Larval Exposure to Polychlorinated Biphenyl-126 Led to a Long-Lasting Decrease in Immune Function in Postmetamorphic Juvenile Northern Leopard Frogs, Lithobates pipiens.

Amphibian populations are decreasing worldwide, and pollution is a contributing factor. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants known to exert immunotoxicity. To assess impacts of PCBs on frogs, we exposed Lithobates pipiens tadpoles to a diet of PCB-126 (0-5 ng PCB-126/g wet food) through metamorphic climax. Postmetamorphic frogs were immunized with keyhole limpet hemocyanin (KLH); then production of KLH-specific IgY, as well as total IgY and IgM, was measured (Trial I). A second larval study (0 and 7.3 ng PCB-126/g wet food) was performed to investigate whether PCB altered antigenic responses in prometamorphic tadpoles (Gosner Stage 36-39), and to measure the innate immune response of postmetamorphic frogs (Trial II). After larval PCB-126 exposure, both KLH-specific IgY levels and complement activity were reduced. Because postmetamorphic frogs carried a body burden of PCB-126 (2.4 ng/g or less), we wanted to determine whether the effect on immune response was due to larval exposure or to the resulting body burden as frogs. To test this, we reared tadpoles under control conditions (no PCB), and limited PCB exposure to postmetamorphosis only by injecting 2-week-old frogs with 10 ng PCB-126/g (Trial III). The resulting body burden (3.4 ng/g) was similar to that of frogs in Trial I, but we no longer detected suppression of KLH-specific IgY or hemolytic activity. These results suggest life-stage-specific immune responses; however, because we administered PCB-126 differently between trials, it is premature to conclude that these differences are intrinsically life stage dependent, and further study is warranted. Regardless, our study demonstrated a long-lasting effect of larval PCB-126 exposure that persisted through metamorphosis and suppressed frog immunity. Environ Toxicol Chem 2022;41:81-94. © 2021 SETAC.

Resources for Teaching and Assessing the Vision and Change Biology Core Concepts.

The Vision and Change report called for the biology community to mobilize around teaching the core concepts of biology. This essay describes a collection of resources developed by several different groups that can be used to respond to the report's call to transform undergraduate education at both the individual course and departmental levels. First, we present two frameworks that help articulate the Vision and Change core concepts, the BioCore Guide and the Conceptual Elements (CE) Framework, which can be used in mapping the core concepts onto existing curricula and designing new curricula that teach the biology core concepts. Second, we describe how the BioCore Guide and the CE Framework can be used alongside the Partnership for Undergraduate Life Sciences Education curricular rubric as a way for departments to self-assess their teaching of the core concepts. Finally, we highlight three sets of instruments that can be used to directly assess student learning of the core concepts: the Biology Card Sorting Task, the Biology Core Concept Instruments, and the Biology-Measuring Achievement and Progression in Science instruments. Approaches to using these resources independently and synergistically are discussed.

A Biology Core Concept Instrument (BCCI) to Teach and Assess Student Conceptual Understanding.

Instruments for teaching and assessing student understanding of the five core concepts in biology from Vision and Change are needed. We developed four Biology Core -Concept Instruments (BCCIs) that teach and assess students' ability to describe a concept in their own words, identify concepts represented in biological phenomena, and make connections between concepts. The BCCI includes a narrative, followed by a series of 10 true-false/identify (TF/I) and three open-ended questions. The TF/I questions are aligned with Cary and Branchaw's Conceptual Elements Framework and were iteratively developed with feedback from biology experts and student performance and feedback obtained during think-aloud interviews. A component scoring system was developed to discriminate between a student's ability to apply and identify each core concept from his or her ability to make connections between concepts. We field-tested the BCCIs (n = 152-191) with students in a first-year course focused on learning the five core concepts in biology and collected evidence of interrater reliability (α = 0.70) and item validity. With component scoring, we identified examples in which students were able to identify concepts singularly, but not make connections between concepts, or were better able to apply concepts to one biological phenomenon than another. Identifying these nuanced differences in learning can guide instruction to improve students' conceptual understanding.

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.

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.

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.

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.

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.

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

Population consequences of fipronil and degradates to copepods at field concentrations: an integration of life cycle testing with leslie matrix population modeling.

The predominant data used in ecological risk assessment today are individual-based rather than population-based; yet environmental policies are usually designed to protect populations of threatened species or communities. Most current methods in ecotoxicology are limited by largely logistic/ technology-driven requirements that yield data for a relatively small number of test species and end points that focus on acute lethality or sublethal nonproduction-based parameters (e.g., biomarkers, mutagenesis, genetic change, physiological condition). A contrasting example is presented here showing the predictive ability of meiobenthos-based full life cycle toxicity testing to extrapolate multi-generational effects of chemicals on variables of import to population growth and maintenance. Less than 24-h-old larvae of a meiobenthic copepod were reared individually in 96-well microplate exposures to parent and degradates of the phenylpyrazole insecticide fipronil. Survival, development rates, sex ratio change, fertility, fecundity, and hatching success were tracked daily for 32 d through mating and production of three broods in spiked seawater. These data were then inserted in a Leslie (Lefkovitch) matrix stage-based population growth model to predict relative rates of population increase (lambda) and changes in net population growth with time and toxicant concentration. Field-reported test concentrations produced strong reproductive (52-88%) and net production (40-80%) depressions for parent (at 0.25 and 0.5 microg/L), desthionyl (0.25 and 0.5 microg/L), and sulfide (0.15 microg/L) moieties as compared to controls. Spiked sediment exposures of 65-300 ng of fipronil/g of dry sediment yielded significantly reduced production rates per female that were 67-50% of control production. The consistent reproductively linked impacts of fipronil and its degradation products at the population maintenance levels suggest risks to sediment-dwelling crustaceans at concentrations well below noneffects for most aquatic test species based on risk assessment data from primarily acute and sub-life cycle toxicity tests.

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.

Phenylpyrazole insecticide fipronil induces male infertility in the estuarine meiobenthic crustacean Amphiascus tenuiremis.

Copepods are the most abundant arthropods on earth and are often the most important secondary producers in estuarine/marine food webs. The new GABA (gamma-aminobutyric acid)-disrupting insecticide fipronil (FP) induces unique sex-specific reproductive dysfunction in male meiobenthic copepods, leading to trans-generational population depression at environmentally realistic concentrations (0.63 microg/L). Using a newly developed 96-well microplate lifecycle bioassay, more than 700 individual Stage-I juveniles were reared to adulthood in as short as 12 days in only 200 microL of control (CTL) or 0.63 microg-FP/L seawater solution. Individual virgin male: female pairs were then cross-mated for all possible combinations within and across rearing treatments and allowed to mate for an additional 12 days in CTL or 0.63 microg-FP/L solution. FP at 0.63 microg/L caused no significant lethality to any mating combinations but evoked 73% or 89% inhibition of reproduction when FP-reared males were mated with either a control- or FP-reared female in FP solution, respectively. In contrast, when CTL-reared males were mated with FP-reared females in FP solution, there was no difference in reproductive success compared to FP-free controls. When FP-reared males were mated with either female group in FP-free solution, these mating pairs displayed a 3-day delay in time to brood sac extrusion but ultimately did reproduce. As fipronil (1) has a high K(ow), (2) is persistent in sediments where meiobenthic copepods live, and (3) has been detected in estuarine waters >0.7 microg/L, it may pose high risk to copepod production in estuarine systems.