Dissertations / Theses on the topic 'Amphibian metamorphosis'

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (May 24, 2006) Vita. Includes bibliographical references.

For nearly a century, amphibian metamorphosis has served as an important model of how thyroid hormones regulate vertebrate development. Consequently metamorphosis has been studied in a number of ways including: morphologically, developmentally, ecologically, and from an endocrine perspective. Over the last two decades, much has been learned about the molecular basis of anuran (frog) metamorphosis. However, very little is known about the molecular underpinnings of urodele (salamander) metamorphosis. Using the axolotl and axolotl hybrids as models, I present some of the first studies on the gene expression changes that occur during urodele metamorphosis. In Chapter 1, the motivation for the research described in the subsequent chapters is presented and the literature is briefly reviewed. In Chapter 2, the first microarray analysis of urodele metamorphosis is presented. This analysis shows that hundreds of genes are differentially expressed during thyroid hormone-induced metamorphic skin remodeling. Chapter 3 extends the analysis presented in Chapter 2 by showing that the transcriptional patterns associated with metamorphic skin remodeling are robust even when the concentration of thyroid hormone used to induce metamorphosis is varied by an order of magnitude. Chapter 4 makes use of the differentially expressed genes identified in Chapters 2 and 3 to articulate the first model of urodele metamorphosis to integrate changes in morphology, gene expression, and histology. In addition, Chapter 4 outlines a novel application for piecewise linear regression. In turn, Chapter 5 makes use of the model presented in Chapter 4 to demonstrate that full siblings segregating profound variation in metamorphic timing begin to diverge in phenotype early during larval development. In Chapter 6 the conclusions drawn from the research are summarized and future directions are suggested.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb length, the highest treatment was more developed compared to the control. There were no differences in tail resorption rates between the treatments and controls. At relevant concentrations, neither TCS nor TCC were lethal to Xenopus prior to metamorphosis. Exposure to relatively high doses of both compounds resulted in stunted growth, which would most likely not be evident at lower concentrations. TCS and TCC accumulated in Xenopus, indicating that the compound has the potential to bioaccumulate through trophic levels. Although TCS may increase the rate of metamorphosis in terms of developmental stage, it did not disrupt thyroid function and metamorphosis in regards to limb development and tail resorption.

The ability of animals to vary growth, development rate and behaviour in response to environmental conditions has been well documented, particularly during the larval phase in animals with complex life cycles. The evolution and maintenance of plasticity in response to environmental conditions is likely to be adaptive in animals that face unpredictable environments. However, there are two aspects of life histories in animals with complex life cycles, which would be expected to favour plasticity, that have received limited attention: traits during metamorphic climax and variation in the life history phase at which temperate species spend the winter. Therefore the aims of this thesis were to consider the environmental factors that are likely to result in plasticity in the timing and duration of metamorphic climax and contribute to variation in the over-wintering life stage, using amphibians as study animals. To assess the ability of animals to respond to environmental conditions during metamorphic climax conditions were manipulated during metamorphosis independent of larval treatment. Accordingly all larvae entered metamorphic climax having experienced the same conditions. The African clawed toad, Xenopus laevis, was used. I examined the influence of environmental temperature, predation risk and starting body size on several traits during the transitional stage (e.g. mass, snout-vent length (SVL), head width, tail morphology, duration and locomotor performance). Morphological measures and the duration of the life stage were shown to vary with temperature and predation risk. As predicted, higher temperatures and the risk of predation resulted in faster development through metamorphosis and smaller sizes on completion. The acceleration of metamorphosis was demonstrated to have potential costs, not in the form of reduced locomotor performance as predicted, but in a reduction in juvenile size as a result of faster metamorphic development. This suggests that, during this potentially vulnerable stage, it would be advantageous to take more time to complete in the absence of predators. Greater body size at the onset of metamorphosis requires a longer time to complete metamorphic climax suggesting that having a greater quantity of tissue to reconfigure during metamorphosis takes more time. Therefore, the conditions experienced during metamorphosis may have important implications for juvenile fitness and should be considered in studies of life history plasticity. In many temperate species with complex life cycles, the life history stage at which a species can survive the winter is generally fixed, imposing time limits on the timing of development. Most of these species must therefore often modify developmental rate to reach the appropriate stage or size at the onset of winter, usually at a cost to other traits. However, variation in the stage or developmental group that some amphibian, fish and insect species spend the winter has been observed, such as in the common frog Rana temporaria in the UK, which can spend the first winter as either a tadpole or as a juvenile frog. To investigate the factors that contribute to this variation in life history, I examined the influence of environmental temperature, food availability and water depth on the rate of larval development and growth. Data on development, growth and environmental temperature of a field population of R. temporaria, which have been observed to over-winter as larvae, were collected to determine how and when the two divergent early life history patterns of development were established. Development rate was slowed by reduced temperatures and food availability and greater water depth during rearing. Temperature and food availability also had a significant impact on the proportion of larvae that over-wintered, but in the field other factors are likely to contribute to the within-population variation in wintering strategy. While a greater water depth did prolong larval development, as predicted, this does not appear to be due to the cost of surfacing to respire acting as a constraint on development, since a similar slowing in development was observed in the lung-less Bufo bufo tadpoles. The results of these studies did not allow a definitive assessment of whether over-wintering as larvae represents an adaptive strategy or occurs as the result of developmental constraints. There is some evidence that over-wintering as larvae might be adaptive, since on completion of metamorphosis individuals that wintered as larvae were larger than those that completed metamorphosis late in the summer. Further work is necessary to identify other factors contributing to the over-wintering of larvae in Rana temporaria and to determine the adaptive significance, if any, of the alternative life history patterns.

Amphibians have been facing global declines over the last decades due to direct and indirect effects of anthropogenic activities. One of the leading causes is environmental contamination, particularly that of waterbodies which are used by many amphibian species for reproduction, development, and adult life. An important source of contamination comes from agricultural runoffs of pesticides such as neonicotinoids, which are known to alter anuran survival, behaviour, predation stress response, and development. However, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors which could alter the strength and direction of observed neonicotinoid effects. This study investigated how a concentration of imidacloprid (a neonicotinoid) measured in surface waters interacted with high population density, an important environmental stressor, to influence behaviour and development across metamorphosis in wood frogs (Lithobates sylvaticus) known to breed in agricultural landscapes. I reared tadpoles in a fully crossed design experiment, between two densities (0.33 and 1 tadpole/L) and clean vs contaminated water (10 µg/L imidacloprid). Behaviours were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. I found that imidacloprid did not interact with population density or independently affect behaviours in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural behavioural response (i.e., “freezing”) of tadpoles to predation cues. Both treatments also slightly accelerated metamorphosis while only density altered final mass at metamorphosis. Finally, I found that distance travelled was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. This study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioural assays and stressors in studies of amphibian ecotoxicology.

Les connexions de l'aire sensorielle octavolaterale du systeme nerveux central (aire primaire des fonctions acoustiques, vestibulaires et de la ligne laterale) ont ete tracees a l'aide de la peroxydase du raifort (hrp) chez trois amphibiens anoures au cours de leur developpement, le xenope, le discoglosse et la gastrotheque. Cette etude a permis de determiner les caracteristiques temps de latence au prelevement et vitesse de transport de la hrp dans le systeme nerveux central des anoures en developpement. En debut de metamorphose, le plan d'organisation des connexions de l'aire octavolaterale est similaire chez les trois anoures etudies. Il consiste en une connexion bilaterale de l'aire avec elle-meme et avec la formation reticulee et en une projection bilaterale vers l'olive superieure et vers le tegmentum: tores semicirculaires et region oculomotrice. Au cours de l'ontogenese, le contingent de connexions ipsilaterales tegmentales regresse chez le xenope et la gastrotheque et au dernier stade de la metamorphose, le plan d'organisation du systeme octavolateral correspond a celui etabli comme typique des anoures selon les donnees de la litterature. Cependant le discoglosse ne s'inscrit pas dans ce schema car l'ensemble des connexions tegmetales disparait a la fin de la metamorphose. La comparaison de l'evolution de la cytoarchitecture et des connexions du systeme octavolateral au cours de l'ontogenese chez les trois anoures etudies est discutee d'un point de vue phylogenetique et eco-ethologique

Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

Analyse de l'expression du génome des épithéliocytes intestinaux au cours de la métamorphose naturelle ou induite par la triiodothyronine par électrophorèse bidimensionnelle suivie de fluorographie. La même étude a été réalisée sur les protéines extraites de fractions enrichies en bordure en brosse intestinale au cours de la métamorphose naturelle ou induite ; la villine, protéine du cytosquelette des bordures en brosse a été mise en évidence dans l'intestin des amphibiens. Cette protéine a été purifiée, les caractères et l'expression post-embryonnaire de la villine intestinale d'amphibiens ont été analysés

La métamorphose des amphibiens est le processus rapide et irréversible par lequel un têtard aquatique se transforme en une grenouille respirant à la surface. Cette transition écologique, réminiscente de la période périnatale chez les mammifères, s'accompagne de changements spectaculaires (régime alimentaire, organes locomoteurs, système respiratoire...). Ces modifications morphologiques et physiologiques nécessitent la réponse concertée à un signal hormonal, les hormones thyroïdiennes (HT), de différents tissus vers des destin parfois opposés : apoptose (dans la queue), prolifération (dans les pattes), et remodelage (dans les intestins et le système nerveux central). Toutefois, la synchronisation de la réponse des différents tissus fait appel à d'autres signaux hormonaux, et notamment les glucocorticoïdes (GC). Ces derniers sont également les médiateurs principaux de la réponse au stress. Les processus endocriniens de la métamorphose et la réponse au stress sont fortement couplés. Les GC peuvent ainsi jouer le rôle d'interface permettant l'intégration de signaux environnementaux au niveau de réseaux de régulation. Dans le cadre de mon doctorat, j'ai analysé les transcriptomes des bourgeons de membres postérieurs et de l'épiderme caudal de têtards de Xenopus tropicalis traités ponctuellement avec des HT et / ou des GC. La comparaison de ces deux tissus a permis de caractériser la diversité des profils d'expression des gènes cibles des HT et des GC.Il en ressort plusieurs résultats majeurs. Tout d'abord, la diversité des profils d'interaction entre ces deux voies est limitée, et la majorité des types de profils sont communs aux deux tissus. Indépendamment du tissu, certains profils sont caractéristiques de fonctions biologiques spécifiques comme le remodelage de la matrice extracellulaire et le système immunitaire. Les gènes impliqués dans ces fonctions communes aux deux tissus sont cependant différents. Enfin, plusieurs facteurs impliqués dans la méthylation de l'ADN sont régulés par les deux hormones
Amphibian metamorphosis is the rapid and irreversible process during which an aquatic tadpole transforms into an air breathing adult frog. This ecological transition, reminiscent of the mammalian perinatal period, comes with spectacular changes (diet, locmotor organs, respiratory system...). These morphological and physiological modifications necessitate the properly timed response to a single hormonal signal, the thyroid hormones (TH), in various tissues to lead them to sometimes opposite fates : apoptosis (in the tail), cell prolifération and differenciation (in the limbs) and remodeling (in the intestine and the central nervous system).However, TH do not act alone. In particular, glucocorticoids (GC) play important roles during this process. They also are the main mediator of the stress response. Endocrine processes of the metamorphosis and the stress response are deeply intertwined. GC can thus act as an interface to integrate environmental inputs into regulatory networks.During my doctorate, I analyzed the possible transcriptional crosstalks between TH and GC in two larval tissues : the tailfin (TF) and the hindlimb buds (HLB). Comparing these two tissues allowed me to caracterize the diversity of TH and GC target gene expression profiles. This resulted in several major results. First, the diversity of the profiles of crosstalk between these two pathways is limited, and the majority of the types of profiles is common to both tissues. Next, independently ofthe tissues, some profiles are caracteristic of spécific biological functions such as extracellular matrix remodeling and the immune system. Yet, the genes involved in these shared functions are different between the TF and the HLB. Finally, several factors involved in DNA methylation are subject to a crosstalk between the two hormones

Nanoparticles (NPs) are engineered in the nanoscale (<100nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver (nAg) is the most prevalent nanoparticle in consumer products due to its strong antimicrobial action and can be released to the environment during product manufacture, usage and disposal. The predicted environmental concentrations are within the North American guidelines for the protection of aquatic life and in drinking water. While nAg toxicity at high concentrations has been well described, the sublethal effects at environmentally-relevant concentrations are relatively unknown. Initial screening in our lab showed nAg was a potential endocrine disrupting chemical (EDC). Amphibian metamorphosis is mediated by thyroid hormone (TH), and nAg perturbed TH-dependent transcriptional responses in the tailfin of bullfrog (Rana catesbeiana) tadpoles. The primary objective of this thesis was to further investigate and characterize the effects of low, environmentally relevant concentrations of nAg on TH-dependent metamorphosis in R. catesbeiana and Xenopus laevis. Two chronic, 28 day in vivo exposures at 0.06 and 6µg/L nAg were conducted with premetamorphic R. catesbeiana tadpoles using TH to induce precocious metamorphosis. Ionic silver (iAg) was also examined to control for the complete dissolution of Ag. Analysis of metamorphic stage progression demonstrated nAg-induced acceleration of hindlimb growth and development. After 6 days of nAg exposure, analysis with quantitative real-time polymerase chain reaction (QPCR) demonstrated nAg-induced disruption of TH-responsive transcripts in a tissue-specific manner. Furthermore, the nAg effects could not be fully explained by iAg, indicating NP-specific disruption. Two chronic, 28 day exposures to 0.018-1.8 µg/L nAg were conducted on X. laevis premetamorphic and prometamorphic tadpoles. nAg was found to significantly bioaccumulate in tadpole tissue after 28 days. Furthermore, nAg increased the hindlimb length during early premetamorphosis and in post-metamorphic juvenile tadpoles. Using an in-house MAGEX microarray and QPCR transcriptional analysis, 7 biomarkers of nAg exposure were validated. Five of these targets showed disruption to their TH-response. Furthermore, the increased mRNA abundance of two peroxidases indicated that nAg generated reactive oxygen species (ROS) even at low, environmental concentrations. This thesis demonstrates that nAg has consistent EDC actions across two distinct amphibian species, and the data suggest that regulatory guidelines for silver may need revision. A X. laevis derived fibroblast-like TH-responsive cell line, XTC-2, was used in conjunction with the 7 biomarkers of nAg exposure to gain mechanistic insight into the role of ROS in TH signaling disruption. Monocultures were created and validated to increase the specificity of TH-response. While the monocultures were successfully created, the biomarkers were not responsive to nAg in this cell line. Additional investigations were made into the relationship between genetic sex and responsiveness to TH. Genetic sexing methods were used to investigate transcriptional differences between males and females during natural and TH-induced metamorphosis. The sexing protocol was optimized and validated successfully. The genetic sex was determined for premetamorphic and prometamorphic X. laevis tadpoles exposed to TH for 48 h. QPCR and microarray analysis were used to identify three markers that demonstrated transcriptional sex-bias during early gonadal differentiation stages.
Graduate
0307
0383
0487
amanda_carew14@yahoo.ca

The anuran Xenopus tropicalis tadpole is an attractive model animal in toxicological evaluation of suspected thyroid disrupting xenobiotics. Due to its reliance of a functioning hypothalamic-pituitary-thyroid (HPT) axis for normal metamorphosis, effects on the HPT axis produces apical endpoints, which are easy to measure. More sensitive endpoints of mRNA expression and histological evaluation of the thyroid gland itself provide strong indications of in vivo thyroid endocrine disruption. X. tropicalis is traditionally exposed in groups of 20 animals in four replicates for each treatment group. However, exposing tadpoles individually can provide stronger statistics and a reduction of total animal sacrifice. In this study we have developed and optimized an individual exposure system by a method development study. This method was then applied in an exposure experiment of a suspected thyroid endocrine disruptor, propylparaben (PrP). Prometamorphotic (NF stage 51) X. tropicalis tadpoles were distributed in three treatment groups (0.05, 0.5 and 5.0 mg PrP/L water) and maintained semi-statically for 14 days. Afterwards, apical measurements (body weight (BW), total body length (BL), snout to vent length (SVL) and hind limb length (HLL)) and reached developmental stage were assessed. In addition, mRNA expression of HPT axis relevant genes encoding deiodinase 2 (D2, hepatic tissue), deiodinase 3 (D3, hepatic and tail tissue) and transthyretin (Ttr, hepatic tissue) were measured by quantitative reverse transcription PCR (qRT-PCR). The PrP exposure did not affect general growth and development, but it did cause a downregulation of dio3 and ttr. The downregulation of dio3 could possibly be associated with a reduced serum content of thyroid hormone, while ttr might be connected to a previously described xenoestrogenic effect of PrP in vitro and in fish.

Cannibalism is a seemingly aberrant interaction, appearing counter to the fitness of individuals. Yet cannibalism is not overly uncommon, and naturally occurs among aquatic organisms, including larval amphibians. In temporary wetlands larval amphibians are in a race to complete metamorphosis before their aquatic habitat disappears. When intraspecific competition intensifies, eating conspecifics may represent a beneficial if not necessary strategy. The research presented within this thesis aims to characterize factors that influence cannibalism within populations of larval amphibians. Wood frog tadpoles (Lithobates sylvaticus) were used to test potential benefits of cannibalism as a diet, determine if dietary quality and nutritional stress influence cannibalism, and investigate the roles of competition and chemical cues in influencing cannibalism. Larval long-toed salamanders (Ambystoma macrodactylum), and ringed salamanders (A. annulatum) were used to investigate a functional link between trophic polymorphism and cannibalism in natural populations. Results suggest that perceived increases in competition may stimulate some individuals to become less risk averse, and more aggressive, which may in turn facilitate cannibalistic behaviour. Cannibalism itself provided only conditional benefits to larval wood frogs, rather than the optimal growth that would be expected from an ideal diet. However, this may have been the result of individual variation in response to the diet and/or conspecific cues as opposed to a nutritional deficit. In conditions where tadpoles could perceive increased competition they altered their behaviour and morphology in ways that may improve their foraging success and potentially promote cannibalism. Finally, a functional link appears to exist between head morphology and cannibalism in natural wetlands. However, the appearance of this morphology appears related to conditions that may facilitate increased population densities through rapid pond drying.

Many organisms with complex life cycles undergo transition periods associated with increased vulnerability to predation. Several evolutionary adaptations have been proposed as antipredator defenses for organisms during risky transition periods. These include: shortening of the transition period, parental care, cryptic coloration, and synchrony of risky transitions with large numbers of conspecifics. The results of my research support the hypothesis that synchrony of metamorphosis and emergence from the water and aggregation during the period of transformation may be antipredator defenses for the western toad (Bufo boreas). For some anuran species, synchronous metamorphosis may function as an antipredator adaptation by swamping predators during the period of transformation. I examined the levels of synchrony of emergence from the water of metamorphosing western toads (Bufo boreas) in the presence and absence of a live snake predator, the common garter snake (Thamnophis sirtalis) in a laboratory experiment. To compare between the treatments, I measured the time to emergence from the water, the number of metamorphs emerging together, and the level of aggregation (before and during emergence) of the toads in each treatment. There was a difference between the treatments when all three factors were considered. I attributed these differences to a behavioral response in which B. boreas emerged sooner in the presence of the predator, regardless of whether individual toads had reached the point at which they were physically better suited to the terrestrial environment than the larval environment. Since the Pacific treefrog (Hyla regilla) is also preyed upon by T. sirtalis during the vulnerable period of metamorphosis, I conducted a laboratory experiment to test the effects of the presence of T. sirtalis on 1) aggregation of larval and metamorphosing H. regilla, 2) time to metamorphosis, 3) synchrony of metamorphosis, 4) time to emergence from the water and 5) synchrony of emergence from the water. The only significant effect observed in this experiment was a difference between aggregation levels of H. regilla throughout the experiment. There was, however, a strong trend in which the variances around the mean times to metamorphosis and emergence of the frogs in the control treatments were larger than those in the predator treatments. This could indicate a trend toward synchrony of metamorphosis and emergence for H. regilla in the presence of snake predators.
Graduation date: 1998

博士
東海大學
生命科學系
97
The phenomenon that amphibians breed in brackish water is more commonly than originally thought, and they exhibit interesting physiological and ecological adjustments to increased salinity stress. I used Fejervarya limnocharis tadpoles living in brackish water and designed three experiments to study effects of salinity on tadpole survival, growth, development, and metamorphosis and to identify the possible osmoregulatory mechanisms. In the first experiment, I studied salinity tolerance and salinity effects on growth, development, and metamorphosis of F. limnocharis tadpoles. Specifically, I examined if tadpoles exhibited adaptive developmental plasticity when exposed to different salinities. Tadpoles collected on Green and Orchid Islands off southeastern Taiwan were assigned to different salinities: 0, 3, 5, 7, 9, 11, and 13 parts per thousand (ppt). Daily survival, weekly growth, and development of tadpoles were recorded until metamorphosis. More than 50% survived in 9 ppt for over a month. A few tadpoles survived 11 ppt for 20 days, suggesting saline tolerance of F. limnocharis tadpoles is better than most tadpole species studied to date. Tadpoles at 9 ppt had lower survivorship and retarded growth and development (from Gosner stage 26 to 35) compared to other treatments. Tadpoles metamorphosed early at a smaller size when salinity increased, suggesting existence of adaptive developmental plasticity in F. limnocharis in response to osmotic stress. In the second experiment, I studied how changes in salinity affected tadpole survival, growth, development, and metamorphosis. Tadpoles of Gosner stage 26 were initially assigned to either low or high salinities (3 ppt and 9 ppt, respectively). I switched salinity when tadpoles reached Gosner stage 30 (early salinity switch) or 38 (late salinity switch). I had a total of six treatments: constant low salinity (LLL), low salinity with early switch to high salinity (LHH), low salinity with late switch to high salinity (LLH), constant high salinity (HHH), high salinity with early switch to low salinity (HLL), and high salinity with late switch to low salinity (HHL). I recorded tadpole daily survivorship, weekly growth, and development until metamorphosis. Tadpoles that initially assigned to low salinity (LLL, LLH, and LHH treatments) had high survival rates whereas tadpoles that reared at high salinity before Gosner stage 38 (HHH and HHL treatments) had poor survival, even if later switched to low salinity. Growth and development of tadpoles after transfer to low salinity was partially reversible (HLL vs. LLL), indicating tadpoles did not completely recover growth and development after osmotic stress release. In addition, tadpoles of LHH grew and developed better than that of HLL, suggesting initial salinities were important for the growth and development. Tadpoles in the LLH treatment underwent metamorphosis more quickly and at a smaller size than those in the LLL treatment, indicating that tadpoles exhibited phenotypic plasticity in size and age at metamorphosis in response to salinity variation. In contrast, there were no differences in metamorphic traits among the initially high salinity treatments, suggesting the timing of switching from high salinity to low salinity did not affect tadpole metamorphic traits. My results suggest that initial salinities were important for the survival, growth, development, and metamorphic traits of F. limnocharis tadpoles. Furthermore, the growth and development of tadpoles are partially reversible after salinity stress is released. My third experiment was designed to study the effects of salinity switching on tadpole survival, osmolality, water content, and gill Na+, K+-ATPase expression. Tadpoles collected from Green Island were reared in 3 ppt for at least one week before transfer to: 1) 11 ppt; 2) 7 ppt for 24 h and then 11 ppt.; and 3) 7 ppt for 48 h and then 11 ppt. Results showed that tadpoles did not survive >12 h if directly transferred to 11 ppt whereas tadpoles pre-acclimated for 48 h in 7 ppt survived at least 48 h. Then, I reared tadpoles in 3 ppt for at least one week before transfer to: 1) 3 ppt (control); 2) 11 ppt; and 3) 7 ppt for 48 h and then 11 ppt. In the first 6 h after transfer to 11 ppt, tadpole osmolality sharply increased and tadpole water content sharply decreased. Tadpoles pre-acclimated for 48 h in 7 ppt were able to maintain lower and more stable osmolality 3 h after transfer. These tadpoles immediately lost water content, but over the next 6 h gradually regained water and stabilized. These tadpoles had a higher level of relative protein abundance than other treatments. Results suggest that tadpoles that were pre-acclimated to 7 ppt for 48 h was sufficient to activate NKA expression, resulting in increased survivorship and reduced dehydration when tadpoles were transferred to 11 ppt. In conclusion, my results documented high saline tolerance of F. limnocharis tadpoles and demonstrate that salinity could induce adaptive developmental plasticity in tadpoles. Phenotypic plasticity in age and size at metamorphosis in response to salinity may provide a way for tadpoles to adapt to the unpredictable variation in salinity in coastal rock pools. In addition, my results also showed that initial salinities were important for the survival, growth, development, and metamorphic traits of tadpoles. Finally, my results showed pre-acclimation to a medium salinity for 48 h was sufficient to activate osmoregulatory mechanisms that increased survivorship and reduced dehydration when tadpoles were transferred to a high salinity. My results not only shed light on how amphibians adapt to salinity when they face natural or anthropogenic salinisation of wetlands, but also provide important information for the future study of physiological mechanisms of tadpole osmoregulation.

The ING (INhibitor of Growth) tumor suppressor genes are conserved from yeast to humans and are implicated in several processes important to cell proliferation and apoptosis. ING proteins contain a plant homeodomain (PHD) finger that suggests these proteins may modulate transcription factor-mediated pathways. Little is known about the mechanism of action of INGs, especially in the context of normal development. The ING family of proteins includes at least five different genes, ING1-ING5, with evidence for alternate promoter usage and splicing that generate multiple isoforms. To elucidate the role of ING in different tissues to modulate function, I used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates apoptosis, proliferation, and remodeling in the tail, hindlimb, and brain, respectively. I discovered seven ING1 and three ING2 transcript variants in Xenopus laevis and investigated their expression patterns. High expression levels of most variants were found in adult brain, testis, and eye. During natural metamorphosis or precocious metamorphosis induced by treating tadpoles with exogenous TH, ING1 and ING2 transcript variant levels were differentially regulated in a tissue-specific manner. Some variant levels increased with the induction of apoptosis of the tail, while levels of the same variants decreased upon induction of proliferation and differentiation in the hindlimb. Although levels of all INC variants were relatively high in whole brain, they did not change during metamorphosis or TH treatment. Given that ING has previously been shown to modulate apoptosis, it is likely that upregulation of specific isoforms may contribute to the tissue-specific TH-mediated response in the tail, and that downregulation facilitates proliferation of the hindlimb. To further investigate the hypothesis that ING is regulated by TH, an analysis of 1NG1 and ING2 genomic sequences was carried out. Promoter sequences for each variant were determined and putative thyroid hormone response elements (TREs) located. To test whether thyroid hormone receptors associate with these elements, chromatin immunoprecipitations (ChIP) assays were done on tail homogenates from premetamorphic tadpoles treated with TH or vehicle control. Both thyroid hormone receptor α (TRα) and thyroid hormone receptor β (TRβ) differentially associate with ING1 and ING2 promoter regions. TR association increased significantly on promoters for ING variant transcripts that increase upon TH treatment, and decreased significantly on promoters for ING variant transcripts that decrease upon TH treatment. ChIPs also showed that ING associates with TH-regulated promoters including TRβ, TH-Responsive Basic Leucine Zipper Transcription Factor (TH/bZIP), ING1 and ING2. Furthermore, TR and ING were shown to co-immunoprecipitate with both purified proteins and using total tail homogenates from metamorphic tadpoles. The antibodies used for these experiments were made against Xenopus TRβ and ING2 and were characterized as part of this thesis. Bioinformatics revealed that TREs are present in promoters of ING genes for other species including human, mouse, and a related frog species, Xenopus tropicalis; therefore, it is likely that modulation by TH is a conserved mechanism of ING regulation. These data suggest that there may be antagonistic regulation of ING transcript variants by TH that correlates with tissue fate. TRs associate with ING promoters, and ING is associated with TR-regulated promoters. Moreover, TR and ING proteins co-immunoprecipitate. It is therefore likely that TR and ING are co-regulators of gene expression during TH-dependent tadpole metamorphosis. This thesis contributes to the understanding of ING which is relevant to elucidating many disease states, as well as being critical in understanding the role of this tumor suppressor in the context of TH regulation and normal development.