Transcriptomics investigation of thyroid hormone disruption in the olfactory system of the Rana [Lithobates] catesbeiana tadpole.

Thyroid hormones (THs) regulate vertebrate growth, development, and metabolism. Despite their importance, there is a need for effective detection of TH-disruption by endocrine disrupting chemicals (EDCs). The frog olfactory system substantially remodels during TH-dependent metamorphosis and the objective of the present study is to examine olfactory system gene expression for TH biomarkers that can evaluate the biological effects of complex mixtures such as municipal wastewater. We first examine classic TH-response gene transcripts using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in the olfactory epithelium (OE) and olfactory bulb (OB) of premetamorphic Rana (Lithobates) catesbeiana tadpoles after 48 h exposure to biologically-relevant concentrations of the THs, 3,5,3'-triiodothyronine (T3) and L-thyroxine (T4), or 17-beta estradiol (E2); a hormone that can crosstalk with THs. As the OE was particularly sensitive to THs, further RNA-seq analysis found >30,000 TH-responsive contigs. In contrast, E2 affected 267 contigs of which only 57 overlapped with THs suggesting that E2 has limited effect on the OE at this developmental phase. Gene ontology enrichment analyses identified sensory perception and nucleoside diphosphate phosphorylation as the top affected terms for THs and E2, respectively. Using classic and additional RNA-seq-derived TH-response gene transcripts, we queried TH-disrupting activity in municipal wastewater effluent from two different treatment systems: anaerobic membrane bioreactor (AnMBR) and membrane enhanced biological phosphorous removal (MEBPR). While we observed physical EDC removal in both systems, some TH disruption activity was retained in the effluents. This work lays an important foundation for linking TH-dependent gene expression with olfactory system function in amphibians.

Behavioral and molecular analyses of olfaction-mediated avoidance responses of Rana (Lithobates) catesbeiana tadpoles: Sensitivity to thyroid hormones, estrogen, and treated municipal wastewater effluent.

Olfaction is critical for survival, facilitating predator avoidance and food location. The nature of the olfactory system changes during amphibian metamorphosis as the aquatic herbivorous tadpole transitions to a terrestrial, carnivorous frog. Metamorphosis is principally dependent on the action of thyroid hormones (THs), l-thyroxine (T4) and 3,5,3'-triiodothyronine (T3), yet little is known about their influence on olfaction during this phase of postembryonic development. We exposed Taylor Kollros stage I-XIII Rana (Lithobates) catesbeiana tadpoles to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) for 48h and evaluated a predator cue avoidance response. The avoidance response in T3-exposed tadpoles was abolished while T4- or E2-exposed tadpoles were unaffected compared to control tadpoles. qPCR analyses on classic TH-response gene transcripts (thra, thrb, and thibz) in the olfactory epithelium demonstrated that, while both THs produced molecular responses, T3 elicited greater responses than T4. Municipal wastewater feed stock was spiked with a defined pharmaceutical and personal care product (PPCP) cocktail and treated with an anaerobic membrane bioreactor (AnMBR). Despite substantially reduced PPCP levels, exposure to this effluent abolished avoidance behavior relative to AnMBR effluent whose feed stock was spiked with vehicle. Thibz transcript levels increased upon exposure to either effluent indicating TH mimic activity. The present work is the first to demonstrate differential TH responsiveness of the frog tadpole olfactory system with both behavioral and molecular alterations. A systems-based analysis is warranted to further elucidate the mechanism of action on the olfactory epithelium and identify further molecular bioindicators linked to behavioral response disruption.

The North American bullfrog draft genome provides insight into hormonal regulation of long noncoding RNA.

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research.

De novo assembly of the ringed seal (Pusa hispida) blubber transcriptome: A tool that enables identification of molecular health indicators associated with PCB exposure.

The ringed seal, Pusa hispida, is a keystone species in the Arctic marine ecosystem, and is proving a useful marine mammal for linking polychlorinated biphenyl (PCB) exposure to toxic injury. We report here the first de novo assembled transcriptome for the ringed seal (342,863 transcripts, of which 53% were annotated), which we then applied to a population of ringed seals exposed to a local PCB source in Arctic Labrador, Canada. We found an indication of energy metabolism imbalance in local ringed seals (n=4), and identified five significant gene transcript targets: plasminogen receptor (Plg-R(KT)), solute carrier family 25 member 43 receptor (Slc25a43), ankyrin repeat domain-containing protein 26-like receptor (Ankrd26), HIS30 (not yet annotated) and HIS16 (not yet annotated) that may represent indicators of PCB exposure and effects in marine mammals. The abundance profiles of these five gene targets were validated in blubber samples collected from 43 ringed seals using a qPCR assay. The mRNA transcript levels for all five gene targets, (Plg-R(KT), r2=0.43), (Slc25a43, r2=0.51), (Ankrd26, r2=0.43), (HIS30, r2=0.39) and (HIS16, r2=0.31) correlated with increasing levels of blubber PCBs. Results from the present study contribute to our understanding of PCB associated effects in marine mammals, and provide new tools for future molecular and toxicology work in pinnipeds.

A multi-omic approach to elucidate low-dose effects of xenobiotics in zebrafish (Danio rerio) larvae.

Regulatory-approved toxicity assays such as the OECD Fish Embryo Toxicity Assay (TG236) allow correlation of chemical exposure to adverse morphological phenotypes. However, these assays are ineffective in assessing sub-lethal (i.e. low-dose) effects, or differentiating between similar phenotypes induced by different chemicals. Inclusion of multi-omic analyses in studies investigating xenobiotic action provides improved characterization of biological response, thereby enhancing prediction of toxicological outcomes in whole animals in the absence of morphological effects. In the current study, we assessed perturbations in both the metabolome and transcriptome of zebrafish (Danio rerio; ZF) larvae exposed from 96 to 120h post fertilization to environmental concentrations of acetaminophen (APAP), diphenhydramine (DH), carbamazepine (CBZ), and fluoxetine (FLX); common pharmaceuticals with known mechanisms of action. Multi-omic responses were evaluated independently and integrated to identify molecular interactions and biological relevance of the responses. Results indicated chemical- and dose-specific changes suggesting differences in the time scale of transcript abundance and metabolite production. Increased impact on the metabolome relative to the transcriptome in FLX-treated animals suggests a stronger post-translational effect of the treatment. In contrast, the transcriptome showed higher sensitivity to perturbation in DH-exposed animals. Integration of 'omic' responses using multivariate approaches provided additional insights not obtained by independent 'omic' analyses and demonstrated that the most distinct overall response profiles were induced following low-dose exposure for all 4 pharmaceuticals. Importantly, changes in transcript abundance corroborated with predictions from metabolomic enrichment analyses and the identified perturbed biological pathways aligned with known xenobiotic mechanisms of action. This work demonstrates that a multi-omic toxicological approach, coupled with a sensitive animal model such as ZF larvae, can help characterize the toxicological relevance of acute low-dose chemical exposures.

Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.

Rethinking the biological relationships of the thyroid hormones, l-thyroxine and 3,5,3'-triiodothyronine.

Thyroid hormones (THs), l-thyroxine (T4) and 3,5,3'-triiodothyronine (T3), are essential for vertebrate growth and development. Classically, T4 is 5'-deiodinated to the active hormone, T3, in target tissues which then binds nuclear TH receptors (TRs) and regulates gene transcription. However, it is possible that T4 acts directly on target tissues. Frog metamorphosis is a powerful TR-dependent model for studying TH action. Premetamorphic Rana (Lithobates) catesbeiana tadpoles were injected with 0.1-50 T3 or 0.5-250T4pmol/gbodyweight to account for their 5-fold difference in biological activity and the mRNA profiles in six tissues from well-characterized TH-responsive genes were evaluated after 48h using quantitative real time polymerase chain reaction. 5'-deiodinase-poor tissues should produce superimposable dose-response curves if T4 does not require conversion to T3. This was the case in lung and tail fin; the latter tissue recapitulating these responses in organ culture. 5'-deiodinase-rich tissues should convert T4 to T3. Because T3 has a higher affinity to TRs, a 5-fold higher T4 dose compared to T3 should produce greater transcript induction. This was observed in the brain and for most intestinal transcripts. However, some gene transcripts in the intestine and all transcripts in the back skin produced superimposable response curves suggesting that a direct mode of T4 action is plausible in these tissues. While the liver showed results consistent with its 5'-deiodinase-poor status, we found evidence of an alternate, non-genomic mechanism for two gene transcripts. Therefore, mechanisms not requiring T4 conversion to T3 may play a far greater role than previously thought.

Cadmium-induced olfactory dysfunction in rainbow trout: Effects of binary and quaternary metal mixtures.

A functioning olfactory response is essential for fish to be able to undertake essential behaviors. The majority of work investigating the effects of metals on the olfactory response of fish has focused on single-metal exposures. In this study we exposed rainbow trout to cadmium, copper, nickel, zinc, or a mixture of these four metals at or below the current Canadian Council of Ministers of the Environment guidelines for the protection of aquatic life. Measurement of olfactory acuity using an electro-olfactogram demonstrated that cadmium causes significant impairment of the entire olfactory system, while the other three metals or the mixture of all four metals did not. Binary mixtures with cadmium and each of the other metals demonstrated that nickel and zinc, but not copper, protect against cadmium-induced olfactory dysfunction. Testing was done to determine if the protection from cadmium-induced olfactory dysfunction could be explained by binding competition between cadmium and the other metals at the cell surface, or if the protection could be explained by an up-regulation of an intracellular detoxification pathway, namely metallothionein. This study is the first to measure the effects of binary and quaternary metal mixtures on the olfactory response of fish, something that will aid in future assessments of the effects of metals on the environment.

Identification of organ-autonomous constituents of the molecular memory conferred by thyroid hormone exposure in cold temperature-arrested metamorphosing Rana (Lithobates) catesbeiana tadpoles.

Environmental temperature modulates thyroid hormone (TH)-dependent metamorphosis in some amphibian species. The North American bullfrog--Rana (Lithobates) catesbeiana - tadpole is naturally adapted to a wide range of temperatures over multiple seasons. Cold temperatures delay while warmer temperatures accelerate metamorphosis. Exogenous TH exposure of premetamorphic tadpoles results in a rapid precocious induction of metamorphosis at warm temperatures (20-25 °C). The same exposure at cold temperatures (4-5 °C) does not elicit an overt metamorphic response. However, a molecular memory of TH exposure is established such that cold, TH-exposed tadpoles returned to permissive warm temperatures will rapidly execute TH-induced genetic programs. Previous mRNA profiling has identified TH-regulated transcription factors encoded by thra, thrb, thibz, klf9, and cebp1 as components of the molecular memory after one week post-exposure. However, a further hierarchy may exist within the initiation phase since many gene transcripts demonstrated tissue-specific patterns. Whether the molecular memory is organ autonomous or requires additional modulating factors is unknown. Herein we examine tail fin and back skin and determine that thibz is the only transcript that is TH-responsive after 2 days post-exposure at low temperature in both tissues in the intact animal. In back skin, cebp1 is also TH-responsive under these conditions. Serum-free tail fin organ culture (C-Fin) reveals that the thibz response is organ autonomous whereas cultured back skin (C-Skin) results suggest that thibz and cebp1 require an additional factor for induction from elsewhere within the intact animal. Subsequent investigations are now possible to identify endogenous factors that modulate the molecular memory in intact animals.

Comparison of thyroid hormone-dependent gene responses in vivo and in organ culture of the American bullfrog (Rana (Lithobates) catesbeiana) lung.

Postembryonic frog development requires a thyroid hormone (TH)-dependent metamorphic transition from an aquatic larva to a terrestrial frog. Such change in environment involves lung maturation in preparation for breathing air. However, little is known regarding the underlying molecular events and the role of THs in this process. Using quantitative real-time polymerase chain reaction, we evaluated Rana (Lithobates) catesbeiana lung mRNA transcripts representing key elements of TH and oxidative stress signaling pathways during natural and TH-induced precocious metamorphosis. TH induction was evaluated in two ways: 1) in vivo through interperitoneal injection of 10pmol/g body weight of 3,3', 5-triiodothyronine (T3) into premetamorphic tadpoles and analysis after 48h, and 2) in serum-free organ culture in the presence of 10nM T3 after 48h. Abundance of transcripts encoding the transcriptional regulators TH receptors α and ß, TH-induced bZip protein, and CCAAT/enhancer binding protein 1 was increased during postembryonic development and following administration of exogenous THs to premetamorphic tadpoles in vivo and culture. In contrast, mRNA representing Krüppel-like factor 9 and cold-inducible RNA binding protein revealed differential effects between natural and precocious metamorphosis. Elevated levels of catalase and Cu/Zn superoxide dismutase mRNA were observed at the end of metamorphosis with transcript levels displaying minimal TH-dependency. No change in stress-responsive heat shock protein 30 mRNA abundance was noted. The results support a role for TH-dependent reprogramming of the lung transcriptome during frog development and reveal a requirement for increased antioxidant capacity following anuran metamorphosis.

De novo Transcriptome Assemblies of Rana (Lithobates) catesbeiana and Xenopus laevis Tadpole Livers for Comparative Genomics without Reference Genomes.

In this work we studied the liver transcriptomes of two frog species, the American bullfrog (Rana (Lithobates) catesbeiana) and the African clawed frog (Xenopus laevis). We used high throughput RNA sequencing (RNA-seq) data to assemble and annotate these transcriptomes, and compared how their baseline expression profiles change when tadpoles of the two species are exposed to thyroid hormone. We generated more than 1.5 billion RNA-seq reads in total for the two species under two conditions as treatment/control pairs. We de novo assembled these reads using Trans-ABySS to reconstruct reference transcriptomes, obtaining over 350,000 and 130,000 putative transcripts for R. catesbeiana and X. laevis, respectively. Using available genomics resources for X. laevis, we annotated over 97% of our X. laevis transcriptome contigs, demonstrating the utility and efficacy of our methodology. Leveraging this validated analysis pipeline, we also annotated the assembled R. catesbeiana transcriptome. We used the expression profiles of the annotated genes of the two species to examine the similarities and differences between the tadpole liver transcriptomes. We also compared the gene ontology terms of expressed genes to measure how the animals react to a challenge by thyroid hormone. Our study reports three main conclusions. First, de novo assembly of RNA-seq data is a powerful method for annotating and establishing transcriptomes of non-model organisms. Second, the liver transcriptomes of the two frog species, R. catesbeiana and X. laevis, show many common features, and the distribution of their gene ontology profiles are statistically indistinguishable. Third, although they broadly respond the same way to the presence of thyroid hormone in their environment, their receptor/signal transduction pathways display marked differences.

Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog.

Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring of endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17ß-estradiol (E2; 10(-9)M) or 4-tert-octylphenol (OP; 10(-9)M, 10(-8)M, and 10(-7)M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10(-7)M OP increased testicular transcript levels. Treatment with 10(-9) and 10(-8)M OP, but not 10(-7)M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within the environment influence the levels of gonadal mRNA encoding key proteins involved in gonadal differentiation.

Influence of temperature on thyroid hormone signaling and endocrine disruptor action in Rana (Lithobates) catesbeiana tadpoles.

Thyroid hormones (THs) are essential for normal growth, development, and metabolic control in vertebrates. Their absolute requirement during amphibian metamorphosis provides a powerful means to detect and assess the impact of environmental contaminants on TH signaling in the field and laboratory. As poikilotherms, frogs can experience considerable temperature fluctuations. Previous work demonstrated that low temperature prevents precocious TH-dependent induction of metamorphosis. However, a shift to a permissive higher temperature allows resumption of the induced metamorphic program regardless of whether or not TH remains. We investigated the impact of temperature on the TH-induced gene expression programs of premetamorphic Rana (Lithobates) catesbeiana tadpoles following a single injection of 10pmol/g body wet weight 3,3',5-triiodothyronine (T3). Abundance profiles of several T3-responsive mRNAs in liver, brain, lung, back skin, and tail fin were characterized under permissive (24°C), nonpermissive (5°C), or temperature shift (5-24°C) conditions. While responsiveness to T3 was retained to varying degrees at nonpermissive temperature, T3 modulation of thibz occurred in all tissues at 5°C suggesting an important role for this transcription factor in initiation of T3-dependent gene expression programs. Low temperature immersion of tadpoles in water containing 10nM T3 and the nonsteroidal anti-inflammatory drug, ibuprofen, or the antimicrobial agent, triclosan, perturbed some aspects of the gene expression programs of tail fin and back skin that was only evident upon temperature shift. Such temporal uncoupling of chemical exposure and resultant biological effects in developing frogs necessitates a careful evaluation of environmental temperature influence in environmental monitoring programs.

PCB related effects thresholds as derived through gene transcript profiles in locally contaminated ringed seals (Pusa hispida).

Causal evidence linking toxic injury to polychlorinated biphenyl (PCB) exposure is typically confounded by the complexity of real-world contaminant mixtures to which aquatic wildlife are exposed. A local PCB "hotspot" on the Labrador coast provided a rare opportunity to evaluate the effects of PCBs on the health of a marine mammal as this chemical dominated their persistent organic pollutant (POP) burdens. The release of approximately 260 kg of PCBs by a military radar facility over a 30 year period (1970-2000) contaminated some local marine biota, including the ringed seal (Pusa hispida). The abundance profiles of eight health-related gene transcripts were evaluated in liver samples collected from 43 ringed seals in the affected area. The mRNA transcript levels of five gene targets, including aryl hydrocarbon receptor (Ahr), interleukin-1 ß (Il1b), estrogen receptor α (Esr1), insulin like growth factor receptor 1 (Igf1), and glucocorticoid receptor α (Nr3c1) correlated with increasing levels of blubber PCBs. PCB threshold values calculated using best-fit hockey-stick regression models for these five genes averaged 1,680±206 ng/g lw, with the lowest, most conservative, being 1,370 ng/g lw for Il1b. Approximately 14% of the seals in the region exceeded this threshold. The dominance of PCBs in the seals studied enabled an assessment of the effects of this chemical on gene transcripts involved in regulating the health of a highly mobile predator, something that is rarely possible in the world of complex mixtures.

Distinctive metabolite profiles in in-migrating Sockeye salmon suggest sex-linked endocrine perturbation.

The health of Skeena River Sockeye salmon (Onchorhychus nerka) has been of increasing concern due to declining stock returns over the past decade. In the present work, in-migrating Sockeye from the 2008 run were evaluated using a mass spectrometry-based, targeted metabolomics platform. Our objectives were to (a) investigate natural changes in a subset of the hepatic metabolome arising from migration-associated changes in osmoregulation, locomotion, and gametogenesis, and (b) compare the resultant profiles with animals displaying altered hepatic vitellogenin A (vtg) expression at the spawning grounds, which was previously hypothesized as a marker of xenobiotic exposure. Of 203 metabolites monitored, 95 were consistently observed in Sockeye salmon livers and over half of these changed significantly during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health of migrating Skeena Sockeye.

Effects of acute exposure to the non-steroidal anti-inflammatory drug ibuprofen on the developing North American Bullfrog (Rana catesbeiana) tadpole.

A variety of pharmaceutical chemicals can represent constituents of municipal effluent outflows that are dispersed into aquatic receiving environments worldwide. Increasingly, there is concern as to the potential of such bioactive substances to interact with wildlife species at sensitive life stages and affect their biology. Using a combination of DNA microarray, quantitative real-time polymerase chain reaction, and quantitative nuclease protection assays, we assessed the ability of sub-lethal and environmentally relevant concentrations of ibuprofen (IBF), a non-steroidal anti-inflammatory agent and prevalent environmental contaminant, to function as a disruptor of endocrine-mediated post-embryonic development of the frog. While the LC50 of IBF for pre-metamorphic Rana catesbeiana tadpoles is 41.5 mg/L (95% confidence interval: 32.3-53.5 mg/L), exposure to concentrations in the ppb range elicited molecular responses both in vivo and in organ culture. A nominal concentration of 15 µg/L IBF (actual = 13.7 µg/L) altered the abundance of 26 mRNA transcripts within the liver of exposed pre-metamorphic R. catesbeiana tadpoles within 6 d. IBF-treated animals demonstrated subsequent disruption of thyroid hormone-mediated reprogramming in the liver transcriptome affecting constituents of several metabolic, developmental, and signaling pathways. Cultured tadpole tail fin treated with IBF for 48 h also demonstrated altered mRNA levels at drug concentrations as low as 1.5 µg/L. These observations raise the possibility that IBF may alter the post-embryonic development of anuran species in freshwater environs, where IBF is a persistent or seasonal pollutant.

Development of a non-lethal method for evaluating transcriptomic endpoints in Arctic grayling (Thymallus arcticus).

With increases in active mining and continued discharge associated with former mine operations, evaluating the health of watersheds in the Canadian Yukon Territory is warranted. Current environmental assessment approaches often employ guidelines established using sentinel species not relevant to Arctic monitoring programs. The present study focused on the successful development of a quantitative real-time polymerase chain reaction (qPCR) assay directed towards the indigenous Arctic grayling (Thymallus arcticus) and examines the feasibility of using non-lethal sampling from the caudal fin as a means for evaluation of mRNA abundance profiles reflective of environmental conditions. In a proof of concept study performed blind, qPCR results from animals in an area with elevated water concentrations of cadmium (Cd) and zinc (Zn) and higher body burdens of Cd, Zn, and lead (Pb) were compared to a reference location in the Yukon Territory. Lower condition factor and a higher abundance of hepatic and caudal fin gene transcripts encoding the metallothionein isoforms (mta/mtb), in addition to elevated heat shock protein 70 (hsp70) and catalase (cat) mRNAs in liver, were observed in fish from the test site. The strong positive correlation between metal body burden and caudal fin mta/mtb mRNA abundance demonstrates a high potential for use of the Arctic grayling assay in non-lethal environmental monitoring programs.

Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species.

Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available.

PCBs are associated with altered gene transcript profiles in arctic Beluga Whales (Delphinapterus leucas).

High trophic level arctic beluga whales (Delphinapterus leucas) are exposed to persistent organic pollutants (POP) originating primarily from southern latitudes. We collected samples from 43 male beluga harvested by Inuvialuit hunters (2008-2010) in the Beaufort Sea to evaluate the effects of POPs on the levels of 13 health-related gene transcripts using quantitative real-time polymerase chain reaction. Consistent with their role in detoxification, the aryl hydrocarbon receptor (Ahr) (r(2) = 0.18, p = 0.045 for 2008 and 2009) and cytochrome P450 1A1 (Cyp1a1) (r(2) = 0.20, p < 0.001 for 2008 and 2009; r(2) = 0.43, p = 0.049 for 2010) transcripts were positively correlated with polychlorinated biphenyls (PCBs), the dominant POP in beluga. Principal Components Analysis distinguished between these two toxicology genes and 11 other genes primarily involved in growth, metabolism, and development. Factor 1 explained 56% of gene profiles, with these latter 11 gene transcripts displaying greater abundance in years coinciding with periods of low sea ice extent (2008 and 2010). δ(13)C results suggested a shift in feeding ecology and/or change in condition of these ice edge-associated beluga whales during these two years. While this provides insight into the legacy of PCBs in a remote environment, the possible impacts of a changing ice climate on the health of beluga underscores the need for long-term studies.

Phenotypic plasticity in the hepatic transcriptome of the European common frog (Rana temporaria): the interplay between environmental induction and geographical lineage on developmental response.

Phenotypic plasticity might facilitate adaptation to new environmental conditions through the enhancement of initial survival of organisms. Once a population is established, further adaptation and diversification may occur through adaptive trait evolution. While several studies have found evidence for this mechanism using phenotypic traits, much less is known at the level of gene expression. Here, we use an islands system of frog populations that show local adaptation and phenotypic plasticity to pool drying conditions in development time until metamorphoses. We examined gene expression differences in Rana temporaria tadpole livers with respect to pool drying at the source population and in response to simulated pool drying in the laboratory. Using a MAGEX cDNA microarray and quantitative real-time polymerase chain reaction (qPCR), we identified an increase in several gene transcripts in response to artificial pool drying including thyroid hormone receptor alpha and beta, carbamoyl phosphate synthetase 1, ornithine transcarbamylase and catalase. In addition, these gene transcripts also showed greater abundance in island populations that developed faster. Hence, the gene transcripts were related to both constitutive response (higher levels in island populations that developed faster) and plastic response (increased abundance under decreasing water levels). This pattern is in accordance with genetic accommodation, which predicts similarities between plastic gene expression and constitutive expression in locally adapted populations.