Tadpole Metamorphosis Research Articles

Characterizing the composition, metabolism and physiological functions of the fatty liver in Rana omeimontis tadpoles

BackgroundFat storage is required for the life cycle of many organisms. The primary fat depot for most vertebrates is white adipose tissue. However, in primitive vertebrates (e.g., agnathan group and elasmobranchs), the liver is usually responsible for fat storage. Among the vertebrates, amphibians have a unique status, as their larvae live in the water and exhibit some primitive traits that are similar to fish. Although it has been recognized that adult frogs use their abdominal white adipose tissue as a primary fat depot, how tadpoles store their fat is still inconclusive. The metabolic traits and physiological functions of primitive fat depots may have wide-ranging implications on the pathology of abnormal lipid deposition in mammals and the evolution of fat storage.ResultsRana omeimontis tadpoles used their liver as the primary fat depot. In sufficiently fed tadpoles at stage 30–31, the hepatosomatic index (HSI) reached up to 7%, and triglycerides (TG) accounted for 15% of liver weight. Their liver resembled white adipose tissue in histological morphology, characterized by polygonal hepatocytes filled with fat. Their liver metabolic composition was unique, characterized by the dominance of maltotriose, arachidonic acid and dipeptides in soluble carbohydrates, free fatty acids and amino acids. Hepatic fat was the major metabolic fuel of fasted R. omeimontis tadpoles, which had similar reserve mobilization and allocation patterns as mammals. From a developmental perspective, hepatic fat was important to fuel late metamorphic climax. Interestingly, starvation induced accelerated metamorphosis in tadpoles with high HSI (4.96 ± 0.21%). However, this phenomenon was not observed in tadpoles with low HSI (2.71 ± 0.16%), even though they had similar initial body weight and developmental stage. Hepatic fat abundance was the most prominent difference between the two groups.ConclusionTo the best of our knowledge, this is the first report that liver can be the primary fat depot in vertebrates with higher evolutionary status than bony fish. The unique hepatic histological and metabolic traits likely either guard their liver against lipotoxicity or make their hepatocytes adapt to fat accumulation. This fatty liver could be a primitive counterpart of mammalian white adipose tissue (WAT). In addition, our study showed that the hepatic reserves of tadpoles, especially TG content, may provide body condition signals to modulate metamorphosis.

Changes in intestinal microbial community of Rana chensinensis tadpoles during metamorphosis

Intestinal microbiota plays important roles in animal development, but little is known about intestinal microbial community changes of amphibian during metamorphosis. In our research, we investigated the intestinal bacteria community in 120 tadpoles of Rana chensinensis (R. chensinensis) at five key developmental time points via 16S rRNA amplicon sequencing. We counted morphological parameters of the body and intestine of R. chensinensis tadpoles at varying developmental stages. Our survey revealed that intestinal length and intestinal weight were reduced by 89% and 83.2% during metamorphosis, respectively. The microbial diversity and composition of R. chensinensis intestine exhibited marked differences along with the process of metamorphosis. Compared to the aquatic stages, terrestrial stages showed major shifts in microbial composition, including reduction in Bacteroides, increases in Shewanella and Cetobacterium. The remodeling of intestinal microbiota is accompanied by the transition of diet type, host habitat and gut. Our evidence supports the idea that the composition of intestinal microbiota changed along with tadpole metamorphosis.

Modulation of Metamorphic and Regenerative Events by Cold Atmospheric Pressure Plasma Exposure in Tadpoles, Xenopus laevis

Atmospheric pressure plasma has found wide clinical applications including wound healing, tissue regeneration, sterilization, and cancer treatment. Here, we have investigated its effect on developmental processes like metamorphosis and tail regeneration in tadpoles. Plasma exposure hastens the process of tail regeneration but delays metamorphic development. The observed differences in these two developmental processes following plasma exposure are indicative of physiological costs associated with developmental plasticity for their survival. Ultrastructural changes in epidermis and mitochondria in response to the stress of tail amputation and plasma exposure show characteristics of cellular hypoxia and oxidative stress. Mitochondria show morphological changes such as swelling with wide and fewer cristae and seem to undergo processes such as fission and fusion. Complex interactions between calcium, peroxisomes, mitochondria and their pore transition pathways are responsible for changes in mitochondrial structure and function, suggesting the subcellular site of action of plasma in this system.

Thyroid Hormone Receptor Alpha Is Required for Thyroid Hormone-Dependent Neural Cell Proliferation During Tadpole Metamorphosis.

Thyroid hormone (T3) plays several key roles in development of the nervous system in vertebrates, controlling diverse processes such as neurogenesis, cell migration, apoptosis, differentiation, and maturation. In anuran amphibians, the hormone exerts its actions on the tadpole brain during metamorphosis, a developmental period dependent on T3. Thyroid hormone regulates gene transcription by binding to two nuclear receptors, TRα and TRβ. Our previous findings using pharmacological and other approaches supported that TRα plays a pivotal role in mediating T3 actions on neural cell proliferation in Xenopus tadpole brain. Here we used Xenopus tropicalis (X. tropicalis) tadpoles with an inactivating mutation in the gene that encodes TRα to investigate roles for TRα in mitosis and gene regulation in tadpole brain. Gross morphological analysis showed that mutant tadpoles had proportionally smaller brains, corrected for body size, compared with wildtype, both during prometamorphosis and at the completion of metamorphosis. This was reflected in a large reduction in phosphorylated histone 3 (pH3; a mitosis marker) immunoreactive (ir) nuclei in prometamorphic tadpole brain, when T3-dependent cell proliferation is maximal. Treatment of wild type premetamorphic tadpoles with T3 for 48 h induced gross morphological changes in the brain, and strongly increased pH3-ir, but had no effect in mutant tadpoles. Thyroid hormone induction of the direct TR target genes thrb, klf9, and thibz was dysregulated in mutant tadpoles. Analysis of gene expression by RNA sequencing in the brain of premetamorphic tadpoles treated with or without T3 for 16 h showed that the TRα accounts for 95% of the gene regulation responses to T3.

Early Spring and Early Vanishing Wetlands as Harbingers of the Future? The Climate Change Trap for Ephemeral Pond-Breeding Frogs

The decline of the northern red-legged frog, Rana aurora, in the Pacific Northwest has been attributed to invasive species, habitat loss, and climate change. Rana aurora lay eggs on emergent vegetation, and larval development occurs in shallow, often ephemeral wetlands. In 2015 and 2016 we investigated how habitat and water quality parameters influence breeding productivity and development of R. aurora. Early season temperatures in southwestern British Columbia during both years were warmer than in the previous two decades and egg-laying occurred much earlier than any previous records, allowing us to follow the development of R. aurora under unusual climatic conditions. We monitored 43 wetlands in the Little Campbell River Watershed and nearby areas in 2015 and selected six sites to monitor tadpole metamorphosis. Peak egg mass abundance was inversely proportional to concentrations of nitrates and orthophosphates, and colder water appeared to support greater egg mass abundance. Although warmer ambient temperatures facilitated earlier onset of R. aurora breeding, we observed some egg mortality due to subsequent frost. Sites with earlier peak egg mass abundance dates did not exhibit earlier peak metamorph dates, nor did warmer water appear to facilitate quicker larval development. In 2015, one wetland dried up before tadpoles completed metamorphosis. The frog's reproductive cycle may adapt to warmer temperatures to some degree, but erratic precipitation or frost events could reduce R. aurora survivorship beyond recovery. The disruption of larval development we observed under unusually warm spring conditions provides a useful vantage point for predicting future impacts of climate change.

Contaminant and Environmental Influences on Thyroid Hormone Action in Amphibian Metamorphosis.

Aquatic and terrestrial environments are increasingly contaminated by anthropogenic sources that include pharmaceuticals, personal care products, and industrial and agricultural chemicals (i. e., pesticides). Many of these substances have the potential to disrupt endocrine function, yet their effect on thyroid hormone (TH) action has garnered relatively little attention. Anuran postembryonic metamorphosis is strictly dependent on TH and perturbation of this process can serve as a sensitive barometer for the detection and mechanistic elucidation of TH disrupting activities of chemical contaminants and their complex mixtures. The ecological threats posed by these contaminants are further exacerbated by changing environmental conditions such as temperature, photoperiod, pond drying, food restriction, and ultraviolet radiation. We review the current knowledge of several chemical and environmental factors that disrupt TH-dependent metamorphosis in amphibian tadpoles as assessed by morphological, thyroid histology, behavioral, and molecular endpoints. Although the molecular mechanisms for TH disruption have yet to be determined for many chemical and environmental factors, several affect TH synthesis, transport or metabolism with subsequent downstream effects. As molecular dysfunction typically precedes phenotypic or histological pathologies, sensitive assays that detect changes in transcript, protein, or metabolite abundance are indispensable for the timely detection of TH disruption. The emergence and application of ‘omics techniques—genomics, transcriptomics, proteomics, metabolomics, and epigenomics—on metamorphosing tadpoles are powerful emerging assets for the rapid, proxy assessment of toxicant or environmental damage for all vertebrates including humans. Moreover, these highly informative ‘omics techniques will complement morphological, behavioral, and histological assessments, thereby providing a comprehensive understanding of how TH-dependent signal disruption is propagated by environmental contaminants and factors.

Tail Resorption During Metamorphosis in Xenopus Tadpoles.

Tail resorption in anuran tadpoles is one of the most physically and physiologically notable phenomena in developmental biology. A tail that is over twice as long as the tadpole trunk is absorbed within several days, while concurrently the tadpole's locomotive function is continuously managed during the transition of the driving force from the tail to hindlimbs. Elaborate regulation is necessary to accomplish this locomotive switch. Tadpole's hindlimbs must develop from the limb-bud size to the mature size and the nervous system must be arranged to control movement before the tail is degenerated. The order of the development and growth of hindlimbs and the regression of the tail are regulated by the increasing levels of thyroid hormones (THs), the intracellular metabolism of THs, the expression levels of TH receptors, the expression of several effector genes, and other factors that can modulate TH signaling. The tail degeneration that is induced by the TH surge occurs through two mechanisms, direct TH-responsive cell death (suicide) and cell death caused by the degradation of the extracellular matrix and a loss of cellular anchorage (murder). These pathways lead to the collapse of the notochord, the contraction of surviving slow muscles, and, ultimately, the loss of the tail. In this review, I focus on the differential TH sensitivity of the tail and hindlimbs and the mechanism of tail resorption during Xenopus metamorphosis.

Reproduction and metamorphosis in the Myristica Swamp tree frog, Mercurana myristicapalustris (Anura: Rhacophoridae).

The reproductive biology of the Myristica Swamp tree frog (Mercurana myristicapalustris), a monotypic rhacophorid frog endemic to the foothills of the Western Ghats mountains of India, has remained unknown since the description of the genus and species. We monitored individuals from parental generation amplexus to the completion of offspring generation tadpole metamorphosis. Surprisingly, our observations revealed that this species exhibits many previously unknown characteristics, including the first ever record of the female, and a diverse call repertoire, consisting of five different call types (the functions of which remain incompletely known). We were also able to determine that reproductive activity peaked during the late pre-monsoon season, that males engaged in intraspecific aggressive encounters to occupy and to defend desirable territories, and that oviposition took place in terrestrial nests made by females. Embryonic development in the unattended nest was followed by tadpole development, which concluded within 40 days. The specific breeding mode employed by Mercurana, which restricts its range to the endangered Myristica swamp ecosystem, likely renders it susceptible to multiple threats, which should be considered jointly in future conservation planning.

Development and evaluation of gene expression biomarkers for chemical pollution in common frog (Rana temporaria) tadpoles

Pollutants have been proposed as one factor in the worldwide declines of amphibian species and populations. Applying gene expression analysis of liver RNA in tadpoles would be a possible approach for biomarker measurements to increase knowledge of ecological health in amphibian populations. The major aim of this study was to explore the relevance of applying gene expression analyses of cytochrome p450 (cyp1a), metallothionein (mt), and vitellogenin (vtg) in Rana temporaria tadpoles. Therefore, tadpoles were exposed for 1 week to β-naphthoflavone (BNF), cadmium chloride (CdCl2), and ethinylestradiol (EE2). Primers were developed for RT-qPCR to analyze gene expression in livers. The result showed that the methods for gene expression analyses of cyp1a, mt, and vtg as well as the reference gene β-actin (bact) were successful not only in R. temporaria but also in another amphibian, Rana arvalis. The gene expression of cyp1a was induced by BNF and the gene expression of mt was induced by CdCl2 but no significant induction was recorded in vtg expression after exposure to EE2. Gene expressions varied throughout the tadpole metamorphosis development, in particular for vtg. Overall, the use of gene expression of cyp1a and mt as biomarkers in wild tadpoles seems promising while the use of vtg seems less relevant due to high natural variation and low background expression. The study shows that variations in gene expressions between tadpoles of different genetic origin are important to consider when evaluating the data. The present study has thus increased the background knowledge about gene expression applicability as biomarker for tadpoles.

The observed decline of Western Toads (<i>Anaxyrus boreas</i>) over several decades at a novel winter breeding site

The Western Toad (Anaxyrus boreas) population of the Atlin Warm Springs in northwestern British Columbia has persisted since at least 1924. An extraordinary feature of the population has been winter breeding in late February to early March, while nearby cold-water populations breed in late-May. Metamorphosis of tadpoles, enhanced by the warm water, occurs as early as late-March. In 2008, Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) was documented in toadlets at the warm springs. Until 2005, as many as eight egg clutches and 25 breeding adults had been observed at the warm springs, after which the population declined. In 2017, novel spring breeding occurred in a cooler pond in the spring complex. Future observations will help determine whether the population is recovering and whether breeding phenology and habitat use have changed.

Effect of Two Levels of Temperature and Natural and Artificial Photoperiod on Growth and Metamorphosis of Bullfrog Tadpoles in an Intensive Rearing System

AbstractThe effect of two levels of temperature and natural and artificial photoperiods on the growth and metamorphosis of tadpoles of the American bullfrog Rana catesbeiana were evaluated in an intensive‐rearing culture environment. Four groups of tadpoles at Gosner stage 24–25 were subjected for 50 d to four different treatments: (1) Tadpole culture with temperature control system (25.9 ± 0.34°C) and natural photoperiod of 12.25 h light : 11.75 h dark (treatment T), (2) an artificial photoperiod of 10 h light : 14 h dark using a white LED lamp and no temperature control system (18.7 ± 1°C) (treatment P), (3) the combination of temperature control system (26.01 ± 0.44°C) and an artificial photoperiod (10 h light : 14 h dark) (treatment T/P), and (4) culture with no temperature control system (21.87 ± 1.09°C) and a natural photoperiod (12.25 h light : 11.75 h dark) (treatment N/S). The experiment was performed under greenhouse conditions. Tadpoles in treatments T and T/P had the best growth performance as measured by total length (45.34 ± 1.89 mm [mean ± SE] for T/P, 44.72 ± 2.13 mm for T), head length (17.15 ± 0.67 mm for T/P, 18.34 ± 0.70 for T), and weight (1.00 ± 0.03 g for T/P, 1.14 ± 0.013 g for T). Tadpoles reared using white light LED lamps had reduced growth. The best results for tadpole metamorphosis were obtained in treatment T/P (25.9%), while treatment P presented the highest survival (36.2%). This experiment demonstrates that having temperature regulation is important to achieve better tadpole growth in intensive rearing systems. The regulation of both variables (T and P) is recommended for activating metamorphosis in the final phases of the tadpole development.

Following Endocrine-Disrupting Effects on Gene Expression in Xenopus laevis.

Endocrine-disrupting chemicals (EDCs), found in all categories of chemicals, are suspected to be a cause of declining well-being and human health, both as single molecules and as mixtures. It is therefore necessary to develop high throughput methods to assess the endocrine-disrupting potential of multiple chemicals currently on the market that are as yet untested. An advantage of in vivo chemical screening is that it provides a full spectrum of physiological impacts exerted by a given chemical. Xenopus laevis is an ideal model organism to test thyroid axis disruption in vivo as thyroid hormones (THs) are highly conserved across vertebrates and orchestrate tadpole metamorphosis. In particular, NF stage 45 Xenopus laevis are most apt for in vivo screening as at this stage the tadpoles possess all the main elements of thyroid hormone signaling (thyroid receptors, deiodinases transporters) and are metabolically competent, while fitting into multiple well plates, allowing the use of small amounts of test chemicals. One way to assess the endocrine-disrupting potential of chemicals or mixtures thereof is to analyze gene expression in organisms after a short time exposure to the chemical(s). Here we describe a protocol using Xenopus laevis embryos to detect endocrine disruption of the thyroid axis by analysis of gene expression and an alternative protocol for fluorescence read-out using a transgenic GFP-expressing Xenopus laevis line. Taken together, these methods allow detection of subtle changes in TH signaling by EDCs that either activate or inhibit TH signaling in vivo.

Effect of the Metabolic Modification of Environmental Chemicals on Endocrine-disrupting Activity

The endocrine-disrupting activities of various environmental chemicals are metabolically activated. For example, diphenyls, styrene oligomers, chalcones, trans-stilbene and 2-nitrofluorene are not estrogens, but after incubation with liver microsomes, their metabolites show estrogenic activities. Thus, these chemicals are estrogenically activated by the cytochrome P450 system. In contrast, the antiandrogenic activity of fenthion, an organophosphorus insecticide, is abolished after metabolism to sulfoxide and sulfone derivatives. Structural requirements of twenty bisphenol A related compounds, as well as various benzophenones, for estrogenic and antiandrogenic activities have been investigated. The estrogenic and antiandrogenic activities of Benzophenone 3, a representative UV absorbant, are activated by oxidative metabolism. Parabens (used as antimicrobial agents) exhibit estrogenic activity, and their potency shows a bell-shaped curve between C1 (methylparaben) and C12 (dodecylparaben) parabens. The AhR ligand activity of indirubin is decreased by metabolism. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDE) are activated by hydroxylation to show estrogenic and thyroid hormone-disrupting activities. Halogen adjacent to a hydroxyl group is essential for thyroid hormone-disrupting activity. Tetrabromobisphenol A, tetrachlorobisphenol A and tetramethylbisphenol A also exhibit thyroid hormone-disrupting activity. Amphibian metamorphosis of tadpoles to frogs is affected by hydroxylated PCB, hydroxylated PBDE and bisphenol A derivatives. These chemicals suppress thyroid hormone-dependent metamorphosis, acting as antagonists of thyroid hormone. Thus, metabolic modification can have a dramatic impact on the endocrine-disrupting activities of environmental chemicals.

Photic and non-photic regulation of growth, development, and metamorphosis in giant tree frog (Rhacophorus maximus) tadpoles

Amphibians are seasonal breeders living in seasonal environments having time constraints to achieve their annual life history states. Their survival is strongly dependent on successful timing of certain critical life history decisions. Larval amphibian development, growth, and metamorphosis are strongly time-limited due to variations in the seasonal factors. Photoperiod influences wide range of physiological processes in seasonal breeder. Being ectothermic, temperature may also play critical role in development of amphibian larvae. Food also acts as an ultimate factor in many organisms. Most studies of rearing conditions on growth, development, and metamorphosis of amphibian’s tadpoles have been focused on laboratory species; genus Xenopus. Less is known about other seasonally breeding amphibians. In present study, we addressed the effects of photic (light quality) and non-photic cues (temperature and food quality) on growth, development and metamorphosis of giant tree frog (Rhacophorus maximus). Three experiments have been performed. In experiment one effect of light quality (light wavelength), in experiment two effect of food quality and in experiment three effect of temperature on growth, development, and metamorphosis of giant tree frog tadpoles were studied. Our results suggest that light and food quality and high temperature promotes faster growth, development, and metamorphosis in R. maximus tadpoles.

Full compensation of growth in salt‐tolerant tadpoles after release from salinity stress

AbstractThe benefit of compensatory growth is to minimize the negative effects of stress experienced by organisms early in development once the conditions improve. Tadpoles of crab‐eating frog Fejervarya cancrivora are euryhaline, have the highest salt tolerance among reported anuran species and may exhibit a different pattern of compensatory growth after stress release. We examined how F. cancrivora tadpoles responded and possibly recovered from salinity stresses. Specifically, we tested whether tadpoles expressed compensatory growth, what degree of compensation they expressed after release from salinity stress and whether the stage at and duration during which they experienced salt stress influenced the size at and time to metamorphosis of tadpoles. We found that the effects of salinity stress on the growth and development of tadpoles are quite different between early and late exposure to high salinity (LHH vs. LLH). Exposure to high salinity late in development (LLH) reduced growth but not the rate of development, resulting in smaller metamorphs but a similar time to metamorphosis when compared to the control treatment (LLL). By contrast, exposure to high salinity early in development (LHH) retarded rates of growth and development of tadpoles, but the resulting prolonged larval period enabled tadpoles to catch up and reach similar metamorphic sizes to LLL treatment metamorphs. Most importantly, tadpoles released from salinity stress (LHL) exhibited similar time to and size at metamorphosis when compared to the LLL tadpoles, suggesting that they can exhibit full compensation of growth. Compensatory growth may be a useful mechanism for F. cancrivora tadpoles inhabiting brackish habitats, allowing them to overcome the effects of dramatically increased salt levels by expressing catch‐up growth when the salt stress ceases.

CORRIGENDUM.

Molecular EcologyVolume 26, Issue 14 p. 3851-3851 CORRIGENDUMFree Access CORRIGENDUM This article corrects the following: Fine-grained adaptive divergence in an amphibian: genetic basis of phenotypic divergence and the role of nonrandom gene flow in restricting effective migration among wetlands Alex Richter-Boix, María Quintela, Marcin Kierczak, Marc Franch, Anssi Laurila, Volume 22Issue 5Molecular Ecology pages: 1322-1340 First Published online: January 7, 2013 First published: 04 July 2017 https://doi.org/10.1111/mec.14161AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat ALEX RICHTER - BOIX, MARIA QUINTELA, MARCIN KIERCZAK, MARC FRANCH, ANSSI LAURILA In the published paper (Richter-Boix et al. 2013), we incorrectly stated that we sequenced the protein-coding region of the thyroid hormone receptor gene (TRβ). The gene sequenced was in fact the gene C/EBP-1, a thyroid hormone receptor gene and transcription factor. As a consequence, instead of TRβ, it should read “the thyroid hormone receptor gene C/EBP-1” throughout the paper and supporting information of the article online. This error, however, does not strongly affect the main interpretation of our results. The C/EBP-1 gene, as the TRβ gene, is a thyroid hormone receptor with the dual function of repressing or activating thyroid-induced gene expression before or during tadpole metamorphosis. The C/EBP-1 gene, like TRβ, is a primary early TH-response gene during metamorphosis (Becker, Schneider, Davey, & Galton, 1995; Chen & Atkinson, 1997; Mochizuki, Goda, & Yamauchi, 2012; Murata & Yamauchi, 2005). It is not a general TH-receptor, but a specific one involved in the reprogramming of gene expression in the liver during amphibian metamorphosis (Chen & Atkinson, 1997; Chen, Hu, & Atkinson, 1994). The C/EBP-1 gene is a homologue to the mammalian C/EBP-α gene, which is known to play an essential role in cell cycle, hematopoiesis, differentiation and host immune response (Tsukada, Yoshida, Kominato, & Auron, 2011). Metamorphosis of a tadpole to a frog involves coordination between environmental cues, endocrine system and tissue-specific developmental programmes, like the one described in the tadpole liver tissue, which orchestrate changes in form and function required in the transition from aquatic to terrestrial lifestyle. We would like to thank Yvonne Meyer-Lucht for pointing this out. References Becker, K. B., Schneider, M. J., Davey, J. C., & Galton, V. A. (1995). The type III 5-deiodinase in Rana catesbeiana tadpoles is encoded by a thyroid hormone-responsive gene. Endocrinology, 136, 4424– 4431. Chen, Y., & Atkinson, B. G. (1997). Role for the Rana catesbeiana homologue of C/EBP α in the reprogramming of gene expression in the liver of metamorphosing tadpoles. Developmental Genetics, 20, 152– 162. Chen, Y., Hu, H., & Atkinson, B. G. (1994). Characterization and expression of C/EPB-like genes in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis. Developmental Genetics, 15, 366– 377. Mochizuki, K., Goda, T., & Yamauchi, K. (2012). Gene expression profile in the liver of Rana catesbiana tadpoles exposed to low temperature in the presence of thyroid hormone. Biochemical and Biophysical Research Communications, 420, 845– 850. Murata, T., & Yamauchi, K. (2005). Low-temperature arrest of the triiodothyronine-dependent transcription in Rana catesbeiana red blood cells. Endocrinology, 146, 256– 264. Richter-Boix, A., Quintela, M., Kierczak, M., Franch, M., & Laurila, A. (2013). Fine-grained adaptive divergence in an amphibian: Genetic basis of phenotypic divergence and the role of nonrandom gene flow in restricting effective migration among wetlands. Molecular Ecology, 22, 1322– 1340. Tsukada, J., Yoshida, Y., Kominato, Y., & Auron, P. E. (2011). The CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation. Cytokine, 54, 6– 19. Volume26, Issue14July 2017Pages 3851-3851 ReferencesRelatedInformation

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, Euphlyctis cyanophlyctis.

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32 degrees C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32 degrees C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32 degrees C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32 degrees C) during stages 7-42 produced male-biased sex ratio. Rearing of tadpoles at 32 degrees C during stages 25-42 produced malebiased sex ratio, while exposure during stages 7-25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22 degrees C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.

116 INCORPORATION AND DEVELOPMENTAL TOXICITY OF QUANTUM DOT NANOPARTICLES IN AMPHIBIAN LARVAE

The use of nanoparticles both commercially and pharmaceutically has increased over the past decade, including fluorescent quantum dot nanoparticles (QD) in biochemical research for in vivo imaging. Previous studies have reported the toxic effects of nanoparticles, but their effects on larval metamorphosis and animal development and growth have not been thoroughly examined. Additionally, the method of uptake of nanoparticles by larval systems is unknown. Amphibian larvae are an ideal model for assessing toxicity because of their sensitivity to environmental contaminants and rapid and easily observable developmental stages. We used Anaxyrus fowleri tadpoles to investigate QD (≤ 25 nm diameter) integration into larvae and possible deleterious effects on their growth and development. Tadpoles (A. fowleri; n = 5/group) were placed in 24-well plates containing 1 mL of distilled water and increasing concentrations of QD (0, 1, and 2 nM) 72 h post-hatch. The fluorescence emission of QD in wells was detected at various time points (1, 2, 24, 48, and 72 h) using the in vivo imaging system (IVIS). A subset of tadpoles was killed (MS-222) and sectioned for histopathology. Remaining tadpoles were monitored throughout development. Fluorescence emission of QD in sectioned tadpoles was visualised using an EVOS Cell Imaging System. Developmental metrics of living tadpoles were recorded until metamorphosis. Fluorescence intensity between controls and dosage groups were analysed by ANOVA-1, followed by Student’s l.s.d. test to evaluate the effects of QD concentration and exposure time. The threshold of significance was P < 0.05. The rate of incorporation of QD into tadpoles was determined using the equation y = C + Ao*2(–x/t1/2), where t1/2 is the half-life of QD remaining in solution. The IVIS imaging revealed a rapid decrease of QD fluorescence (total flux) signals from the aqueous tadpole environment. Decreases in fluorescence occurred within 1 h post-exposure and appeared dose and time dependent, with signal nearly gone within 48 h. Half-life of total flux (time necessary for tadpoles to absorb half of the QD in solution) is 20.75 h (R2 = 0.92) and 2.54 h (R2 = 0.96) for 1 nm and 2 nm QD in solution, respectively. The EVOS imaging revealed integration of QD and localization into tadpole tissues. Fluorescence was exclusively found within the mouth, gills, and sections of the intestinal lumen of exposed tadpoles within the first hour. Dose-dependent increases in fluorescence within tissue were observed at each time-point. No signal was observed in controls. In remaining live tadpoles, QD treated tadpoles were smaller in size [t(34) = 2.35, P = 0.024] than controls. Findings reveal that (1) A. fowleri tadpoles integrate and accumulate nanoparticles, without detectable excretion within 72 h post-exposure, and (2) nanoparticles impede normal tadpole development. Ongoing studies are determining the effects of QD exposure on complete tadpole metamorphosis. The work was supported by USDA-ARS Biophotonics Initiative grant #58–6402–3-018.

Salinity increases total body prolactin and gill and skin prolactin receptor expression in the Chinese edible frog,Hoplobatrachus rugulosus, tadpole

Water salinity has effects on growth and metamorphosis of anuran species, including Hoplobatrachus rugulosus. Previously, we reported that cultured H. rugulosus tadpoles at low salinities (2–4‰) were of bigger size and grew faster than those in fresh water (FW). However, at a higher salinity level of 6‰, their sizes were reduced and the metamorphosis was delayed. It was therefore hypothesized that high salinity-induced osmotic stress affected secretion of prolactin (PRL), which acts as osmoregulatory hormone and a regulator of metamorphosis in amphibians. In this study, transferring tadpoles into 4‰ and 6‰ brackish water increased the PRL levels by ~1.2- and ~twofold, respectively, as compared to FW group. These osmotic challenges also increased the total body fluid osmolality and levels of Na+, Cl− and Ca2+. The contents of triiodothyronine (T3) were significantly reduced in 4‰ and 6‰ groups, but not 2‰ group. Three sizes (49, 29 and 23 kDa) of H. rugulosus PRL receptors (PRLRs) were detected, and their protein expression was found in the skin, gill, tail fin, brain, intestine, heart, liver and kidney. The expression of PRLR-49k protein was significantly higher in both skin and gills in 2–4‰ groups, whereas that of PRLR-29k and PRLR-23k were higher in gills and lower in skin of the 6‰ group than FW group. In conclusion, salinity challenge, particularly 4‰ and 6‰, increased the levels of PRL, while decreasing the T3 levels, which could explain why salinity markedly modulated growth, metamorphosis and survival of tadpoles.