Dissertations / Theses on the topic 'Tadpoles'

The investigation of new threats to amphibian conservation is a priority of researchers and wildlife managers. Emerging infectious diseases are one of the most threatening processes to wildlife around the world including amphibians. Australian frogs have suffered large scale declines and extinctions from pathogens such as chytrid fungus (Batrochochytrium dendrobatidis). The once overly abundant Green and golden bell frog (Litoria aurea) has declined over 90% of its range with disease listed as a key threat. A routine pathogen screen of tadpoles from a captive breeding population of Green and golden bell frogs found an unknown parasitic infection in the brains, bile ducts and gallbladders of tadpoles (later confirmed as Myxosporea). It was this preliminary identification that was the impetus for my thesis. Myxosporean parasites found in Australian frog gallbladders were thought to be Cystodiscus immersus from Central and South America. The parasite was assumed to have been introduced to Australia with the Cane toad (Rhinella marina, syn. Bufo marinus) in 1935. Cystodiscus immersus was supposed to have translocated with the Cane toad from native Brazil and was infecting a broad range of Australian frog species with no apparent host impact. The aim of this thesis was to challenge all aspects of this assumption, to establish the true myxosporean species present in Australian frogs, if it was an exotic infection, if it was an emerging pathogen and what threat did it pose to hosts. The parasite found in Australian frogs and tadpoles was examined using histological and ultrastructural morphology as well as molecular identification. This revealed there was in fact two myxosporean species in multiple species of frogs and tadpoles. Sequencing of partial small iv subunit and large subunit as well as the internal transcribed spacers (ITS1 and ITS2) of ribosomal DNA regions confirmed these were distinct species (differing 9%, 7%, 34% and 37% at each region respectively). They were in fact separate species infecting multiple Australian host species,samples of C. immersus from Brazil were compared to establish if either of these was the exotic C. immersus. We found instead that these were completely unrelated despite sharing similar spore morphology. In addition the Brazil material revealed the cryptic diversity of myxosporea in frog gallbladders around the world and the ambiguous identity of Cystodiscus immersus. To determine if the parasites were emerging, the presence of these parasites in 130 years was established using voucher specimens. Four native species and the exotic Cane toad were examined for gallbladder myxospores from 1879 to 2009, the first positive was in 1966 in Green tree frog outside of Sydney. The prevalence of myxosporea increased in both native and exotic species over the last 40 years including the threatened Green and golden bell frog. The emergence of these parasites is a cause for concern, especially when found in endangered species. Close scrutiny of these parasites prompted the formal description of Cystodiscus axonis and Cystodiscus australis, resurrecting the original genus name proposed by Lutz in 1889. The description revealed diagnostic features previously unknown for frog gallbladder myxosporea species. Scanning electron microscopy revealed filiform polar appendages on the myxospores of C. axonis, absent in C. australis. In addition, histological examination showed unique developmental stages in the nervous tissue of tadpoles and frog hosts infected with C. axonis, these stages are only found in hosts infected with this parasite. The use of development stages as a species diagnostic character has not been previously reported for any amphibian myxosporea as yet. The genetic and morphological differences between C. axonis and C. australis prompted the development of a species specific multiplex PCR using ITS1, 5.8S and ITS2 ribosomal DNA. Multifaceted diagnostic tools (morphology and species specific PCR) demonstrated significant disease associated with these pathogens in native and exotic frog species. Seven host species were morphologically examined for Cystodiscus infection showing endangered species to have significant disease associated with infection. Cystodiscus axonis was linked to neurological signs,haemorrhage, necrosis and gliosis in Southern bell frogs (Litoria raniformis), Green and golden bell frogs (Litoria aurea), Booroolong frogs (Litoria booroolongensis), and the recently rediscovered Yellow spotted bell frogs (Litoria castanea). Cystodiscus spp. infection in the bile ducts of tadpoles was associated with biliary hyperplasia, loss of hepatocytes and inflammation. These lesions were statistically associated with the presence of infection in the Green and golden bell frog. This research disproved an assumption about myxosporean parasites that had held for 25 years. The disease found with Cystodiscus parasite infection in Australian frogs and tadpoles, as well as the previously unknown biodiversity in this cryptic species complex, highlight a number of areas in need of further research. This work has provided insight into the importance of multifaceted approaches to species identification and amphibian pathogen surveillance which uncovered a threat to endangered Australian amphibians, now listed as one of the key disease threats to frogs by the Department of Environment and Health (Australia) in 2011.

Mutations of Drosophila bagpipe, an NK3 class homeobox gene, result in failure of visceral mesoderm to differentiate into stomach tissue. Thus bagpipe, in association with other factors, is a good candidate for specification of visceral mesoderm in Drosophila. The cloning and characterisation of a Xenopus bagpipe homologue was therefore of great interest. This study describes the isolation of a full length Xenopus cDNA clone that on the basis of database analysis and sequence comparisons has been assigned as Xenopus bagpipe (XBap). Previous studies had revealed that the majority of homeoproteins recognise DNA sites with a 5'-TAAT-3' core but the NK class of homeoproteins had been shown to bind specifically to sites containing a 5'-CAAG-3' core. Experiments described here, however, show the XBap DNA binding site to be an even more divergent, 5'-TTAAGTGG-- TTAAGTGG-3'. A series of mutant oligonucleotides revealed that the `T' of the 5'- T_{1}A_{2}A_{3}G_{4}-3' core, as well as the presence of two such cores, are indeed essential for optimal XBap DNA binding. The murine NK3 class homeoproteins, Nkx-3.1 and Bapxl, are demonstrated to have the same requirements for optimal DNA binding as XBap. Drosophila Bagpipe, however, was found to have a less stringent requirement for a `T' at position one of the core, binding equally well to a 'C' in this position, but the presence of two such cores is still necessary for optimal DNA binding. Preliminary studies using site directed mutagenesis attempted to define the amino acids responsible for the differences. The effect of XBap on transcription was studied using a Xenopus oocyte assay and two cell transfection assays. XBap was not found to act as a transcriptional activator in any of these assays but evidence was obtained to suggest that a C-terminal truncation of XBap could act as a repressor of transcription.

Methylmercury (MeHg) is a toxic heavy metal and a health threat to wildlife and humans, however nothing is known about its effects on amphibians. MeHg is produced from inorganic Hg in the aquatic environment, and bioaccumulates in the food chain. This exposes tadpoles to elevated levels of MeHg in their diet, and may pose a risk to development. Tadpoles of the North American species Bufo americanus and Rana pipiens as well as the African frog model species Xenopus tropicalis were subchronically exposed to dietary McHg ranging in concentration from 1ng/g to 1000 ng/g to determine LC50s and species sensitivity differences. A developmental differences study was also performed with B. americanus. The 33-day LC50 estimates indicate that Gosner stage 25 tadpoles of both B. americanus and R. pipiens were the most sensitive, and they exhibited a similar sensitivity to McHg toxicity. The X. tropicales LC50 estimate is significantly higher (p=0.05) than those calculated for B. americanus and R. pipiens Gosner stage 25, and the developmentally advanced B. americanus Gosner stage 27-30 LC50 estimate is also significantly higher (p=0.05) than the B. americanus Gosner stage 25 LC50. (Abstract shortened by UMI.)

This work centered on the tadpoles in a temporaray pond in the middle of Kenilworth racecourse, Cape Town, South Africa. Trapping was carried out over two wet seasons and five species were found. The racecourse was selected to investigate the tadpole community occupying temporary winter pools. The main focus of this study was the community of tadpoles that occur in the ephemeral ponds in the centre of Kenilworth Racecourse. This study was a very broad insight into tadpole ecology in the Western Cape.

“Issues on tadpoles and vacuum redefinitions in String Theory” Marco Nicolosi Questa Tesi di dottorato è dedicata al problema dei “tadpoles” di NS-NS, funzioni ad un punto di campi bosonici assorbiti dal vuoto che tipicamente emergono nella Teoria delle Stringhe in seguito alla rottura della supersimmetria. Queste teorie contengono campi bosonici in due settori, quello di NS-NS e quello di R-R. Mentre i “tadpoles” di R-R tipicamente segnalano un’inconsistenza, come la presenza di anomalie quantistiche nel caso di spazi interni compatti, e quindi in generale devono essere cancellati, i “tadpoles” di NS-NS sono associati ad una ridefinizione del vuoto, come indicato per la prima volta da Fischler e Susskind negli anni ottanta. In particolare, nella stringa di Tipo I i “tadpoles” di NS-NS emergono già a livello del disco e, da un punto di vista spazio temporale, corrispondono a una configurazione di D-brane e piani di orientifold con tensione non nulla che danno luogo ad una netta attrazione gravitazionale che curva lo spazio tempo di “background”. Fino ad oggi siamo capaci di fare calcoli di stringa in maniera efficiente solo nel “background” piatto di Minkowski, un caso che è permesso e protetto dalla supersimmetria. Quindi, le divergenze infrarosse che emergono nelle ampiezze di stringa (canale chiuso) dopo la rottura della supersimmetria, dovute alla propagazione di stati non massivi di NS-NS che sono assorbiti dai “tadpoles” a impulso nullo, sono proprio il segnale che il “background” piatto di Minkowski non è più un vuoto della teoria. In questo contesto la nostra proposta è di continuare a quantizzare la stringa nel “background” di Minkowski e recuperare il risultato corretto risommando opportunamente i “tadpoles” in modo da cancellare le divergenze infrarosse. Questa procedura è comunque molto difficile da attuare nella Teoria delle Stringhe, perché le correzioni di “tadpoles” di ordine più grande corrispondono a superfici di Riemann di genere crescente, mentre si riescono a fare calcoli di stringa essenzialmente fino a genere uno (ampiezze ad un “loop”). Inoltre, nella maggior parte dei modelli che realizzano la rottura di supersimmetria, i “tadpoles” emergono già a livello del disco, e quindi, perfino in una regione perturbativa di piccola costante di accoppiamento di stringa, le prime correzioni di “tadpole” possono essere grandi. Pertanto, è interessante cercare modelli con “tadpoles” piccoli. Esempi di questo tipo sembrano essere forniti da modelli con opportuni flussi interni, per i quali risultati perturbativi credibili possono essere ottenuti considerando solo le prime correzioni di “tadpole”. Un’altra linea che si può perseguire è quella di cercare quantità che sono protette dalle divergenze infrarosse. Un esempio di questo tipo è dato dalle correzioni di stringa ad un “loop” alle costanti di accoppiamento di “gauge”, comunemente conosciute come “correzioni di soglia”, per modelli con supersimmetria rotta e con brane parallele, un caso che è stato ampiamente discusso in questa Tesi. Questa Tesi è organizzata nel seguente modo. Iniziamo con un’Introduzione generale sulla Teoria delle Stringhe, dove riportiamo le principale idee della Teoria, provando ad evidenziarne successi e problemi. Nel primo Capitolo richiamiamo le proprietà di base degli spettri di stringa e discutiamo qualche semplice esempio di compattificazione toroidale e di “orbifold”. Il secondo Capitolo è dedicato a riassumere differenti meccanismi di rottura di supersimmetria. Nel terzo Capitolo iniziamo ad analizzare il nostro programma di risommazione per diversi modelli giocattolo in Teoria dei Campi, provando a recuperare i giusti risultati, almeno a livello classico, a partire da un “vuoto sbagliato”. I casi di potenziale cubico e quartico sono semplici ed interessanti e mostrano alcune caratteristiche generali riguardanti le risommazioni dei “tadpoles” e i domini di convergenza intorno ai punti di flesso del potenziale, dove l’espansione nei “tadpoles” viene meno. La nostra analisi mostra che, partendo da un valore iniziale arbitrario del campo, la risommazione dei “tadpoles” a livello classico tipicamente guida le quantità che stiamo calcolando verso un estremo del potenziale, non necessariamente un minimo. Inoltre, nel caso del potenziale quartico troviamo alcuni punti molto speciali di “non-rinormalizzazione” per i quali tutte le correzioni di “tadpole” di ordine superiore si cancellano. Analizziamo poi la nostra procedura per un modello giocattolo inspirato dalla Teoria delle Stringhe, con “tadpoles” localizzati su D-brane di dimensione più bassa, calcolando le risommazioni esplicitamente. L’introduzione della gravità, che dovrebbe introdurre ulteriori complicazioni legate al termine di massa del gravitone, sembra non alterare sostanzialmente il nostro programma, ed infatti le risommazioni dei “tadpoles” continuano a funzionare ancora anche in questo caso. In fine, nel quarto Capitolo, iniziamo a trattare il problema dei “tadpoles” nella Teoria delle Stringhe. Nel primo Paragrafo, descriviamo un esempio dove la ridefinizione del vuoto può essere capita non solo a livello della teoria effettiva di bassa energia, ma anche a livello della stringa. In particolare, mostriamo che il vuoto di un “orientifold” di un modello di Tipo II con una dimensione compatta e “tadpoles” locali è un orientifold di Tipo 0 senza dimensioni compatte. Questi risultati sono contenuti in un articolo pubblicato in Nuclear Physics B. In fine, nell’ultimo Paragrafo, iniziamo l’analisi delle “correzioni di soglia” ad un “loop” in diversi modelli con rottura di supersimmetria e brane parallele, privi di tachioni chiusi che si propagano nel “bulk”. Il risultato è che le “correzioni di soglia” ad un “loop” in tutti questi casi sono sempre finite nell’infrarosso (canale chiuso), nonostante la presenza dei “tadpoles” di NS-NS. Questi risultati saranno inclusi in un articolo attualmente in preparazione.
“Issues on tadpoles and vacuum redefinitions in String Theory” M. Nicolosi This Thesis is devoted to the problem of NS-NS tadpoles, bosonic one-point functions going into the vacuum that typically emerge in String Theory after supersymmetry breaking. These theories contain bosonic fields in two sectors, commonly denoted with NS-NS and R-R. While R-R tadpoles typically signal an inconsistency, like the presence of quantum anomalies in the case of a compact internal space, and thus in general must be cancelled, NS-NS tadpoles are associated to redefinitions of the background, as first stressed by Fischler and Susskind in the eighties. In particular, in Type I String Theory NS-NS tadpoles emerge already at the disk level and, from a space-time viewpoint, correspond to configurations of D-branes and orientifold planes with a non-vanishing tension giving rise to a net gravitational attraction that curves the background space-time. Up to now one is able to perform efficient string computations only in a flat Minkowski background, a case that is allowed and protected by supersymmetry. Hence, the (closed) infrared divergences emerging after supersymmetry breaking in string amplitudes, due to the propagation of NS-NS massless states that are absorbed by tadpoles at vanishing momentum, are just the signal that the flat Minkowski background is no more a vacuum of the theory. In this context our proposal is to keep quantizing the string around the Minkowski background, recovering the proper results after suitable tadpole resummations that cancel the infrared divergences. This procedure is still very difficult to carry out in String Theory, because the higher-order tadpole corrections correspond to Riemann surfaces of increasing genus, and efficient calculations can be only carried out up to genus one (one-loop amplitudes). Moreover, in most models that realize supersymmetry breaking, tadpoles arise already at the disk level, and thus, even in a perturbative region of small string coupling, the first tadpole corrections can be large. Hence, it is interesting to search for models with “small” tadpoles. Examples of this kind seem are provided by models with suitable internal fluxes, for which reliable perturbative results can be recovered just considering the first tadpole corrections. Another line that one can pursue is to search for quantities that are protected against the infrared divergences. An example of this kind is provided by the one-loop string corrections to gauge couplings, commonly known as threshold corrections, for supersymmetry breaking models with parallel branes, a case that we have widely discussed in this Thesis. The Thesis is organized in the following way. There is a general Introduction to String Theory, where we summarize the main ideas of the Theory, trying to underline its successes and its open problems. Then in the first Chapter we recall the basic properties of string spectra and discuss some simple examples of toroidal and orbifold compactifications. The second Chapter is devoted to reviewing a number of different mechanisms to break supersymmetry. In the third Chapter we begin to analyze our resummation program in a number of field theory toy models, trying to recover the right results, at least at the classical level, starting from a “wrong vacuum”. The cases of cubic and quartic potentials are simple and interesting, and display some general features concerning tadpole resummations and convergence domains around inflection points of the potential, where the tadpole expansion breaks down. Our analysis shows that, starting from an arbitrary initial value of the field, classical tadpole resummations typically drive the quantities we are computing towards an extremum of the potential, not necessary a minimum. In addition, for the case of a quartic potential we find some very special “non-renormalization” points for which all higher order tadpole corrections cancel. We then analyze our procedure for a sting-inspired toy model with tadpoles localized on lower dimensional D-branes, performing explicitly the resummations. We also consider the introduction of gravity, that should give further complications related to the graviton mass terms, but seems to not affect substantially our program, and indeed tadpole resummations prove still to work in this case. Finally, in Chapter four we begin to face the tadpole problem in String Theory itself. In the first Section, we describe an example where the vacuum redefinition can be understood not only at the level of the low energy effective field theory, but also at the full string theory level. In particular, we show that the vacuum of a Type II orientifold with a compact dimension and local tadpoles is a Type 0 orientifold without compact dimensions. These results are contained in a paper to appear in Nuclear Physics B. Finally, in the last Section we begin the analysis of one-loop threshold corrections in a number of models with supersymmetry breaking with parallel branes and no closed tachyons propagating in the bulk. The result is that the one-loop threshold corrections in all these cases are always (closed) infrared finite, in spite of the presence of NS-NS tadpoles. These computations will be included in a paper that is currently in preparation.

Effective movement is central to survival and it is essential for all animals to react in response to changes around them. In many animals the rhythmic signals that drive locomotion are generated intrinsically by small networks of neurons in the nervous system which can be switched on and off. In this thesis I use a very simple animal, in which the behaviours and neuronal networks have been well characterised experimentally, to explore the salient features of such networks. Two days after hatching, tadpoles of the frog Xenopus laevis respond to a brief touch to the head by starting to swim. The swimming rhythm is driven by a small population of electrically coupled brainstem neurons (called dINs) on each side of the tadpole. These neurons also receive synaptic input following head skin stimulation. I build biophysical computational models of these neurons based on experimental data in order to address questions about the effects of electrical coupling, synaptic feedback excitation and initiation pathways. My aim is better understanding of how swimming activity is initiated and sustained in the tadpole. I find that the electrical coupling between the dINs causes their firing properties to be modulated. This allows two experimental observations to be reconciled: that a dIN only fires a single action potential in response to step current injections but the population fires like pacemakers during swimming. I build on this hypothesis and show that long-lasting, excitatory feedback within the population of dINs allows rhythmic pacemaker activity to be sustained in one side of the nervous system. This activity can be switched on and off at short latency in response to biologically realistic synaptic input. I further investigate models of synaptic input from a defined swim initiation pathway and show that electrical coupling causes a population of dINs to be recruited to fire either as a group or not at all. This allows the animal to convert continuously varying sensory stimuli into a discrete decision. Finally I find that it is difficult to reliably start swimming-like activity in the tadpole model using simple, short-latency, symmetrical initiation pathways but that by using more complex, asymmetrical, neuronal-pathways to each side of the body, consistent with experimental observations, the initiation of swimming is more robust. Throughout this work, I make testable predictions about the population of brainstem neurons and also describe where more experimental data is needed. In order to manage the parameters and simulations, I present prototype libraries to build and manage these biophysical model networks.

Body size is important in determining the outcome of competition and predator-prey interactions. Size structure of a population (i.e. relative proportion of large and small conspecifics) may be particularly important in organisms with prolonged breeding periods and rapid growth where populations may have multiple cohorts at different stages of development competing for one resource. Both the consumptive and nonconsumptive effects of predators can also be size-dependent and can alter competitive interactions. Here we study the importance of size structure in the Neotropical leaf-breeding tree frog, Agalychnis callidryas. This species is a prolonged breeder such that multiple overlapping cohorts of differing sizes are common. Specifically, we examine size-specific intraspecific competition between A. callidryas tadpoles and then explore how predation affects these interactions. To determine the strength of inter-cohort competition, we manipulated the density and relative proportion of large and hatchling tadpoles in a response surface design and quantified growth. We then observed the effect of a dragonfly larvae predator (Anax amazili) on tadpole growth and survival at different size-structured treatments. Large tadpoles were greater per individual competitors while hatchlings were greater per gram competitors. When predators were added, dragonflies reduced survival and growth of hatchlings substantially, but had no effect on large tadpoles. Further, dragonflies reduced hatchling growth more when other hatchlings were present. The predator effect on hatchling growth was 23% larger than the effect of competition with large tadpoles, such that the importance of size structure for A. callidryas may be mediated more through predation than intercohort competition.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Raymond D. Semlitsch. Vita. Includes bibliographical references.

A variety of aquatic habitats with different levels of potential predators are available to larval amphibians in Central Amazon rainforest. The anuran community at Reserva Florestal Adolfo Ducke, 25 km east of Manaus, Amazonas, Brasil, was studied to determine which species have eggs and/or larvae in water and how those larvae are distributed in time and space. The temporal and spatial distribution of potential predators as well as abiotic characteristics of these waterbodies were determined simultaneously to test for correlations with the distribution of tadpoles. The distribution of tadpoles was strongly related to fish predation pressure. Several tadpole species were found only in waterbodies with high fish abundance and thus have the ability to survive with fish. Most of these tadpoles were found to be unpalatable in controlled experiments. Unpalatability is the major adaptation allowing the coexistence of tadpoles and fish and is thus a major factor affecting tadpole community composition in this system. Controlled experiments showed that fish do not eat anuran eggs while the tadpoles of Leptodacrylus knudseni and Osteocephalus taurinus ate all types of eggs offered. The percentage of anurans with aquatic oviposition was positively related to fish abundance and negatively related to the occurrence of species of tadpole that ate eggs in experiments. These findings suggest that the present patterns of anuran distribution represent an evolutionary response to predation on the eggs and larvae. Contrary to the models of Heyer et al. (1975) and Wilbur (1984), desiccation and predation-pressure were not the major factors affecting species richness within waterbodies of the RFAD rainforest. In support of the model of Heyer et al. (1975), anuran species richness was correlated with the size of the waterbody. This could be because the size of the waterbody is related to increased complexity and availability of microhabitats. The range of volumes of waterbodies was also found to directly affect species richness of the RFAD community. While predation appeared to have a limited effect on species richness of individual ponds, predation-pressure was found to have a major influence on species composition. Anuran eggs and larvae survived with specific predators by possessing particular survival-traits (e.g. unpalatability and oviposition strategies). However, survival-traits were not effective against all predators in all habitats. The distribution of different predators among ponds provides a patchy environment on a local scale (i.e. within ponds). When combined with the variety of survival-traits exhibited by the anuran species, this spatial patchiness in predation contributes towards species richness within the anuran community of the RFAD rainforest.

This work examines the ecological, social and genetic factors that predispose amphibians to infection. In the last 30 years many amphibian populations have declined due to infectious disease, although few researchers have studied the factors involved in mediating amphibian infection. My research is designed to explore some of these factors. I first examined the effects of crowding, kin composition (the relatedness of individuals in a group), and habitat complexity on the growth and survival of Xenopus laevis tadpoles exposed to the bacterial pathogen Aeromonas hydrophila. In tadpoles, stress, and in particular corticosterone, a hormone associated with stress, is known to inhibit growth. Crowding, kin composition, and habitat complexity have all been linked to tadpole growth. As corticosterone exposure is also linked to reduced immune function, I examined how these ecological factors influence tadpoles' disease resistance. Tadpoles exposed to the bacterium were significantly smaller and more likely to die than control tadpoles. Tadpoles reared only with siblings (pure sibship groups) were larger, less variable in size, and had lower mortality rates than tadpoles reared in mixed sibship groups. The size difference between pure and mixed sibship groups was greatest when they were exposed to the pathogen. Habitat complexity reduced size variation within tanks but did not affect mean tadpole size. Mixed kinship composition and high tadpole density can increase competition, reduce growth, and increase disease susceptibility. These results indicate that growth was inhibited by pathogen exposure but kin association may ameliorate this effect. The Major Histocompatibility Complex (MHC) is an integral part of the vertebrate adaptive immune system. To determine the importance of the MHC in conferring disease resistance in amphibians, I challenged X. laevis tadpoles, bearing different combinations of four MHC haplotypes (f, g, j, and r), with A. hydrophila in two experiments. Exposure to A. hydrophila affected the growth and survival of these tadpoles and that the MHC moderated these effects. Tadpoles with two MHC haplotypes (r and g) were susceptible to this pathogen and tadpoles with the other two haplotypes (f and j) were resistant. Heterozygous tadpoles with both susceptible and resistant haplotypes were always intermediate to either homozygotes in size and survival. These results demonstrate that MHC genotype plays a major role in determining the impact of bacterial pathogens on the growth and survival of X. laevis tadpoles. To test whether the effect of exposure to pathogens differs according to the similarity of the hosts I challenged tadpoles with natural levels of the microorganisms associated with different MHC genotypes by exposing the tadpoles to water preconditioned by adults of different MHC genotypes. If the pathogens are adapted to the MHC genotype of their hosts, tadpoles exposed to water from adults with which they shared MHC haplotypes would be more susceptible than those exposed to water from MHC-dissimilar adults. Alternatively, if the hosts are adapted to their pathogens tadpoles may be more resistant to pathogens from MHC-similar frogs than those from MHC-dissimilar frogs. I found that tadpoles exposed to water from MHC-dissimilar animals developed faster, but without increased growth, and were more likely to die than those exposed to water from MHC-similar animals. Furthermore, there was an optimal difference between the tadpoles’ and the donors’ MHC where tadpoles were sufficiently different to the donor to defend against its locally adapted pathogens, and sufficiently similar to not be exposed to especially virulent foreign pathogens. Finally, I present an inventory of bacteria found in the gut and skin (nonsystemic sites) and heart, muscle, and abdominal cavity (systemic sites) of captive frogs. I found several species of bacteria previously identified as amphibian pathogens and many bacteria in systemic sites that have not been considered pathogenic to amphibians. None of the frogs tested positive for the amphibian chytrid fungus, Batrachochytrium dendrobatidis. I discuss the potential importance of these species of bacteria as amphibian pathogens and as protective probiotics, using New Zealand frogs as a case study. In its sum, this work describes some of the factors that can affect amphibians’ ability to resist disease. I show that the genetic constitution of an individual, specifically in terms of the MHC, affects the impact of a disease, and so too does its social and ecological conditions, including the level of crowding, the kinship of its groupmates and the specific microbial challenges of its immediate environment. I also show that many of the factors linked to tadpole growth and development that are well described in other amphibians also affect Xenopus tadpoles.

A variety of aquatic habitats with different levels of potential predators are available to larval amphibians in Central Amazon rainforest. The anuran community at Reserva Florestal Adolfo Ducke, 25 km east of Manaus, Amazonas, Brasil, was studied to determine which species have eggs and/or larvae in water and how those larvae are distributed in time and space. The temporal and spatial distribution of potential predators as well as abiotic characteristics of these waterbodies were determined simultaneously to test for correlations with the distribution of tadpoles. The distribution of tadpoles was strongly related to fish predation pressure. Several tadpole species were found only in waterbodies with high fish abundance and thus have the ability to survive with fish. Most of these tadpoles were found to be unpalatable in controlled experiments. Unpalatability is the major adaptation allowing the coexistence of tadpoles and fish and is thus a major factor affecting tadpole community composition in this system. Controlled experiments showed that fish do not eat anuran eggs while the tadpoles of Leptodacrylus knudseni and Osteocephalus taurinus ate all types of eggs offered. The percentage of anurans with aquatic oviposition was positively related to fish abundance and negatively related to the occurrence of species of tadpole that ate eggs in experiments. These findings suggest that the present patterns of anuran distribution represent an evolutionary response to predation on the eggs and larvae. Contrary to the models of Heyer et al. (1975) and Wilbur (1984), desiccation and predation-pressure were not the major factors affecting species richness within waterbodies of the RFAD rainforest. In support of the model of Heyer et al. (1975), anuran species richness was correlated with the size of the waterbody. This could be because the size of the waterbody is related to increased complexity and availability of microhabitats. The range of volumes of waterbodies was also found to directly affect species richness of the RFAD community. While predation appeared to have a limited effect on species richness of individual ponds, predation-pressure was found to have a major influence on species composition. Anuran eggs and larvae survived with specific predators by possessing particular survival-traits (e.g. unpalatability and oviposition strategies). However, survival-traits were not effective against all predators in all habitats. The distribution of different predators among ponds provides a patchy environment on a local scale (i.e. within ponds). When combined with the variety of survival-traits exhibited by the anuran species, this spatial patchiness in predation contributes towards species richness within the anuran community of the RFAD rainforest.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Division of Australian Environmental Studies
Science, Environment, Engineering and Technology
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The widespread use and subsequent release of pesticides into aquatic environments have sparked concerns about how organisms within these aquatic systems are affected by pesticide pollution. While many studies have examined the effects of pesticides on individual organisms, in a series of experiments, I investigated the effects of a pesticide mixture on members of a complex host-parasite system and on host susceptibility to infection. In my first experiment in the laboratory, I examined changes in survivorship when trematode parasites (Echinostoma trivolvis) and their first intermediate host, Planorbella trivolvis snails, were exposed to a low concentration (10 ppb: 15 ppb) and high concentration (85 ppb: 100 ppb) mixture of atrazine and metolachlor, respectively. There was a significant decline in parasite survivorship in the high concentration treatment at 14 hours, while snail survivorship was unaffected across all treatments. In my second experiment, prior to infection, I exposed the parasites and/or second intermediate hosts, Rana clamitans and Rana sylvatica tadpoles, to the pesticide mixtures and examined subsequent infection levels in the tadpoles. The atrazine and metolachlor mixtures had no significant effects on parasite load in the laboratory. Newly shed parasites were more likely than 10 hours old parasites to infect tadpoles, regardless of pesticide exposure. In my final experiment, I utilized outdoor mesocosms to expose parasites, snail hosts, and Rana sylvatica tadpoles to the pesticide mixture, and I examined differences in parasite load within the tadpoles after two weeks. The pesticides had no significant effect on parasite loads in the field. Overall, my findings suggest the atrazine and metolachlor mixtures used in this study had no significant effects on disease dynamics in a system involving Echinostome parasites, snails, and tadpoles.
Master of Science

Kin-recognition abilities, first demonstrated 25 years ago in toad tadpoles, now appear to be widespread among amphibians. In some vertebrates kin recognition is based, at least in part, on highly polymorphic major histocompatibility complex (MHC) genes. Besides protecting animals from disease resistance, MHC genes regulate social behaviour. They allow relatives to recognise one another so that they can cooperate for mutual benefit. These two seemingly distinct functions of MHC genes may be integrally related, because animals need to outbreed to optimise the immune systems of their offspring. The ability to discriminate MHC-type is therefore likely to facilitate kin discrimination in tadpoles. I tested association preferences of African clawed-frog (Xenopus laevis) tadpoles in a laboratory choice apparatus. As in other anuran species, I found that tadpoles at earlier developmental stages preferentially associate with unfamiliar siblings over unfamiliar non-siblings but that this preference reverses during development. Tadpoles approaching metamorphosis demonstrated a reversal in their preference; they preferentially school with non-kin rather than kin. The ontogenetic switch in larval schooling preferences coincides with the onset of thyroid hormone (TH) controlled development and may be indicative of decreased fitness benefits associated with schooling with kin at later developmental stages. These may result from an increase in intraspecific competition, predation, or disease susceptibilities of prometamorphic individuals. Alternatively, the kin avoidance behaviours observed at later larval stages might reflect disassociative behaviour that facilitates inbreeding avoidance at reproductive maturity. This is the first study to find a shift from an association preference for kin to non-kin during amphibian larval development. Using allele-specific PCR techniques to MHC-type tadpoles, I tested association preferences among siblings based on shared MHC haplotypes. By using only full siblings in experimental tests, I controlled for genetic variation elsewhere in the genome that might influence schooling preferences. I found that X. laevis tadpoles discriminate among familiar full siblings based on differences at MHC genes. Subjects from four families preferentially schooled with MHC-identical siblings over those with which they shared no or one haplotype. Furthermore, the strength of tadpoles’ MHC-assortative schooling preferences significantly correlated with amino acid differences in the peptide-binding region (PBR) of both the MHC class I and II loci. Since MHC-PBR polymorphisms determine the pool of peptides that can serve as ligands for MHC molecules, these findings support the hypothesis that MHC peptide ligands mediate MHC type discrimination. As test subjects were equally familiar with all stimulus groups, tadpole discrimination appears to involve a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics. I also found that non-MHC-linked genetic differences contribute to tadpole association preferences in tests that contrast MHC and kinship. Tadpoles did not discriminate between MHC-similar non-siblings and MHC-dissimilar siblings and preferentially associated with MHC-dissimilar non-siblings rather than MHC-similar non-siblings. Although the MHC may be not solely responsible for the genetically determined cues that direct tadpole association preferences, it certainly is important in facilitating discrimination among conspecifics in X. laevis tadpoles. MHC-based discrimination may be retained through ontogeny and serve to maintain MHC-polymorphisms by facilitating disassortative mating.

Using spawn collected from the north Merseyside sand dune system, the effects of temperature on growth, development, metabolism and metamorphosis in B. bufo and B. calamita tadpoles were investigated, together with a limited study on the effects of L-thyroxine. The effects of density on growth, developteI1t and metamorphosis were examined. Physiological and behavioural effects of B. bufo tadpoles on B. calamita tadpoles were also studied. B. calamita spawn and tadpoles survive at higher temperatures than B. bufo. Below20°CB. bufo spawn, and below 15°Ctheir t.adpcl.es, develop at a greater rate than B. calamita. B. calamita spawn and tadpoles' growth and development is faster at higher temperatures. B. calamita tadpoles have higher metabolic rates than B. bufo across the temperature range 15-30°C. Faster rates of metabolism and development in B. calamita result in smaller tadpoles and toadlets than B. bufo. B. calamita may compensate for this with increased metamorphic efficiency in terms of energy. It is speculated that differences in tadpole thyroid physiology could account for observed differences. metamorphic efficiency was greatest, and rates of development and growth maximised/at the tadpoles' preferred body temperature. Increased density reduced growth-and developteI1t of tadpoles, but influenced different stages in the two species. metamorphosis in all B. bufo tadpoles was delayed, whereas in B. calamita a proportion of the population metamorphosed apparently unaffected by increased density. The responses of tadpoles to temperature and density were related to the species' preferred spawning environments. In the presence of B. bufo tadpoles, B. calamita growth, development and metabolism was suppressed. The pattern of development and timing of metamorphosis in B. calamita became similar to that of B. bufo. Inhibition was not relieved by L-thyroxine, and did not effect tadpole behaviour. Mass specific food consumption was increased. It is speculated that the inhibitor is a parasite or a substance which affects assimilation.

To investigate the effects of methylmercury (MeHg) on development, Xenopus tropicalis tadpoles were exposed to MeHg diets with concentrations of 0.42 (control), 44 (low), and 270 (high) ng/g wet weight. Increased mortality, decreased metamorphosis, increased size, and a greater number of days between tadpoles reaching metamorphosis were observed in the high MeHg group, suggesting disruption to the thyroid axis. Triiodothyronine levels, however, were not significantly different between groups. In both the control and low MeHg groups, total Hg and MeHg body burdens increased rapidly, reached a plateau and eventually declined with a lower percent MeHg body burden. In the high MeHg group, body burden concentrations increased throughout the experiment. This study demonstrates that at low dosages of MeHg, elimination may prevent toxic exposure whereas at high dosages, demethylation and selective excretion mechanisms may be overwhelmed and disruption of development or death may occur in tadpoles.

Amphibians are particularly sensitive to environmental degradation and, therefore, serve as effective environmental quality indicators. Research has suggested that amphibian declines are exacerbated by manmade environmental toxicants, especially those found in high concentrations in urban areas. The NIH has pinpointed genotoxicity as a major route of cancer causation, and has since developed stringent testing procedures for potentially hazardous chemicals. One such method, recognized for its simplicity and economy, is the micronucleus assay. A study was conducted assessing the genotoxicity of the widely used antimicrobial agent Triclosan to American Bullfrog tadpoles. Lithobates catesbeianus tadpoles were reared in glass aquaria containing ultra-high purity water and were dosed with nominal concentrations of 2.3 µg/L, 23 µg/L, and 230 µg/L Triclosan, reflecting 1x, 10x, and 100x concentrations of the compound as found in US surface waters. Eight replicates of each of the three levels of Triclosan contamination were prepared, as well as eight replicates per control group. Each replicate contained three tadpoles in a glass aquarium, from which one tadpole per tank was sampled after 1, 8, or 15 days following initial exposure to test compounds. Erythrocytes were prepared on slides and scored for micronucleus presence under 1000x magnification. Triclosan induced significant micronucleus formation after only 24 hours in all treatments relative to the negative control and exhibited a maximum of 15 micronuclei per 2,000 erythrocytes scored. Modeling of MN induction dynamics by treatment suggested that the best predictor of micronucleus induction was the acute TCS exposure level, as described by a linear mixed effects model including a binomial term of time exposed. Micronucleus induction was TCS concentration dose-dependent. This study supports that Triclosan induces significant genetic damage at environmentally relevant concentrations. It is clear that the effects of genotoxic agents must be certified so proper regulatory protocols can be developed and enforced, in order to conserve wildlife and promote human health.

The effects of AroclorRTM1242 exposure on developing amphibians were examined by exposing southern leopard frogs, Rana sphenocephala, to a contaminated diet throughout the larval period. Treatment groups consisted of an untreated control, a solvent control, 17beta-estradiol, and three PCB doses (10, 100, and 1000 mug/g). PCB accumulation and biotransformation were examined. Effects of PCB exposure on metamorphosis and sexual differentiation were assessed. PCB accumulation was not statistically different between the egg masses. Levels of PCBs in the tissues differed in a dose related manner. Within treatments, body weight appeared to be the major factor correlated with PCB concentration. Congener patterns varied between AroclorRTM1242 and tadpole tissues. Hydroxybiphenyls were not detected in individual tadpoles, but were present in pooled samples. Mortality was similar among the treatment groups. The untreated food control had slightly higher mortality. The effects of AroclorRTM 1242 exposure differed between the egg masses. From the first egg mass, there were no treatment differences in the proportion of survivors metamorphosing. Treatment with 17beta-estradiol affected sexual differentiation relative to the controls. Exposure to AroclorRTM1242 had no effect. This may be due to the nature of gonadal maturation in this population, which was entirely female. In the second egg mass, PCB treatment was associated with an increased rate of metamorphosis in the highest dose. The high dose significantly (alpha = .05) altered sexual differentiation relative to the solvent control (88% female). This effect was similar to 17beta-estradiol treatment (94% female). Exposure to high and medium doses of PCBs produced five individuals with asynchronous gonads. The effect of body weight on PCB levels may be a result of greater ingestion of the contaminated diet by larger animals, relative to their smaller siblings. The apparent stimulation of metamorphosis in the second egg mass by PCB exposure may be due to an endocrine disrupting effect on the corticosterone "stress" axis. A thyroid-mediated mechanism of effect is also possible. While the effects on sexual differentiation may be due to estrogenic activity, a corticosterone-like effect cannot be ruled out. Higher level endocrine function, such as feedback regulation, could also be affected.

This research is designed to test how sublethal exposure to copper affects tadpole predator-escape behavior and how quickly tadpoles recover. After exposure, tadpoles were separated. Escape behavior was recorded for two-thirds of exposed tadpoles while one-third of the exposed population was measured weekly to determine growth and recovery. Control tadpoles were consumed within 15 minutes whereas those exposed to higher concentrations were consumed at a slower rate, which does not support the hypotheses. Although the rate of predation was lower, tadpoles exposed to higher Cu concentrations were on average, 1.47 cm in total body length. Those exposed to 0.93 mg/L averaged 0.86 cm. After being placed into clean water, treatment tadpoles recovered after 20 days.

Understanding relationships between biodiversity and ecosystem function is a critical challenge, particularly in freshwater ecosystems where species losses are occurring at unprecedented rates. There is a particular need to examine these relationships in natural settings at large spatial scales. Ongoing, disease-driven amphibian declines may influence the structure and function of stream ecosystems, but little is known of the potential roles of stream-dwelling tadpoles in consumer-resource dynamics, ecosystem functions such as decomposition, and ecosystem-level biogeochemical cycling. Tadpoles in tropical streams likely regulate flows and ratios of nitrogen (N), phosphorus (P), and carbon (C), influencing ecosystems by altering nutrient supplies to other animals and their food resources. I used ecological stoichiometry as a framework to assess how the sudden loss of consumer biodiversity in neotropical headwater streams affected ecosystem function. I quantified N and P excretion and C:N:P ratios of tadpoles, macroinvertebrates, and food resources in healthy sites (pre-decline) and sites where disease-driven amphibian declines had occurred (post-decline). I tested the hypothesis of consumer homeostasis (i.e., that organisms maintain consistent body nutrient ratios by altering excretion chemistry) over a range of taxa and size classes. I also used mesocosms in a natural stream setting to quantify the effects of grazing tadpoles, shredding macroinvertebrates and a combination of the two on leaf decomposition and associated microbial activity. Finally, I examined macroinvertebrate community structure and quantified biomass and nutrient storage in tadpoles, macroinvertebrates, and basal resources in pre-decline and post-decline sites. I also measured excretion rates, volumetric excretion, and nutrient turnover for both tadpoles and macroinvertebrates. Patterns of consumer-resource stoichiometry varied with the presence or absence of tadpoles. There were higher concentrations of C, N, and P in basal resources in pre-decline sites compared to post-decline sites, but little variation in elemental ratios among sites. Elemental composition and molar ratios in grazers and shredders varied, with pronounced differences in %N for gatherers and filterers across sites. Macroinvertebrate grazer elemental composition was higher for all elements and had lower C:N, N:P, and C:P molar ratios in pre-decline sites compare to grazers in post-decline sites, while shredders showed the opposite pattern. There were differences in both taxon-specific allometric and stoichiometric relationships in tadpoles and macroinvertebrates between pre- and post-decline sites. Body P content was a good predictor of tadpole P excretion and tadpoles in pre-decline sites excreted more P per unit body P than those in post decline sites. Individuals deviated from strict homeostasis, and the degree of deviation varied among taxa. Tadpoles also affected leaf decomposition by influencing microbial communities and altering shredding macroinvertebrate feeding. Higher respiration rates of leaf discs in chambers with tadpoles suggested that tadpoles enhanced microbial activity by excreting nutrients through feeding and excretion. Shredders alone had little effect on respiration rates, indicating that tadpoles play an important and unique role in enhancing microbial activity and litter decomposition. Leaf area loss was greatest when tadpoles and macroinvertebrates were together, indicating facilitation. Macroinvertebrates are important nutrient recyclers in neotropical headwater streams, but their role is greatly decreased in the absence of larval amphibians. I measured ~80% lower standing stocks and storage of C, N, and P in basal resources in post-decline compared to pre-decline sites. Storage of C, N, and P in both tadpoles and macroinvertebrates also decreased in post-decline sites. I also observed 98% decreases in tadpole nutrient excretion and egestion rates, and an additional decrease in macroinvertebrate excretion rates (~80%) for both N and P in post-decline versus pre-decline sites. These decreases led to >8,000% increase in the distance that it took tadpoles to turn over the ambient N pools in post-decline sites, and a 130% increase for macroinvertebrates. Similar patterns were evident for P turnover, with turnover distance increasing by 6,000% and 400% in post-decline sites for tadpoles and macroinvertebrates, respectively. My results indicate that N and P excretion by both tadpoles and macroinvertebrates constitute significant nutrient fluxes in these headwater streams. Both tadpole and macroinvertebrate communities were excreting nutrients at similar rates in pre-decline sites, suggesting that they were playing equally significant roles in their contribution to ecosystem demand. My results demonstrate that tadpoles are important consumers in Neotropical headwater streams and their loss significantly alters stream food webs and ecosystem functions.

Xenopus laevis tadpoles are extensively utilised in scientific research within a number of biological disciplines, but little work has been carried out concerning their behavioural capabilities and their flexibility to different environmental conditions. This has resulted in a near complete lack of data upon which to base recommendations for laboratory maintenance. This thesis aimed to address this problem by investigating the growth, development and behaviour of the tadpoles under a variety of different housing conditions.

My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

A intervenção das atividades antrópicas ao meio ambiente tem repercutido na estrutura e funções dos ecossistemas, sendo que o uso de aditivos químicos, como agrotóxicos e fertilizantes, destacam-se pela parcela de responsabilidade que assumem diante desses desequilíbrios. O agrotóxico Vertimec® 18CE, cuja toxicidade para diversas taxóns já é conhecida, é amplamente utilizado em lavouras agrícolas, e têm-se conhecimento a respeito das intensas práticas de manuseio, muitas vezes irregulares, o que pode fazer com que concentrações muito maiores do que as previstas atinjam o ambiente aquático. Um dos grupos que nos últimos anos vem se destacando pelo declínio crescente associado ao uso destes produtos são os anfíbios. Os anfíbios possuem papel central na cadeia alimentar, e muitos estudos relatam o desequilíbrio ecológico causado pelo declínio desse grupo em relação a adição de contaminantes, incluindo os agrotóxicos. No presente estudo foram desenvolvidos testes laboratoriais a fim de determinar o estágio dos girinos de Lithobates catesbeianus mais suscetível à intervenção do agrotóxico e à concentração capaz de causar mortalidade em 50% dos indivíduos (CL50, 96h). A partir desses resultados, experimentos in situ (mesocosmos) com girinos no estágio mais avançado (25 da tabela de Gosner) foram desenvolvidos com o intuito de comparar as diferenças de toxicidade do Vertimec® 18CE quando manipulado em campo e laboratório, e quais os efeitos causados por uma única aplicação do produto e por aplicações múltiplas. Os testes laboratoriais demonstraram que os girinos em estágios mais precoce (21G) foram mais sensíveis ao agrotóxico Vertimec® 18CE do que àqueles mais avançados (25G). O experimento em campo indicou que a CL50, 96h para L. catesbeianus (25G) foi muito mais agressiva quando próxima às condições naturais, causando a mortalidade de todos os indivíduos no mesmo estágio de desenvolvimento em um período inferior a 24 horas. Entretanto, a concentração inferior à CL50, 96h (0,002 ml de Vertimec.L-1) quando ministrada em campo, assim como no laboratório, não causou mortalidade, mas implicou em atraso no desenvolvimento e maior período larval do que àqueles organismos que não estiveram em contato com o agrotóxico, já que atingiu os recursos disponíveis, promovendo um efeito indireto no sucesso dessa espécie. Não foi possível observar diferença entre as aplicações única e múltipla do Vertimec® 18CE, em decorrência da morte dos girinos do tratamento em dosagem múltipla, porém observou-se desequilíbrio das condições limnológicas do sistema ao sofrer nova intervenção do Vertimec® 18CE. Diante dos resultados obtidos é possível concluir que o Vertimec® 18CE é capaz de afetar diretamente (mortalidade, estágios de desenvolvimento e comportamento) e indiretamente (redução de recursos alimentares) a população de Lithobates catesbeianus, contribuindo para o declínio de anfíbios nos sistemas naturais.
The intervention of anthropogenic activities to the environment has reflected in the structure and functions of ecosystems and the use of chemical additives, such as pesticides and fertilizers, has contributed for the disequilibrium. The Vertimec® 18EC, whose toxicity is described to different taxa, is widely used in agricultural crops, and it has been known about the intense handling practices, often irregular, which can reach higher concentrations than those expected in the aquatic environment. One group that has been highlighted in recent years by the increasing decline associated with the use of these products is the amphibians. Amphibians plays a central role in the food chain and several studies have related the ecological disequilibrium caused by the decline of this group due to the addition of contaminants, including pesticides. In this study, laboratory bioassays were developed to determine the more susceptible stage and the LC50,96h of Lithobates catesbeianus to Vertimec® 18EC. From these results, in situ experiments (mesocosms) were conducted with tadpoles in more advanced stage (25 of Gosner table) in order to compare the differences in toxicity of Vertimec® 18EC when manipulated both in field and in laboratory. Also, the effects caused by a single and multiple application of the pesticides were evaluated. The laboratorial results showed that earlier stages (21G) were more sensitive than more developing tadpoles (25G), and the effects of the pesticide were more aggressive under natural conditions (mesocoms), with 100% of mortality before 24 hours, at the same developmental stage. The results of laboratorial and in situ bioassays indicated that the lower concentration (0.002 ml Vertimec.L-1) didn\'t cause mortality, but alterations on development stage and larval period time were observed, indicating indirect effects (available resources) on the success of this species. On the other hand, no difference was found between the single and multiple applications of Vertimec® 18EC because all organisms died in the unique dosage treatment, but it was possible to verify limnological alterations after the second application of the pesticide. Considering the results obtained we conclude that Vertimec® 18EC can affect directly (mortality, development stages and behavior) and indirectly (food reduction) the population of Lithobates catesbeianus, contributing to the decline of amphibians in the natural systems.

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Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM)
O presente estudo analisou a fauna parasitária de três espécies de anuros desde a fase larval até a adulta, Hypsiboas cinerascens, Hypsiboas geographicus e Rhinella marina da Reserva Florestal Ducke, Amazônia Central. A fauna de parasitas de anuros da região é pouco conhecida, sendo a fase larval a menos estudada. Os dados analisados durante os dois anos de coleta demonstraram que espécies parasitas diferem nas duas fases de vida dos anfíbios anuros. Foram analisados um total de 224 girinos, e 38 indivíduos adultos, em três áreas de amostragem diferentes: poças temporárias, poça permanente, e uma lagoa da reserva. Um total de 827 parasitas foi encontradonosgirinos, e 3.534 nos adultos. Nas fases larvais foram encontrados nematoidespertencentes à espécie Gyrinicola chabaudi, metacercárias de trematódeos de Diplostomídeos e protozoários do gênero Nyctotherus. Nos adultos, a riqueza parasitária foi maior: nestes foram encontrados nematoides, trematódeos, acantocéfalos, carrapatos e larvas de Diptera. As fases larvais das espécies de anuros aqui estudados, representam novos registros de hospedeiros para o nematoide Gyrinicola chabaudi, ampliando o conhecimento de sua distribuição geográfica. Com os dados obtidos, podemos compreender que a riqueza entre as duas fases de desenvolvimento dos anuros são bem distintas. Durante o processo de metamorfose até a fase adulta o animal transporta grupos de parasitas, conectando-os entre o ambiente aquático e o ambiente terrestre. Este trabalho é o primeiro, até o momento, que descreve este tipo de interação na região, contribuindo assim com o aumento de registro de novas informações sobre estudos parasitológicos na Amazônia.
The present study analyzed the parasitic fauna of three species of anurans from the larval to adult phase, Hypsiboas cinerascens, Hypsiboas geographicus, and Rhinella marina of the Ducke Forest Reserve, Central Amazonia. The anurans parasitic’ fauna of the region is little known, being the larval stage less studied. The data analyzed during two years of collection showed that parasite species differ in two life stages of anuran amphibians.A total of 224 tadpoles, and 38 adult individuals, were analyzed in three different sampling areas, temporary pools, permanent pond, and one reserve pond. A total of 827 parasites were found in tadpoles, and 3,534 in adults. In the larval stages nematodes belonging to the species Gyrinicola chabaudi, metacercariae of trematodes of the family Diplostomidae, and protozoa of the genus Nyctotherus sp. In adults, parasite diversity was higher: in these were found nematodes, trematodes, acantocephalus, ticks, and larvae of Diptera. Larval phases of the anuran species studied here represent new host register for the Gyrinicola chabaudi nematode, increasing the knowledge of its geographical distribution.With obtained data we can understand that diversity and richness between the two stages of anuran development are very different. During the process of metamorphosis to adulthood the animal carries groups of parasites, connecting them between the aquatic environment and terrestrial environment. This work is the first, to date, which describes this type of interaction in the region, thus contributing to the increase in the registration of new information on parasitological studies in the Amazon.

Orientador: Luciano Alves dos Anjos
Resumo: O presente estudo analisou a fauna parasitária de três espécies de anuros desde a fase larval até a adulta, Hypsiboas cinerascens, Hypsiboas geographicus e Rhinella marina da Reserva Florestal Ducke, Amazônia Central. A fauna de parasitas de anuros da região é pouco conhecida, sendo a fase larval a menos estudada. Os dados analisados durante os dois anos de coleta demonstraram que espécies parasitas diferem nas duas fases de vida dos anfíbios anuros. Foram analisados um total de 224 girinos, e 38 indivíduos adultos, em três áreas de amostragem diferentes: poças temporárias, poça permanente, e uma lagoa da reserva. Um total de 827 parasitas foi encontradonosgirinos, e 3.534 nos adultos. Nas fases larvais foram encontrados nematoidespertencentes à espécie Gyrinicola chabaudi, metacercárias de trematódeos de Diplostomídeos e protozoários do gênero Nyctotherus. Nos adultos, a riqueza parasitária foi maior: nestes foram encontrados nematoides, trematódeos, acantocéfalos, carrapatos e larvas de Diptera. As fases larvais das espécies de anuros aqui estudados, representam novos registros de hospedeiros para o nematoide Gyrinicola chabaudi, ampliando o conhecimento de sua distribuição geográfica. Com os dados obtidos, podemos compreender que a riqueza entre as duas fases de desenvolvimento dos anuros são bem distintas. Durante o processo de metamorfose até a fase adulta o animal transporta grupos de parasitas, conectando-os entre o ambiente aquático e o ambiente terrestre. Este traba... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

This study resulted from the identification of gaps in the literature pertaining to the morphological descriptions of the tadpoles occurring at high altitudes in the Drakensberg Mountains in South Africa. These tadpoles are exposed to low temperatures, high desiccation risk, elevated ultraviolet radiation, competition, and predation and inhabit the clear, flowing streams and marsh areas of the mountain. Highly varying environmental conditions caused tadpoles to have considerable intraspecific variation. The high degree of plasticity necessitated extensive descriptive studies of tadpole morphology in order to document intraspecific variation and set up reliable keys for species identification. Specified adaptations to the extreme montane conditions are present in tadpoles of certain species. An especially interesting adaptation is the elygium, a hemispherical pigmented area above the eye, which apparently protects the retina from harmful ultraviolet radiation. There are no known studies of elygium plasticity in tadpole eyes in relation to variation in ultraviolet radiation. Particular attention was given to the functionality and cytology of this structure. Detailed measurements of tadpoles of six frog species of the high altitude Drakensberg Mountains were made. Morphological adaptations were described on the basis of these measurements. The cytological origin of the elygium of Amietia vertebralis was revealed through histological and cellular ultrastructure studies. The change in elygium morphology over time was studied as a function of ultraviolet intensity by exposing tadpoles to different levels of ultraviolet radiation. From the detailed morphological descriptions a more reliable binomial key was constructed, which made it possible to distinguish between Amietia umbraculata and A. vertebralis. A new amended definition of the epidermal elygium can now be given as an area of melanophores originating from the pigmented epithelium of the retina, forming a hemispherical shape from the dorsal margin of the iris. It is positioned in such a way as to protect the retina when light enters directly from above. This empirical study of the functional significance of the elygium showed that elygium morphology was considerably plastic, and that there were differences in elygium area and base length in the presence or absence of UVB radiation. In the presence of high UV radiation tadpoles produced an elygium with a broader base rather than longer elygia with a larger area. A wider elygium base shaded the pupil more effectively, thus protecting the retina from harmful UV radiation. The presence of a ventral elygium was also discovered.
Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Resumo: Por utilizarem diversos tipos de habitats e serem relativamente abundantes, girinos de anfíbios anuros são considerados ótimos objetos de estudo para se entender a estruturação das comunidades. A forma do corpo dos girinos varia enormemente de acordo com o tipo de habitat que utilizam e estudos em ecomorfologia podem responder quais características corpóreas são mais importantes para a ocupação do ambiente. Buscando estas respostas foram amostrados mensalmente, ao longo de um ano, nove corpos d'água (três lagoas, três riachos e três poças temporárias) no Parque Estadual de Campos do Jordão (PECJ), Serra da Mantiqueira, SP. O PECJ localiza-se no Bioma de Mata Atlântica e sua vegetação é constituída por Mata de Araucária, Mata Latifoliada de Encosta Atlântica e Campos de Altitude. Foram determinadas a riqueza, a abundância e a diversidade de cada comunidade. A estrutura dos corpos d'água foi determinada por meio de 12 descritores ambientais. Foram registrados girinos de 17 espécies de oito famílias, sendo Hylidae a mais representativa em riqueza de espécies e em abundância total. A maior riqueza de espécies foi registrada nas lagoas permanentes (10 espécies), enquanto as poças temporárias e os riachos apresentaram a mesma riqueza (cinco espécies). A maioria das espécies foi especialista para tipo (riachos, poças temporárias e lagoas) e para uso de hábitat (cada corpo d'água), o que não impediu que a sobreposição no uso de hábitat fosse alta para 88% das 17 espécies registradas. Contudo, essa alta sobreposição ocorreu entre as espécies pertencentes a uma mesma guilda de especialistas para cada tipo de corpo d'água. As espécies de diferentes guildas apresentaram baixa sobreposição no uso de hábitat. Correspondente a isso, a composição de espécies nos corpos d'água amostrados refletiu os três tipos de corpos d'água...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Tadpoles are considered a good model to understand community structure because they use several types of habitats and are relatively abundant. The body shape of tadpoles varies greatly according to the habitat type, and studies in ecomorphology can link body features to the habitat occupation. In this study we selected nine water bodies - three permanent ponds, three permanent streams and three temporary puddles - in the Parque Estadual de Campos de Jordão (PECJ), São Paulo State, southeast Brazil. The PECJ is situated in the Atlantic Rain Forest, and it is characterized by Floresta de Araucária (Araucaria angustifolia), Floresta Latifoliada de Encosta Atlântica and Campos de Altitude. The richness, abundance, diversity and spatial and seasonal occurrences of the species were determined in each habitat, through the samplings, accomplished with deep nets. The structure of the water bodies was determined using 12 environmental descriptors. Tadpoles of 17 species, belonging to eight families, were recorded, being Hylidae the most representative in richness and total abundance. The great species richness was recorded in permanent ponds. Temporary puddles and streams showed the same richness (5 species). Most of the species were specialist to water body type (streams, temporary puddles and permanent ponds) and to habitat use (each water body), did not preventing/avoiding the high overlap in the habitat use for 88% of the 17 recorded species. However, the high overlap occurred among the species belonging to the same guild of specialists for each type of water body. The species of different guilds showed low overlap in habitat use and, corresponding to that, the species composition reflected the three types of sampled water bodies. The flow and duration of the water, the size and the depth of water bodies influenced greatly the tadpoles' spatial distribution. From the 16...(Complete abstract click electronic access below)
Orientador: Denise de Cerqueira Rossa Feres
Coorientador: Itamar Alves Martins
Banca: Arif Cais
Banca: Elieth Floret Spirandelli Cruz
Mestre

Bitumen extraction in the Athabasca oil sands in Alberta releases metals to the region. In this study, I performed an uptake-elimination experiment with wood frog tadpoles (Lithobates sylvaticus) to determine the bioaccumulation potential of metals from exposure to MacKay River sediment, an area affected by oil sands contamination, and to uncontaminated reference sediment. Wood frog tadpoles, Gosner stages 28-32, were exposed to two sediments: (1) MacKay River sediment that is enriched in petrogenic hydrocarbons from natural and anthropogenic sources; and (2) an uncontaminated reference sediment. Tadpole exposures to sediments lasted 4 days, followed by a depuration phase for an additional 4 days where tadpoles were allowed to eliminate excess metals from their bodies. The metal concentrations at various time points during the uptake and elimination phases were determined in order to define toxicokinetic parameters, such as uptake and elimination first order rate constants, accumulation by ingestion, and assimilation efficiencies for specific metals. It was determined that tadpoles exposed to the MacKay sediment had higher concentrations of Al, Co, Cu, Cr, Mg, Ni, Pb, V, and Zn throughout the uptake phase of the study compared to tadpoles exposed to reference sediment. We also observed little to no decrease in concentrations of Al, Co, Cu, Cr, Mg, Ni, Pb, V, and Zn throughout the elimination phase of the study. In addition, biota-sediment accumulation factors (BSAF) revealed that Cu, Zn, Cr, and V had among the highest bioaccumulation potential in our trials. The experiment was subsequently repeated by preventing direct contact of the tadpoles to sediment with a screen, exposing tadpoles only to metals in water. By comparing tadpole exposures to metals from ‘aqueous’ and ‘aqueous +sediment’ in separate trials, and by tracking sediment ingestion rates, I am able to show that sediment ingestion constitutes the primary source of metal bioaccumulation by tadpoles. Not only were metal concentrations higher in tadpoles that were ingesting sediment, but they also had greater metal uptake rates compared to tadpoles that were only exposed to contaminated water. It was also determined that assimilation efficiencies were higher in tadpoles exposed to reference sediment compared to ones exposed to MacKay River sediment. Using toxicokinetic parameters defined by the uptake-elimination experiment, I developed a computational model using STELLATM system dynamics software to accurately estimate first order uptake and depuration rate constants for metals in exposed aquatic animals. The model estimated metal uptake and depuration kinetics with a mean relative error of 2.25 ± 0.93 % (±SE, n=9) for the uptake study and 2.53 ± 2.61 % (±SE, n=9) for the depuration study. With increased oil-sands production anticipated, we recommend continued monitoring of contaminants from oil-sands for the purpose of understanding the potential risks they may have on northern Alberta’s ecosystems.

Burning of fossil fuels by coal-fired power plants (CFPPs) is one of the largest sources of environmental mercury in the United States and there have been conflicting reports of local impacts due to CFPPs in the US. Illinois has 23 coal-fired electrical generating plants that may be contributing to elevated environmental mercury concentrations that have contributed to mercury advisories for 13 bodies of water located throughout the state. The objective of the current study is to determine if there is a pattern of total mercury concentrations in sediment and tadpoles collected from ponds located upwind and downwind of 4 coal-fired electrical generating plants in Illinois. Baldwin, Joppa, Newton, and Southern Illinois Power Cooperative (SIPC) coal-fired power plants were chosen for study. Three sediment samples were collected from ponds located 3-5, 8-10, and 13-15 km downwind and from ponds located 3-5 km upwind of each power plant and analyzed for total mercury concentration. Temperature of overlying water (C), pH, oxidation-reduction potential (mV), and texture were determined for each sediment sample. Bullfrog (Lithobates catesbeiana) or Green frog (Lithobates clamitans) tadpoles were collected opportunistically from 29 of the 44 ponds chosen for study. Each tadpole was identified to species and Gosner stage, and then weighed and measured for total length prior to total mercury analysis. For Baldwin, Joppa, and SIPC power plants, no significant pattern of total mercury could be determined from either sediment or tadpole data. For Newton power plant, total mercury concentrations were significantly greater 3-5 km downwind for sediment, and 8-15 km downwind for tadpoles compared to concentrations at upwind locations. Sediment total mercury concentrations were not significantly correlated with any of the characteristic variables (e.g. pH). Tadpole total mercury was significantly negatively correlated with length and weight, but not significantly correlated with any of the sediment variables. Sediment and tadpole concentrations were not significantly correlated.

Amphibian diversity has declined, in part, due to the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Andean water frogs in the genus Telmatobius are particularly vulnerable to the disease and the genus has been extirpated from Ecuador and in Andean cloud forests, yet populations of species persist in the high Andes of Peru and Bolivia. The Alpaca Water Frog (Telmatobius intermedius), endemic to the Peruvian Andes, can be found infected with Bd. Alpaca Water Frogs inhabit high elevation open canopy freshwater systems. My overall goal was to study the effect of chytrid infection on the trophic ecology of Telmatobius tadpoles. I used stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) to characterize the the trophic structure and energy flow in this system. I observed the values of δ15N were higher for tadpoles than algal material (t-test, t= -8.60, df= 34, p< 0.01), mayfly nymphs (t-test, t= 5.25, df= 30, p< 0.01), and predatory aquatic invertebrates (t-test, t= -4.18, df= 47, p< 0.01). In regard to the δ15N values of tadpoles and frogs, tadpoles had a lower value (t-test, t= -3.0, df= 31, p< 0.01). Values of δ15N in tadpoles were relatively high, signaling the presence of animal tissue in their diet. I also investigated changes in tadpole diet associated with mouthpart deformities caused by the fungal pathogen Bd. There was a positive association between the extent of mouthpart deformity and Bd infection (Fisher’s Exact test, p<0.001). The relative proportions of diatom morphotaxa groups found in the foregut of T. intermedius tadpoles varied in association with the degree of mouthpart deformity, as indicated by an ANOSIM analysis (R=0.875, p<0.001). Finally, in addition to Bd prevalence in adult aquatic frogs, I investigated whether Alpaca Water Frogs and other Andean Water Frogs tested positive for the antifungal bacterium Janthinobacterium lividum (Jliv). My results show that 57% of the sampled frogs were infected with Bd, 12.5% of frogs hosted both Jliv and Bd, while 7.2% hosted just Jliv. We found that the probability of an individual being infected with Bd was independent of the presence of Jliv; however, we did detect a protective effect of Jliv with respect to intensity of infection. My findings demonstrate that the fungal pathogen Bd influences the trophic ecology of tadpoles of Andean water frogs.

PKMz (Protein Kinase M zeta) is a recently identified isoform of Protein Kinase C. It is persistently active upon synthesis because its sequence resembles the catalytic domain of PKC zeta but lacks the auto-inhibitory regulatory domain. Previous studies found that PKMz is critical for LTP maintenance, as well as learning and memory in the adult rat brain. However, it is not known whether and how it functions in developing neural systems. I have identified endogenous PKMz in Xenopus laevis tadpoles brain and found that its expression pattern is temporally and spatially correlated with synaptogenesis and dendritogenesis within tadpole retino-tectal system. By in vivo rapid time-lapse imaging and three-dimensional analysis of dynamic dendritic growth, I find that exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with ZIP (zeta-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Consistent with its established roles in regulating adult glutamatergic synaptic transmission, I also examined role of PKMz in regulating developing synapses, using both immunohistochemistry and in vivo patch clamp recording. Specifically, I find that knocking down endogenous PKMz using a morpholino impairs both transmission and maturation of glutamatergic synapses, and consistently induces promoted dendritic expansion as seen in ZIP treated neurons. The model that PKMz regulates dendritogenesis by regulating glutamatergic synaptic transmission was further investigated using a novel seizure model based on Xenopus tadpoles. I find that PTZ induced seizure activity increases normalized expression level of brain PKMz, which is required for over-stabilization of dendritic filopodia dynamics induced by seizure activity. Based on these findings, together with previous results from other related studies, I have constructed a discreet and stochastic computational model to simulate synaptotropic dendritic growth mechanism. I show that as formation of nascent synapses promotes dendritic expansion into region of synaptic partners by promoting maintenance of dendritic filopodia, synapse maturation drives further dendritic refinement and stabilization of appropriate dendritic structure.

Amphibians are important bioindicators in environmental assessment. This highly diverse group of vertebrates is experiencing unprecedented declines worldwide due to a complex array of factors, including disease, habitat loss, invasive species, and environmental pollution. Heavy metals are especially problematic due to their persistence and ability to present a localized hazard even at sublethal levels. Northern Idaho has a long history of mining activity, and many watersheds have experienced heavy metal contamination. These streams contain many sensitive species, including the Rocky Mountain Tailed Frog, (Ascaphus montanus). While tailed frog populations are known to be especially vulnerable to logging and road building, the effects of local mining have not been documented. In order to assess the vulnerability of this species to heavy metal contamination, tadpoles were collected from three distinct populations across the Idaho Panhandle National Forest. Two sampling sites (Gold Creek and Beauty Creek) were characterized by abandoned mines in the headwaters of the streams, while a stream absent of any local historic mining (Bumblebee Creek) served as a reference site. Whole tadpoles were pooled and treated using EPA Method 3050B and analyzed for dissolved metal concentration (ug/g) using ICP-MS. We found the average metal concentrations in tadpoles (ug/g) at mining sites were generally higher than the reference site. Cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) were significantly higher in tadpoles at Gold Creek when compared with the reference site. These results are consistent with other studies indicating metals from historic mining activity are still evident in these stream communities.