For evolution to occur over time, it is necessary for animals and plants to show phenotypic variation. If the individuals within populations of a species do not show observable differences among themselves, there will be a lack of driving force for natural selection to act on and decide which characteristic gets inherited from one generation to the next. It is hence important to study phenotypic variation, especially against environmental gradients such as latitude and altitude, which gives us an insight into the pattern of change according to essential factors such as temperature and length of seasons. The latter would impose time constraints on growing populations, leading to periods of unfavourable conditions limiting their growth and development. In many cases, such organisms would tend to compensate for the period of slow growth and catch up to the others that did not have to endure the same situation and grow to the same size as them. This study aims to find differences in three key larval life-history traits of the moor frog (Rana arvalis), namely metamorphic mass, larval period, and growth rate, and find what kind of pattern is observed in case of these phenotypic variations. It also aims to find differences in the strength of compensatory response between populations from lower and higher latitudes. A common garden experiment was conducted with populations originating from both sides of the Baltic Sea, ranging from southern Sweden and Latvia to central Finland and northern Sweden. It was expected that the northern populations would grow faster and show a countergradient variation pattern since they are faced with more strict time constraints at higher latitudes, but in most cases, we observed a co-gradient pattern, wherein the environmental effect amplifies the individual’s genetic predisposition instead of opposing it. We also observed a stronger compensatory response in the northern populations as compared to their southern counterparts. Effects of climate change and subsequent rise in temperatures making the environment unpredictable over time could be used to speculate about the reason behind the results obtained. Epigenetics could also be used as an approach to study long lasting changes in an organism’s gene expression to make it adapt better to changing conditions and hence show different patterns of variation from studies in the past. Studying such changes, expected or not, is important to keep up with the needs of the species that require conservation, and will help conservation biologists to formulate strategies that would be effective even in the face of constant change in the world.

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Most vertebrates have separate sexes, and sex-specific traits that are regulated by genes with sex-biased expression patterns. In many species with genetic sex determination system, genetic recombination is suppressed in genomic regions linked to the master regulator of sex determination – the gene or set of linked loci that orchestrate sexual differentiation. Natural selection may favour alleles with sex-specific effects - including those with sexually antagonistic (SA) fitness effects (e.g., beneficial to females but harmful to males) – to become fixed in or be translocated to these non-recombining regions of sex chromosomes, because sex-specific or sex-biased modes of inheritance can resolve genomic conflict associated with SA. Sexually antagonism may also be resolve by sex-biased gene expression, and in theory these two mechanism (sex-linkage and sex-biased gene expression) could operate synergistically. However, there are relatively few empirical studies that test whether genes with sex biased expression patterns are indeed more abundant on sex chromosomes – and especially on newly evolved sex chromosomes. We explored this question with an African frog species Xenopus borealis, whose sex chromosome evolved within the last 25 million years (my) and have a large (~50Mbp) region of suppressed recombination, making it a young sex chromosome system compared to many other intensively studied systems, such as the sex chromosomes of mammals. We tested the possibility that a higher proportion of genes with sex-biased expression would be located on the sex-linked region of the sex chromosome of this species. By examining gene expression in adult liver and gonad and also tadpole gonad/mesenephros at two developmental stages, we found that the sex-linked region of these sex chromosome do have a higher proportion of sex biased genes compared to the non-sex-linked region of the same sex chromosomes, compared to (i) a homeologous genomic region in the tetraploid genome of X. borealis, and also (ii) the autosomes of this species. We did not observe the same pattern in a closely related frog species, Xenopus laevis, which has sex chromosome that are not homologous to those of X. borealis and, unlike X. borealis, lacks a large region of suppressed recombination on its sex chromosome. Using Brownian Motion model, we found as well that expression divergence evolution of genes in the sex-linked region of X. borealis is faster compared to its non-sex-linked homeologs (within X. borealis), and also compared to orthologous regions that are also non-sex-linked. One possible explanation for these observations is that natural selection favoured an expansion of recombination suppression (via unknown mechanisms) on chromosome such that polymorphic regulatory regions became linked (or unlinked) to the sex determining locus in such a way to resolve SA. Alternatively, it is possible that these sex-biased expression pattern evolved rapidly after recombination suppression. / Thesis / Master of Science (MSc) / Sexual selection favours the evolution of distinctive traits in each sex in order to optimize the reproductive success of each one. However, because most of the genome is shared between the sexes, sexual selection may result in genomic conflict when mutations are beneficial to one sex but harmful to the other; this conflict is known as sexual antagonism. Genomic conflict associated with alleles with sexually antagonistic (SA) fitness effects can be resolved via the origin of sex-biased expression patterns and this may be catalyzed by genetic linkage to a sex-determining locus on a sex chromosome. Consequently, one might predict there to be an enrichment of genes with sex-biased expression patterns on the sex chromosome as compared to the autosomes. We tested this expectation in an African frog species Xenopus borealis, which has a relatively young sex chromosomes and a large region of recombination suppression on the female-specific W-chromosome. We found enrichment of sex-biased genes on the nonrecombining region of the sex chromosomes of this species in adult liver and gonad tissue and also tadpole mesenephros/gonads at two developmental stages. Additionally, we found that expression divergence of genes in the non-recombining region have a faster rate of evolution as compared to the rate of expression divergence of genes in other genomic regions. One possible explanation for these observations is that natural selection favours an expansion of recombination suppression (via unknown mechanisms) on sex chromosome such that polymorphic regulatory region become linked (or unlinked) to the sex determining locus in such a way as to resolve SA.

In this study, I used restriction fragment length polymorphism (RFLP) analysis (DNA fingerprinting) to study the genetic population structure of wood frogs, Rana sylvatica, collected as egg masses from vernal pools within the Massachusetts Audubon Society Lincoln Woods Wildlife Sanctuary in Leominster, MA. The average genetic relatedness of sibling individuals, non-sibling individuals from within the same pool, and individuals from pools of close (5 m), far (200 m) and distant (40 km) spatial separations was calculated. The goal was to use genetic relatedness to estimate the breeding patterns of R. sylvatica and use that information to make general management recommendations that could be applied to other vernal pools breeders. I detected relative differences in genetic similarity between individuals from pools only 5 meters apart, however over a larger distance of 200 meters no significant genetic differences were present. This suggests that although wood frogs are known to be highly philopatric, they may use information other than simply proximity to their natal pool when choosing breeding sites. Factors such as species composition, water chemistry and heterogeneity of the landscape between pools may influence breeding site choice. Also, contrary to the findings of recent studies, the distance between vernal pools may not be the best indicator of the genetic similarity of the individuals they host. Pools in close proximity to one another may host genetically distinct populations, and therefore management decisions should be made on a pool-by-pool basis. Consequently, when managing populations of R. sylvatica, and possibly other vernal pool breeders, taking into account parameters other than simply the spatial separation of pools within an array may avoid decisions that could result in the loss of genetic diversity.

Hypothesised causes of the observed world-wide decline of amphibian populations are varied and in some cases contentious. Insufficient information relating to the autecology of many amphibian species can cause erroneous speculations regarding critical habitat requirements and hence management programs designed to enhance population viability are often unsuccessful. Most amphibians display a bi-phasic life history that involves occupation of an aquatic breeding habitat and terrestrial habitats that are used for foraging, and shelter from predation and environmental stress. However, the focus of most amphibian research is centred on the breeding habitat, with limited research being conducted into the terrestrial habitat requirements of most amphibian species. Barakula State Forest is a large continuous area of open woodland situated in the semi-arid region of Queensland. The forest supports 21 species of endemic anurans, many of which use ephemeral waterbodies for breeding. This area is, therefore, an ideal location to test the relative importance of terrestrial habitat on the distribution of a suite of frogs that display different morphological and physiological characteristics. On the landscape scale, the attributes of the terrestrial environment at three survey areas within Barakula were similar. However, at the patch scale, ground truthing showed there were considerable variations in vegetation and ground cover attributes within and between each survey site. Measured properties of the soil also tended to vary within and between sites. Soil texture ranged from sandy to heavy clay, soil pH ranged from 3.9 to 6.4 and soil moisture varied considerably. Agar models, used for testing evaporative moisture loss at different microhabitats, retained significantly higher levels of moisture when positioned in the buried microhabitat during summer, but in winter, models that were placed under leaf litter retained higher levels of moisture. Variations in levels of moisture loss at the five different microhabitats were evident within and between the survey sites. Despite a prolonged drought, 1844 native frogs representing 17 species were pitfall trapped. Members from the family Myobatrachidae comprised 94% of these captures, and burrowing species accounted for 75% of total captures. Species were not randomly distributed within or between the survey sites. Vegetation attributes and soil properties played a significant role in influencing the catch rates and traplines that supported similar vegetation and soil attributes also tended to catch similar species. Capture rates of six of the seven burrowing species were significantly influenced by soil properties. When given a choice of four different microhabitats created in enclosures, individuals from five species showed varying responses to habitat choice during night time activity. During daylight all species tended to avoid bare areas and burrowing species tended to burrow under some form of cover. Pseudophryne bibronii metamorphs showed a significant avoidance to soils with high pH. The number of Limnodynastes ornatus metamorphs was significantly and positively correlated with moisture levels surrounding a breeding area. Limnodynastes ornatus metamorphs tended to avoid areas that did not support some form of cover. Embryos from the terrestrial egg laying P. bibronii translocated to sites with varying levels of soil pH, suffered increased mortality where the soil pH was &gt4.8. In the laboratory, embryonic survival was not significantly different between the four pH treatments. There was a significant influence of fungal infection on survival rates and ranked fungal infection was significantly different between the four pH treatments. The terrestrial environment at the three survey sites has provided sufficient protection from environmental elements to allow a large diversity of anurans to persist for long periods without access to permanent water. Management must consider the importance of the non-breeding habitat when defining buffer zones, restoration programs and conservation strategies to ensure that the complete set of ecological requirements for frog species are provided.

This study attempted to selectively stimulate and record from either the amphibian or basilar papillae of Rana pipiens. Computer-generated, frequency-specific clicks were used to elicit BSER's from either amphibian or basilar papillae. Narrowband noise fatiguers were presented in the frequency region of which each papillae are tuned. It was expected that a threshold shift would be elicited in the papillae that received the acoustic trauma, and that no threshold shift would be observed from the collateral papilla. The results of this experiment indicated that there was no overall difference between the threshold shift of either papilla. Furthermore, the amount of AP threshold shift was relatively constant regardless of whether the fatiguer bandwidth was overloading the amphibian or basilar papillae. By contrast, the amount of BP threshold shift was greater when proceeded by a fatiguer with a bandwidth corresponding to the BP tuning region than by a fatiguer with a bandwidth corresponding to the AP tuning region. Additionally, curare maximized the amount of BP threshold shift following fatiguing noise presented with a bandwidth to which the AP is tuned.

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant

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.

Este trabalho teve como objetivo propor novo método para estudar a permeabilidade de fármacos, utilizando epitélio intestinal de rã-touro (Rana catesbeiana) em método ex vivo, empregando células de Franz. Por utilizar epitélio intestinal, um material de descarte proveniente de um animal utilizado como alimento humano, pode ser considerado um método alternativo, pois não implica no sacrifício de animais. A quantidade de fármaco permeada foi determinada por método de eletroforese capilar com detecção ultravioleta e validado para os antivirais lamivudina, zidovudina e aciclovir, na presença de metoprolol e floridizina. O fármaco escolhido como modelo nos ensaios de permeabilidade foi a lamivudina. Para estabelecimento do protocolo experimental dos estudos de permeabilidade, foi proposta uma análise de variância three-way para verificar a influência na permeabilidade dos fármacos, das seguintes variáveis: secção intestinal, pH da solução de Ringer e temperatura. Foram determinados a quantidade total de fármaco permeado (Qt), o coeficiente de permeabilidade aparente (Papp) e a constante de absorção de primeira ordem (ka). A partir da análise do planejamento experimental, os efeitos das variáveis não foram significativos, exceto para a secção intestinal. Os resultados de coeficiente de permeabilidade aparente (Papp) obtidos foram de 0,09 x 10-4 cm/s para lamivudina e de 0,22 x 10-4 cm/s para o metoprolol. O valor de Papp obtido de para o metoprolol é próximo dos valores encontrados na literatura para outras técnicas. Para a lamivudina, entretanto, a diferença encontrada em comparação às células Caco-2 pode ser devida às diferentes técnicas empregadas. / This work aimed to propose a new method for studying drug permeability using frog intestinal epithelium (Rana catesbeiana) in ex vivo method, using Franz cells. By using intestinal epithelium, a disposal material from an animal used as human food, can be considered an alternative method, because it doesn\'t involve the sacrifice of animals. The amount of permeated drug was determined by capillary electrophoresis method with UV detection and validated for antiviral drugs lamivudine, zidovudine and acyclovir in the presence of metoprolol and floridizina. The drug chosen as a model in permeability studies was lamivudine. To establish the experimental protocol for the permeability studies, a three-way analysis of variance was proposed to check the influence of intestinal section, pH of Ringer\'s solution and the temperature on the permeability. Total amount of drug permeated (Qt), apparent permeability coefficient (Papp) and first-order constant absorption (ka) were determined. By the analysis of experimental design, the effects of the variables were not significant, except for intestinal section. The results of apparent permeability coefficient (Papp) obtained were 0.09 x 10-4 cm/s for lamivudine and 0.22 x 10-4 cm/s for metoprolol. The value of Papp obtained for metoprolol is quite close to the values found in literature for other methods. For lamivudine, however, the difference found in comparison to Caco-2 cells may be due to different techniques.