[show abstract][hide abstract] ABSTRACT: After completion of embryogenesis, many organisms experience an additional obligatory developmental transition to attain a substantially different juvenile or adult form. During anuran metamorphosis, the aquatic tadpole undergoes drastic morphological changes and remodelling of tissues and organs to become a froglet. Thyroid hormones are required to initiate the process, but the mechanism whereby the many requisite changes are coordinated between organs and tissues is poorly understood. Metabolites are often highly conserved biomolecules between species and are the closest reflection of phenotype. Due to the extensive distribution of blood throughout the organism, examination of the metabolites contained therein provides a system-wide overview of the coordinated changes experienced during metamorphosis. We performed an untargeted metabolomic analysis on serum samples from naturally-metamorphosing Rana catesbeiana from tadpoles to froglets using ultraperformance liquid chromatography coupled to a mass spectrometer. Total and aqueous metabolite extracts were obtained from each serum sample to select for nonpolar and polar metabolites, respectively, and selected metabolites were validated by running authentic compounds.
The majority of the detected metabolites (74%) showed statistically significant abundance changes (padj < 0.001) between metamorphic stages. We observed extensive remodelling of five core metabolic pathways: arginine and purine/pyrimidine, cysteine/methionine, sphingolipid, and eicosanoid metabolism and the urea cycle, and found evidence for a major role for lipids during this postembryonic process. Metabolites traditionally linked to human disease states were found to have biological linkages to the system-wide changes occuring during the events leading up to overt morphological change.
To our knowledge, this is the first wide-scale metabolomic study of vertebrate metamorphosis identifying fundamental pathways involved in the coordination of this important developmental process and paves the way for metabolomic studies on other metamorphic systems including fish and insects.
[show abstract][hide abstract] ABSTRACT: 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) (r2=0.18, p=0.045 for 2008 and 2009) and cytochrome P450 1A1 (Cyp1a1) (r2=0.20, p<0.001 for 2008 and 2009; r2=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). δ13C 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: An increasing number of anthropogenic chemicals have demonstrated potential for disruption of biological processes critical to normal growth and development of wildlife species. Both anadromous and freshwater salmon species are at risk of exposure to environmental chemical contaminants that may affect migratory behavior, environmental fitness, and reproductive success. A sensitive metric in determination of the presence and impact of such environmental chemical contaminants is through detection of changes in the status of gene transcript levels using a targeted quantitative real-time polymerase chain reaction assay. Ideally, the wildlife assessment strategy would incorporate conservation-centered non-lethal practices. Herein, we describe the development of such an assay for rainbow trout, Oncorhynchus mykiss, following an acute 96h exposure to increasing concentrations of either 17α-ethinyl estradiol or cadmium. The estrogenic screen included measurement of mRNA encoding estrogen receptor α and β isoforms, vitellogenin, vitelline envelope protein γ, cytochrome p450 family 19 subfamily A, aryl hydrocarbon receptor, and the stress indicator, catalase. The metal exposure screen included evaluation of the latter two mRNA transcripts along with those encoding the metallothionein A and B isoforms. Exposure-dependent transcript abundance profiles were detected in both liver and caudal fin supporting the use of the caudal fin as a non-lethally obtained tissue source. The potential for both transcriptome profiling and genotypic sex determination from fin biopsy was extended, in principle, to field-captured Chinook salmon (Oncorhynchus tshawytscha).
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.
[show abstract][hide abstract] ABSTRACT: The orchestration of anuran metamorphosis is initiated and integrated by thyroid hormones, which change dynamically during larval development and which may represent a target of disruption by environmental contaminants. Studies have found that some anurans experience increased rates of development when exposed to the insecticide carbaryl later in larval development, suggesting that this insecticide could affect thyroid hormone-associated biological pathways. However, the time in development when tadpoles are sensitive to insecticide exposure has not been clearly defined nor has the mechanism been tested. In two separate studies, we exposed recently hatched green frog (Lithobates clamitans) tadpoles to a single, three day carbaryl exposure in the laboratory at either 2, 4, 8, or 16 weeks post-hatching. We examined the impact of carbaryl exposure on mRNA abundance patterns in the brains of frogs following metamorphosis months after a single three day exposure (experiment 1) and in tadpole tails three days after exposure (experiment 2) using cDNA microarrays and quantitative real time polymerase chain reaction (QPCR) analyses. For tadpoles reared through metamorphosis, we measured tadpole growth and development, as well as time to, mass at, and survival to metamorphosis. Although carbaryl did not significantly impact tadpole development, metamorphosis, or survival, clear exposure-related alterations in both tail and brain transcript levels were evident when tadpoles were exposed to carbaryl, particularly in tadpoles exposed at weeks 8 and 16 post-hatching, indicating both short-term and long-term alterations in mRNA expression. These results indicate that carbaryl can have long-lasting effects on brain development when exposure occurs at sensitive developmental stages, which may have implications for animal fitness and function later in the life cycle.
[show abstract][hide abstract] ABSTRACT: Nanoparticles (NPs), materials that have one dimension less than 100 nm, are used in manufacturing, health, and food products, and consumer products including cosmetics, clothing, and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs), thyroxine, and 3,3',5-triiodothyronine (T3), are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs). We used the cultured tailfin (C-fin) assay to examine the exposure effects of 0.1-10 nM (~8-800 ng/L) of three types of ~20 nm TiO2 NPs (P25, M212, M262) and micron-sized TiO2 (μ TiO2) ±10 nM T3. The actual Ti levels were 40.9-64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR) was used to measure the relative amounts of mRNA transcripts encoding TH-responsive THs receptors (thra and thrb) and Rana larval keratin type I (rlk1), as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30), superoxide dismutase (sod), and catalase (cat). The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25, and M212 while no effect was observed with M262 exposure. Therefore, the risk of adversely affecting amphibian tissue by disrupting TH-signaling or inducing cellular stress is low for these compounds relative to other previously-tested NPs.
[show abstract][hide abstract] ABSTRACT: The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5μg/L thyroxine (T(4)), 0.3, 3 and 30μg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and β, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30μg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.
[show abstract][hide abstract] ABSTRACT: The health of sockeye (Oncorhynchus nerka) salmon stocks is of increasing concern; reflecting both a sentinel of human-impacted aquatic environments and as a key fishery for British Columbia, Canada. The spawning migration of Pacific sockeye salmon represents a critical life stage where significant demands are made on animal biology and important BC fisheries are linked to this migration in the Skeena and Fraser River watersheds. These watersheds present very different environments; the former being sparsely populated with little industrial impact, while the latter flows through highly-populated areas. The present study used quantitative real-time PCR analysis of adult sockeye salmon from four 2008 stocks [Fulton River and Pinkut Creek (Skeena) and Weaver Creek and Harrison River (Fraser)] to evaluate ten hepatic gene transcripts associated with reproduction, stress, energy metabolism, and exposure to environmental contaminants. Dynamic changes in mRNA abundance were observed in Fulton River stock animals from the Skeena River mouth to the spawning ground which reflect the physiological demands of in-river migration and reproductive maturation. Inter-stock comparisons of migrants at spawning grounds demonstrated a marked difference in the sex-specific gene hepatic gene expression profiles. Our original hypothesis was that a greater diversity in mRNA profiles is associated with watersheds with higher human impact. However, our observations contradict this posit. Skeena males and females displayed poor definition in their molecular profiles between sexes while the Fraser River fish had very distinctive sex differences that were consistent with the previous year's migration. The genetic sex distribution and ratio of milt versus roe production did not differ between the Skeena and Fraser River spawning site fish. However, a significant percentage of Skeena animals displayed marked discordance of these characteristics with gender-specific hepatic mRNA profiles implying that an alteration in estrogen-mediated signalling has occurred. Continued geospatial and longitudinal assessments will help determine to what extent the dynamic molecular biology of late life-stage sockeye salmon reflects natural variation or modulation by anthropogenic causative agents.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 10/2012; · 2.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many species that contribute to the commercial and ecological richness of our marine ecosystems are harbingers of environmental change. The ability of organisms to rapidly detect and respond to changes in the surrounding environment represents the foundation for application of molecular profiling technologies towards marine sentinel species in an attempt to identify signature profiles that may reside within the transcriptome, proteome, or metabolome and that are indicative of a particular environmental exposure event. The current review highlights recent examples of the biological information obtained for marine sentinel teleosts, mammals, and invertebrates. While in its infancy, such basal information can provide a systems biology framework in the detection and evaluation of environmental chemical contaminant effects on marine fauna. Repeated evaluation across different seasons and local marine environs will lead to discrimination between signature profiles representing normal variation within the complex milieu of environmental factors that trigger biological response in a given sentinel species and permit a greater understanding of normal versus anthropogenic-associated modulation of biological pathways, which prove detrimental to marine fauna. It is anticipated that incorporation of contaminant-specific molecular signatures into current risk assessment paradigms will lead to enhanced wildlife management strategies that minimize the impacts of our industrialized society on marine ecosystems.
Ecotoxicology and Environmental Safety 02/2012; 76(2):23-38. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Because thyroid hormones (THs) are conserved modulators of development and physiology, identification of compounds adversely affecting TH signaling is critical to human and wildlife health. Anurans are an established model for studying disruption of TH signaling because metamorphosis is dependent upon the thyroid system. In order to strengthen this model and identify new gene transcript biomarkers for TH disruption, we performed DNA microarray analysis of Xenopus laevis tadpole tail transcriptomes following treatment with triiodothyronine (T(3)). Comparison of these results with previous studies in frogs and mammals identified 36 gene transcripts that were TH-sensitive across clades. We then tested molecular biomarkers for sensitivity to disruption by exposure to wastewater effluent (WWE). X. laevis tadpoles, exposed to WWE from embryo through metamorphosis, exhibited an increased developmental rate compared to controls. Cultured tadpole tails showed dramatic increases in levels of four TH-sensitive gene transcripts (thyroid hormone receptor β (TRβ), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T(3) and WWE extracts. TRβ, DIO2, and CRHBP were identified as TH sensitive in other studies, while FAPα mRNA transcripts were highly TH sensitive in our array. The results validate the array and demonstrate TH-disrupting activity by WWE. Our findings demonstrate the usefulness of cross-clade analysis for identification of gene transcripts that provide sensitivity to endocrine disruption. Further, the results suggest that development is disrupted by exposure to complex mixes of compounds found in WWE possibly through interference with TH signaling.
General and Comparative Endocrinology 01/2012; 176(3):481-92. · 2.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitrate and nitrite are common aqueous pollutants that are known to disrupt the thyroid axis. In amphibians, thyroid hormone (TH)-dependent metamorphosis is affected, although whether the effect is acceleration or deceleration of this developmental process varies from study to study. One mechanism of action of these nitrogenous compounds is through alteration of TH synthesis. However, direct target tissue effects on TH signaling are hypothesized. The present study uses the recently developed cultured tail fin biopsy (C-fin) assay to study possible direct tissue effects of nitrate and nitrite. Tail biopsies obtained from premetamorphic Rana catesbeiana tadpoles were exposed to 5 and 50 mg/L nitrate (NO(3)-N) and 0.5 and 5 mg/L nitrite (NO(2)-N) in the absence and presence of 10 nM T(3). Thyroid hormone receptor β (TRβ) and Rana larval keratin type I (RLKI), both of which are TH-responsive gene transcripts, were measured using quantitative real time polymerase chain reaction. To assess cellular stress which could affect TH signaling and metamorphosis, heat shock protein 30, and catalase (CAT) transcript levels were also measured. We found that nitrate and nitrite did not significantly change the level of any of the four transcripts tested. However, nitrate exposure significantly increased the heteroscedasticity in response of TRβ and RLKI transcripts to T(3). Alteration in population variation in such a way could contribute to the previously observed alterations of metamorphosis in frog tadpoles, but may not represent a major mechanism of action.
[show abstract][hide abstract] ABSTRACT: Amphibians are important vertebrates in toxicology often representing both aquatic and terrestrial forms within the life history of the same species. Of the thousands of species, only two have substantial genomics resources: the recently published genome of the Pipid, Xenopus (Silurana) tropicalis, and transcript information (and ongoing genome sequencing project) of Xenopus laevis. However, many more species representative of regional ecological niches and life strategies are used in toxicology worldwide. Since Xenopus species diverged from the most populous frog family, the Ranidae, ~200 million years ago, there are notable differences between them and the even more distant Caudates (salamanders) and Caecilians. These differences include genome size, gene composition, and extent of polyploidization. Application of toxicogenomics to amphibians requires the mobilization of resources and expertise to develop de novo sequence assemblies and analysis strategies for a broader range of amphibian species. The present mini-review will present the advances in toxicogenomics as pertains to amphibians with particular emphasis upon the development and use of genomic techniques (inclusive of transcriptomics, proteomics, and metabolomics) and the challenges inherent therein.
[show abstract][hide abstract] ABSTRACT: Killer whales in the NE Pacific Ocean are among the world's most PCB-contaminated marine mammals, raising concerns about implications for their health. Sixteen health-related killer whale mRNA transcripts were analyzed in blubber biopsies collected from 35 free-ranging killer whales in British Columbia using real-time quantitative polymerase chain reaction. We observed PCB-related increases in the expression of five gene targets, including the aryl hydrocarbon receptor (AhR; r(2) = 0.83; p < 0.001), thyroid hormone α receptor (TRα; r(2) = 0.64; p < 0.001), estrogen α receptor (ERα; r(2) = 0.70; p < 0.001), interleukin 10 (IL-10; r(2) = 0.74 and 0.68, males and females, respectively; p < 0.001), and metallothionein 1 (MT1; r(2) = 0.58; p < 0.001). Best-fit models indicated that population (dietary preference), age, and sex were not confounding factors, except for IL-10, where males differed from females. While the population-level consequences are unclear, the PCB-associated alterations in mRNA abundance of such pivotal end points provide compelling evidence of adverse physiological effects of persistent environmental contaminants in these endangered killer whales.
[show abstract][hide abstract] ABSTRACT: The potential impact of commercial salmon aquaculture along the coast of British Columbia on the health of non-target marine wildlife is of growing concern. In the current initiative, the biological effects on gene expression within spot prawn (Pandalus platyceros) exposed to the sea lice controlling agent, emamectin benzoate (EB; 0.1-4.8 mg/kg sediment), were investigated. A mean sediment/water partitioning coefficient (K(p)) was determined to be 21.81 and significant levels of EB were detected in the tail muscle tissue in all exposed animals. Animals selected for the experiment did not have eggs and were of similar weight. Significant mortality was observed within 8 days of EB treatment at concentrations between 0.1 and 0.8 mg/kg and there was no effect of EB on molting. Twelve spot prawn cDNA sequences were isolated from the tail muscle either by directed cloning or subtractive hybridization of control versus EB exposed tissues. Three of the transcripts most affected by EB exposure matched sequences encoding the 60S ribosomal protein L22, spliceosome RNA helicase WM6/UAP56, and the intracellular signal mediator histidine triad nucleotide binding protein 1 suggesting that translation, transcription regulation, and apoptosis pathways were impacted. The mRNA encoding the molting enzyme, β-N-acetylglucosaminidase, was not affected by EB treatment. However, the expression of this transcript was extremely variable making it unsuitable for effects assessment. The results suggest that short-term exposure to EB can impact biological processes within this non-target crustacean.
[show abstract][hide abstract] ABSTRACT: Triclosan (TCS) and triclocarban (TCC) are widely used broad spectrum bactericides that are common pollutants of waterways and soils. Methyl triclosan (mTCS) is the predominant bacterial TCS metabolite. Previous studies have shown that TCS disrupts thyroid hormone (TH) action; however, the effects of mTCS or TCC are not known. The present study uses the cultured frog tadpole tail fin biopsy (C-fin) assay and the TH-responsive rat pituitary GH3 cell line to assess the effects of these three chemicals (1-1000 nM) on TH signaling and cellular stress within 48 h. mRNA abundance of TH receptor β, Rana larval keratin type I (TH-response), heat shock protein 30, and catalase (stress-response) was measured using quantitative real-time polymerase chain reaction in the C-fin assay. The TH-responsive gene transcripts encoding growth hormone, deiodinase I, and prolactin were measured in GH3 cells with the heat shock protein 70 transcript acting as a cellular stress indicator. We found alteration of stress indicators at a wide range of concentrations of TCS, mTCS, and TCC in both test systems. mTCS and TCC affected TH-responsive gene transcripts at the highest concentration in mammalian cells, whereas a modest effect included lower concentrations in the C-fin assay. In contrast, TCS did not affect TH-responsive transcripts. These results identify nontarget biological effects of these bacteriocides on amphibian and mammalian cells and suggest the TH-disrupting effects observed for TCS could be mediated through its metabolite.