparisons, p > 0.05). Frogs previously exposed to 71.4 ng PBDE/g as tadpoles had significantly lower specific anti-KLH IgY compared to control frogs (p = 0.018). (B) Total immunoglobulin (IgY and IgM) levels in juvenile frogs were not statistically different among treatments (p = 0.624 and 0.597, respectively). Data is presented as the mean ± standard error of the mean.

Figure 3. (A) Leukocyte counts measuring the recruitment of neutrophils differed significantly among treatments (analysis of variance, p = 0.022), but PBDE-exposed groups were not significantly different from controls. Treatment groups sharing the same letter are not statistically different from each other (p > 0.05; Tukey post-hoc comparisons). *The highest dose showed a statistical trend for increased neutrophil recruitment compared to the 1.1 ng/g treatment (p = 0.057). (B) Percentage of neutrophils that engulfed beads at different levels [none (0 beads), low (1−2 beads), medium (3−9 beads), or high (10+ beads)] for each treatment group. For all four levels of phagocytosis, the percent phagocytic neutrophils in each level did not differ significantly among treatment groups (analysis of variance, p ≥ 0.107). Data is presented as the mean ± standard error of the mean.

atrophy and splenic depletion were significantly correlated to increased PBDE body burdens.58 Similar suppression of humoral immunity has been shown in frogs exposed to pesticide mixtures, which has also been linked to decreased splenocyte cellularity.44,59 Loss of lymphocytes is likely the cause for the decreased primary antibody responses, and this may be driven by depression of lymphocyte populations in both the spleen and thymus. Because exposed frogs in the present study retained the ability to generate total IgM at the same level as that of unexposed frogs, we do not believe this is the main mode of action for PBDE-induced immunomodulation in L. pipiens at environmentally relevant concentrations. It is possible that with increased PBDE concentration, systematic toxicity leads to apoptosis of lymphocytes and that our present study observed immunomodulation in frogs at a toxin level that did not result in systemic toxicity. However, we are limited in this

discussion as we did not measure lymphoid organ masses (e.g., spleen or thymus) or cellularity in our frogs. The suppression of humoral immunity in frogs exposed to PBDEs is concerning because appropriate antibody responses are necessary for protection against pathogens. Specifically, clearance of ranavirus infections in adult frogs has been linked to humoral immunity,42,60 and antibodies specific to Bd are produced upon infection;48 however, antibody responses to Bd are likely not as protective as innate immune responses due to suppression of the adaptive immune response by the fungus.61,62 Innate Immune Response. In order to provide an accurate assessment of an organism’s immune health, a range of assays to examine the functionality of an immune response is necessary.63 Because of this we also assessed features of the innate immune system in juvenile frogs previously exposed to 5915



Article

Figure 4. Box-whisker plots of (A) the amount of skin peptides extracter per cm2 of skin and (B) the minimum inhibitory concentration (MIC) of skin peptides capable of limiting growth of Batrachochytrium dendrobatidis in vitro. The horizontal line in each box is the median, the top and bottom of the box represent the 75th and the 25th percentile, respectively, and the whiskers define the 5th and 95th percentile observations. The amount of skin peptides extracted per cm2 of skin did not differ among treatments (analysis of variance, p = 0.535), nor did the MIC of skin peptides differ among treatment groups (analysis of variance, p = 0.732). All samples are pooled samples, each representing skin secretions from 3 or 4 frogs.

infecting microbes and protect against external parasites. In our study, frogs fed ≥6.1 ng PBDE/g larval diets, secreted peptides at a level that was 74 to 88% lower than control frogs; however, this decrease was not significantly different likely attributable to a small sample size and limited statistical power. The results of other studies analyzing the impact of pollution on antimicrobial skin peptides offer differing conclusions. For example, Gibble and Baer67 did not detect any effect of the herbicide atrazine on total peptide collection of skin secretions in post-metamorphic X. laevis, while Davidson et al.37 reported that exposure to the insecticide carbaryl significantly reduced the total peptide concentrations recovered from the skin secretions of Rana boylii. Because AMPs act as one of the main barriers against infection by Bd, to the point that a correlation seems to exist between the antifungal efficacy of skin secretion of amphibian species, and their tolerance against the lethal effects of Bd,52,68 it is important to determine how environmental pollutants might contribute to increased Bd infection. Our data suggests that PBDEs may be influencing AMP levels, but additional research is necessary. Additionally, non-native Bd strains (like the strain used in this study) may cause increased adverse effects in native North American frog species.69 For greater ecological realism, future Bd-challenge studies with L. pipiens should use native Bd strains. Lithobates pipiens is among the most tolerant amphibian species against Bd infection,70 in part because of the high efficacy of peptides purified from its skin inhibiting growth of the fungus.71 We should thereby expect a low MIC of peptides extracted from L. pipiens skin compared to other species. However, the average MIC found among control froglets (424 μg/mL) is within the range of, or even higher than that found for other amphibian species (e.g., R. boylii: 25 μg/mL,37 Litoria sp.: 100−200 μg/mL;52 X. laevis: 500 μg/mL72). We did not detect any significant effect of PBDE exposure on the anti-Bd effectiveness of skin peptides, which generally matches with findings of other studies analyzing the impact of environmental pollutants on this innate immunity feature (e.g., refs 37 and 67). Proper immune function is vital for disease resistance, and although the present study found no evidence for increased

PBDEs as tadpoles. To assess innate immunity, we collected peritoneal leukocytes following stimulation with fluid thioglycollate46 and assessed phagocytic activity of the leukocytes. Because neutrophils act as the first responders in the innate immune response, neutrophils were expected to be the representative leukocytes in the peritoneal lavage fluid, and they were the dominating leukocyte type in our samples. Compared to control animals, frogs exposed to DE-71 did not have significantly altered neutrophil recruitment; however, there was a statistically significant increase in neutrophil counts in the 6.1 ng PBDE/g treatment group (p = 0.046) and a trend for an increase in the 634 ng PBDE/g (p = 0.057) when compared to the lowest PBDE-exposed frogs. A dosedependent increase in neutrophils with increasing PBDE levels has also been reported in mammals exposed to PBDEs.30,64 Phagocytic activity of neutrophils did not differ significantly across treatment groups, and this is in contrast to lowered phagocytic activity when northern leopard frogs were exposed to low pH or the pesticide atrazine.46,65 However, it is noteworthy to mention that on average the highest PBDE treatment group had between 29% and 97% more phagocytic cells compared to all other treatment groups. It is possible that our data did not have enough statistical power to determine significant differences in mean percentage phagocytic neutrophils. Because all lavage samples had excess microbeads in the interstitial fluid and were easily distinguishable from engulfed microbeads, we do not think that the delivery of microbeads affected our results. Larval DE-71 exposure did not significantly influence juvenile innate immune function compared to control treated frogs, but because increased neutrophil recruitment occurred with increased PBDE concentration, it is possible that immune stimulation occurred in L. pipiens with increasing PBDE exposure. Because phagocytic cells are precursors to the inflammatory response, increased phagocyte counts are not necessarily indicative of a healthier immune system and, in contrast, may indicate hypersensitive immune reactions, autoimmune disease, and/or a stress response.66 Skin peptides play a major role in amphibian immune defenses, as they contribute to an integument free from 5916



Article

Dawley rats following dietary exposure. Environ. Sci. Technol. 2007, 41, 2371−2377. (11) U.S. Environmental Protection Agency. Deca-BDE Phase-out Initiative http://www.epa.gov/oppt/existingchemicals/pubs/ actionplans/deccabde.html (accessed Jul 8, 2013). (12) Stapleton, H. M.; Baker, J. E. Comparing polybrominated diphenyl ether and polychlorinated biphenyl bioaccumulation in a food web in Grand Traverse Bay, Lake Michigan. Arch. Environ. Contam. Toxicol. 2003, 45, 227−234. (13) Oram, J. J.; McKee, L. J.; Werme, C. E.; Connor, M. S.; Oros, D. R.; Grace, R.; Rodigari, F. A mass budget of polybrominated diphenyl ethers in San Francisco Bay, CA. Environ. Int. 2008, 34, 1137−1147. (14) Qiu, X.; Marvin, C. H.; Hites, R. A. Dechlorane plus and other flame retardants in a sediment core from Lake Ontario. Environ. Sci. Technol. 2007, 41, 6014−6019. (15) Moser, V. C.; Coburn, C. G.; Farmer, J. D.; Jarema, K. A.; MacPhail, R. C.; McDaniel, K. L.; Phillips, P. M.; Kodavanti, P. R. S. Neurobehavioral assessment using a functional observational battery and motor activity in rats perinatally exposed to DE-71. Neurotoxicology 2006, 27, 1166−1166. (16) Yan, T.; Xiang, L.; Xuejun, J.; Chengzhi, C.; Youbin, Q.; Xuelan, Y.; Yang, L.; Changyan, P.; Hui, C. Spatial learning and memory deficit of low level polybrominated diphenyl ethers-47 in male adult rat is modulated by intracellular glutamate receptors. J. Toxicol. Sci. 2012, 37, 223−233. (17) Meerts, I.; van Zanden, J. J.; Luijks, E. a. C.; van Leeuwen-Bol, I.; Marsh, G.; Jakobsson, E.; Bergman, A.; Brouwer, A. Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicol. Sci. 2000, 56, 95−104. (18) Schriks, M.; Roessig, J. M.; Murk, A. J.; Furlow, J. D. Thyroid hormone receptor isoform selectivity of thyroid hormone disrupting compounds quantified with an in vitro reporter gene assay. Environ. Toxicol. Pharmacol. 2007, 23, 302−307. (19) Hallgren, S.; Sinjari, T.; Hakansson, H.; Darnerud, P. O. Effects of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) on thyroid hormone and vitamin A levels in rats and mice. Arch. Toxicol. 2001, 75, 200−208. (20) Balch, G. C.; Vélez-Espino, L. A.; Sweet, C.; Alaee, M.; Metcalfe, C. D. Inhibition of metamorphosis in tadpoles of Xenopus laevis exposed to polybrominated diphenyl ethers (PBDEs). Chemosphere 2006, 64, 328−338. (21) Schriks, M.; Zvinavashe, E.; David Furlow, J.; Murk, A. J. Disruption of thyroid hormone-mediated Xenopus laevis tadpole tail tip r e g r e ss i o n b y h e x a b r o m o c y cl o do d e c a n e ( H B C D ) a n d 2,2′,3,3′,4,4′,5,5′,6-nona brominated diphenyl ether (BDE206). Chemosphere 2006, 65, 1904−1908. (22) Carlsson, G.; Kulkarni, P.; Larsson, P.; Norrgren, L. Distribution of BDE-99 and effects on metamorphosis of BDE-99 and-47 after oral exposure in Xenopus tropicalis. Aquat. Toxicol. 2007, 84, 71−79. (23) Cary Coyle, T. L.; Karasov, W. H. Chronic, dietary polybrominated diphenyl ether exposure affects survival, growth, and development of Rana pipiens tadpoles. Environ. Toxicol. Chem. 2010, 29, 133−141. (24) Stoker, T. E.; Cooper, R. L.; Lambright, C. S.; Wilson, V. S.; Furr, J.; Gray, L. E. In vivo and in vitro anti-androgenic effects of DE71, a commercial polybrominated diphenyl ether (PBDE) mixture. Toxicol. Appl. Pharmacol. 2005, 207, 78−88. (25) Mercado-Feliciano, M.; Bigsby, R. M. Hydroxylated metabolites of the polybrominated diphenyl ether mixture DE-71 are weak estrogen receptor-alpha ligands. Environ. Health Perspect. 2008, 116, 1315−1321. (26) Reistad, T.; Mariussen, E. A commercial mixture of the brominated flame retardant pentabrominated diphenyl ether (DE-71) induces respiratory burst in human neutrophil granulocytes in vitro. Toxicol. Sci. 2005, 87, 57−65. (27) Fair, P. A.; Stavros, H.-C.; Mollenhauer, M. A. M.; DeWitt, J. C.; Henry, N.; Kannan, K.; Yun, S. H.; Bossart, G. D.; Keil, D. E.; PedenAdams, M. M. Immune function in female B6C3F1 mice is modulated

susceptibility to Bd, immunosuppression by environmental pollutants may contribute to declining amphibian populations by increasing susceptible to other pathogens. Immunomodulation in juvenile L. pipiens previously exposed to PBDEs as tadpoles suggests that larval exposure can have long-lasting effects on immune function of frogs. However, at 10 weeks post-metamorphosis, these animals had measurable body burdens of PBDEs,43 so it is not possible to determine from this study whether exposure as a tadpole or whether body burdens of juvenile frogs alone affected immune responses. Further studies are necessary to elucidate whether toxicant exposure at the larval and/or post-metamorphic life-stage results in alterations of the anuran adult immunity.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (608) 263-4344. Fax: (608) 262-9922. E-mail: tcary@ wisc.edu. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Nathan Van Schmidt for assistance with bioassays. The present study was sponsored by the University of Wisconsin Sea Grant Institute under grants from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and from the State of Wisconsin. Federal grant NA16RG2257, project R/EH-2.

■

REFERENCES

(1) Stuart, S. N.; Chanson, J. S.; Cox, N. A.; Young, B. E.; Rodrigues, A. S. L.; Fischman, D. L.; Waller, R. W. Status and trends of amphibian declines and extinctions worldwide. Science 2004, 306, 1783−1786. (2) IUCN. The IUCN Red List of Threatened Species: www. iucnredlist.org (accessed Jul 8, 2013). (3) Hites, R. A. Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. Environ. Sci. Technol. 2004, 38, 945−956. (4) Law, R. J.; Herzke, D.; Harrad, S.; Morris, S.; Bersuder, P.; Allchin, C. R. Levels and trends of HBCD and BDEs in the European and Asian environments, with some information for other BFRs. Chemosphere 2008, 73, 223−241. (5) Voorspoels, S.; Covaci, A.; Jaspers; Neels, H.; Schepens, P. Biomagnification of PBDEs in three small terrestrial food chains. Environ. Sci. Technol. 2007, 41, 411−416. (6) Shaw, S. D.; Berger, M. L.; Brenner, D.; Kannan, K.; Lohmann, N.; Päpke, O. Bioaccumulation of polybrominated diphenyl ethers and hexabromocyclododecane in the northwest Atlantic marine food web. Sci. Total Environ. 2009, 407, 3323−3329. (7) Johnson, P. I.; Stapleton, H. M.; Sjodin, A.; Meeker, J. D. Relationships between polybrominated diphenyl ether concentrations in house dust and serum. Environ. Sci. Technol. 2010, 44, 5627−5632. (8) Stapleton, H. M.; Brazil, B.; Holbrook, R. D.; Mitchelmore, C. L.; Benedict, R.; Konstantinov, A.; Potter, D. In vivo and in vitro debromination of decabromodiphenyl ether (BDE 209) by juvenile rainbow trout and common carp. Environ. Sci. Technol. 2006, 40, 4653−4658. (9) Van den Steen, E.; Covaci, A.; Jaspers, V. L. B.; Dauwe, T.; Voorspoels, S.; Eens, M.; Pinxten, R. Accumulation, tissue-specific distribution and debromination of decabromodiphenyl ether (BDE 209) in European starlings (Sturnus vulgaris). Environ. Pollut. 2007, No. 148, 648−653. (10) Huwe, J. K.; Smith, D. J. Accumulation, whole-body depletion, and debromination of decabromodiphenyl ether in male Sprague5917



Article

by DE-71, a commercial polybrominated diphenyl ether mixture. J. Immunotoxicol. 2012, 9, 96−107. (28) Fowles, J.; Fairbrother, A.; Baechersteppan, L.; Kerkvliet, N. Immunological and Endocrine Effects of the Flame-Retardant Pentabromodiphenyl Ether (de-71) in C57bl/6j Mice. Toxicology 1994, 86, 49−61. (29) Thuvander, A.; Darnerud, P. O. Effects of polybrominated diphenyl ether (PBDE) and polychlorinated biphenyl (PCB) on some immunological parameters after oral exposure in rats and mice. Toxicol. Environ. Chem. 1999, 70, 229−242. (30) Martin, P. A.; Mayne, G. J.; Bursian, S. J.; Tomy, G.; Palace, V.; Pekarik, C.; Smits, J. Immunotoxicity of the commercial polybrominated diphenyl ether mixture DE-71 in ranch mink (Mustela vison). Environ. Toxicol. Chem. 2007, 26, 988−997. (31) Du Pasquier, L.; Robert, J.; Courtet, M.; Mussmann, R. B-cell development in the amphibian Xenopus. Immunol. Rev. 2000, 175, 201−213. (32) Robert, J.; Ohta, Y. Comparative and developmental study of the immune system in Xenopus. Dev. Dyn. 2009, 238, 1249−1270. (33) Rollins-Smith, L. A. Metamorphosis and the amphibian immune system. Immunol. Rev. 1998, 166, 221−230. (34) Carey, C.; Cohen, N.; Rollins-Smith, L. Amphibian declines: an immunological perspective. Dev. Comp. Immunol. 1999, 23, 459−472. (35) Rohr, J. R.; Raffel, T. R.; Halstead, N. T.; McMahon, T. A.; Johnson, S. A.; Boughton, R. K.; Martin, L. B. Early-life exposure to a herbicide has enduring effects on pathogen-induced mortality. Proc. R. Soc. B: Biol. Sci. 2013, 280, 1502. (36) Du Pasquier, L.; Schwager, J.; Flajnik, M. F. The immune system of Xenopus. Annu. Rev. Immunol. 1989, 7, 251−275. (37) Davidson, C.; Benard, M. F.; Shaffer, H. B.; Parker, J. M.; O’Leary, C.; Conlon, J. M.; Rollins-Smith, L. A. Effects of chytrid and carbaryl exposure on survival, growth and skin peptide defenses in foothill yellow-legged frogs. Environ. Sci. Technol. 2007, 41, 1771− 1776. (38) Christin, M. S.; Menard, L.; Giroux, I.; Marcogliese, D. J.; Ruby, S.; Cyr, D.; Fournier, M.; Brousseau, P. Effects of agricultural pesticides on the health of Rana pipiens frogs sampled from the field. Environ. Sci. Pollut. Res. 2013, 20, 601−611. (39) Gosner, K. L. A simplified table for staging Anuran embryos and larvae with notes on identification. Herpetologica 1960, 16, 183−190. (40) Hardy, M. L. The toxicology of the three commercial polybrominated diphenyl oxide (ether) flame retardants. Chemosphere 2002, 46, 757−777. (41) Van Schmidt, N. D.; Cary, T. L.; Ortiz-Santaliestra, M. E.; Karasov, W. H. Effects of chronic polybrominated diphenyl ether exposure on gonadal development in the northern leopard frog Rana pipiens. Environ. Toxicol. Chem. 2012, 31, 347−354. (42) Maniero, G. D.; Morales, H.; Gantress, J.; Robert, J. Generation of a long-lasting, protective, and neutralizing antibody response to the ranavirus FV3 by the frog Xenopus. Dev. Comp. Immunol. 2006, 30, 649−657. (43) Cary, T. L.; Karasov, W. H. Toxicokinetics of polybrominated diphenyl ethers across life stages in the northern leopard frog (Lithobates pipiens). Environ. Toxicol. Chem. 2013, 32, 1631−1640. (44) Gilbertson, M. K.; Haffner, G. D.; Drouillard, K. G.; Albert, A.; Dixon, B. Immunosuppression in the northern leopard frog (Rana pipiens) induced by pesticide exposure. Environ. Toxicol. Chem. 2003, 22, 101−110. (45) Harris, J. R. Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 1999, 30, 597−623. (46) Vatnick, I.; Andrews, J.; Colombo, M.; Madhoun, H.; Rameswaran, M.; Brodkin, M. A. Acid exposure is an immune disruptor in adult Rana pipiens. Environ. Toxicol. Chem. 2006, 25, 199−202. (47) Rollins-Smith, L. A.; Woodhams, D. C.; Reinert, L. K.; Vredenburg, V. T.; Briggs, C. J.; Nielsen, P. F.; Michael Conlon, J. Antimicrobial peptide defenses of the mountain yellow-legged frog (Rana muscosa). Dev. Comp. Immunol. 2006, 30, 831−842.

(48) Ramsey, J. P.; Reinert, L. K.; Harper, L. K.; Woodhams, D. C.; Rollins-Smith, L. A. Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis. Infect. Immun. 2010, 78, 3981−3992. (49) Pask, J. D.; Woodhams, D. C.; Rollins-Smith, L. A. The ebb and flow of antimicrobial skin peptides defends northern leopard frogs (Rana pipiens) against chytridiomycosis. Global Change Biol. 2012, 18, 1231−1238. (50) McClanahan, L., Jr.; Baldwin, R. Rate of water uptake through the integument of the desert toad, Bufo punctatus. Comp. Biochem. Physiol. 1969, 28, 381−389. (51) Brucker, R. M.; Harris, R. N.; Schwantes, C. R.; Gallaher, T. N.; Flaherty, D. C.; Lam, B. A.; Minbiole, K. P. C. Amphibian chemical defense: Antifungal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus. J. Chem. Ecol. 2008, 34, 1422−1429. (52) Woodhams, D. C.; Ardipradja, K.; Alford, R. A.; Marantelli, G.; Reinert, L. K.; Rollins-Smith, L. A. Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Anim. Conserv. 2007, 10, 409−417. (53) Longcore, J. E.; Pessier, A. P.; Nichols, D. K. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 1999, 91, 219−227. (54) Ortiz-Santaliestra, M. E.; Rittenhouse, T. A. G.; Cary, T. L.; Karasov, W. H. Interspecific and post-metamorphic variation in susceptibility of three North American Anurans to Batrachochytrium dendrobatidis. J. Herpetol. 2013, 47, 286−292. (55) Sheafor, B.; Davidson, E. W.; Parr, L.; Rollins-Smith, L. Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens. J. Wildl. Dis. 2008, 44, 226− 236. (56) Welshons, W.; Thayer, K.; Judy, B.; Taylor, J.; Curran, E.; vom Saal, F. Large effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ. Health Perspect. 2003, 111, 994−1006. (57) Zhou, T.; Taylor, M. M.; DeVito, M. J.; Crofton, K. A. Developmental exposure to brominated diphenyl ethers results in thyroid hormone disruption. Toxicol. Sci. 2002, 66, 105−116. (58) Beineke, A.; Siebert, U.; McLachlan, M.; Bruhn, R.; Thron, K.; Failing, K.; Müller, G.; Baumgärtner, W. Investigations of the potential influence of environmental contaminants on the thymus and spleen of harbor porpoises (Phocoena phocoena). Environ. Sci. Technol. 2005, 39, 3933−3938. (59) Christin, M. S.; Ménard, L.; Gendron, A. D.; Ruby, S.; Cyr, D.; Marcogliese, D. J.; Rollins-Smith, L.; Fournier, M. Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens. Aquat. Toxicol. 2004, 67, 33−43. (60) Gantress, J.; Maniero, G. D.; Cohen, N.; Robert, J. Development and characterization of a model system to study amphibian immune responses to iridoviruses. Virology 2003, 311, 254−262. (61) Stice, M. J.; Briggs, C. J. Immunization is ineffective at preventing infection and mortality due to the amphibian chytrid fungus Batrachochytrium dendrobatidis. J. Wildl. Dis. 2010, 46, 70−77. (62) Fites, J. S.; Ramsey, J. P.; Holden, W. M.; Collier, S. P.; Sutherland, D. M.; Reinert, L. K.; Gayek, A. S.; Dermody, T. S.; Aune, T. M.; Oswald-Richter, K.; et al. The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science 2013, 342, 366− 369. (63) Demas, G. E.; Zysling, D. A.; Beechler, B. R.; Muehlenbein, M. P.; French, S. S. Beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts. J. Anim. Ecol. 2011, 80, 710−730. (64) Neale, J. C. C.; Gulland, F. M. D.; Schmelzer, K. R.; Harvey, J. T.; Berg, E. A.; Allen, S. G.; Greig, D. J.; Grigg, E. K.; Tjeerdema, R. S. Contaminant loads and hematological correlates in the harbor seal (Phoca vitulina) of San Francisco Bay, California. J. Toxicol. Environmental Health, Part A 2005, 68, 617−633. 5918



Article

(65) Brodkin, M. A.; Madhoun, H.; Rameswaran, M.; Vatnick, I. Atrazine is an immune disruptor in adult northern leopard frogs (Rana pipiens). Environ. Toxicol. Chem. 2007, 26, 80−84. (66) Latimer, K.; Mahaffey, E.; Prasse, K. Duncan and Prasse’s Veterinary Laboratory Medicine; Clinical Pathology, 4th ed.; Iowa State University Press: Ames, IA, 2003. (67) Gibble, R. E.; Baer, K. N. Effects of atrazine, agricultural runoff, and selected effluents on antimicrobial activity of skin peptides in Xenopus laevis. Ecotoxicol. Environ. Saf. 2011, 74, 593−599. (68) Pask, J. D.; Cary, T. L.; Rollins-Smith, L. A. Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis. J. Exp. Biol. 2013, 16, 2908−2916. (69) Gahl, M. K.; Longcore, J. E.; Houlahan, J. E. Varying responses of northeastern North American amphibians to the chytrid pathogen Batrachochytrium dendrobatidis: Varying susceptibility to Bd. Conserv. Biol. 2012, 26, 135−141. (70) Woodhams, D. C.; Hyatt, A. D.; Boyle, D. G.; Rollins-Smith, L. A. The northern leopard frog Rana pipiens is a widespread reservoir species harboring Batrachochytrium dendrobatidis in North America. Herpetol. Rev. 2008, 39, 66. (71) Rollins-Smith, L. A.; Doersam, J. K.; Longcore, J. E.; Taylor, S. K.; Shamblin, J. C.; Carey, C.; Zasloff, M. A. Antimicrobial peptide defenses against pathogens associated with global amphibian declines. Dev. Comp. Immunol. 2002, 26, 63−72. (72) Gibble, R. E.; Rollins-Smith, L.; Baer, K. N. Development of an assay for testing the antimicrobial activity of skin peptides against the amphibian chytrid fungus (Batrachochytrium dendrobatidis) using Xenopus laevis. Ecotoxicol. Environ. Saf. 2008, 71, 506−513.

5919


Immunomodulation in Post-metamorphic Northern Leopard Frogs

Recommend Documents