Two types of ventilation cycle were recorded in unanaesthetised but restrained frogs (Rana pipiens); one concerned with ventilation of the buccal cavity alone (buccal cycle) and the other with lung ventilation (lung cycle). During the former the nares were open and the glottis closed so that only small pressures were generated by the movement of the buccal floor. The onset of a lung ventilation was signalled by activity in the laryngeal dilator muscle and when the glottis opened lung pressure and volume fell while buccal cavity pressure and volume increased. After narial closure the buccal floor was rapidly raised and gas was forced into the lungs from the buccal cavity. At peak pressure in the lungs and buccal cavity the glottis closed and nares opened, the recovery stroke of the pump being passive. Air flow recordings made at the external nares showed two phases of flow during each buccal cycle, while four phases accompanied each lung ventilation cycle. By plotting pressure/volume loops from the buccal pump an analysis was made of the mechanical work performed in one lung ventilation cycle, and the proportion of this work available for lung inflation after various losses against viscous and flow resistive forces in the pump itself; while measurement of the areas of typical sequences of such loops together with respiratory frequency enabled the mechanical work output of the pump to be determined for frogs ranging in size from 24 to 86 grams. Using Hill's classical equation for muscle efficiency, it was possible to estimate mechanical efficiency for single respiratory cycles by calculating the heat of maintenance and heat of shortening of the buccal floor muscles, while simultaneously measuring mechanical work output. Calculated efficiencies of lung ventilation cycles rose as mechanical work performed increased from 7.4% at 0.65 gram.cm/cycle to 19.3% at 2.73 gram.cm/cycle. Diving apnoea in Rana pipiens was induced by the presence of water at the level of the external nares, at which point the nares closed, no water entering the buccal cavity during the dive. Occasional ventilation cycles occurred during the dive in which gas entered the buccal cavity from the lungs, an equal volume then being pumped back into the lungs, but there was no ventilatory exchange with the external medium. Bilateral section of the trigeminal nerves resulted in an abnormal response to submergence, in that water entered the buccal cavity, and in some cases the lungs, while surfacing often did not result in resumption of ventilation. Skin mechanoreceptors in the region of the external narial openings serving the ophthalmic branch of the trigeminal were found to be capable of responding to the minimum stimulus encountered on submersion, movement of a water meniscus across the narial region, while a tonic response to hydrostatic pressure occurred in some preparations. In control experiments cutaneous mechanoreceptors innervated by the spinal nerves were shown to have no response to a water meniscus passing across their receptive fields, suggesting that they possess higher thresholds than the narial receptors. Periods of apnoea could be induced in air in Rana pipiens by bilateral or unilateral stimulation of the cut peripheral ends of the ophthalmic branch of the trigeminal nerve at threshold voltages as low as 30 mv, at a frequency of 200 Hz. Increase in stimulating voltage resulted in longer periods of apnoea before ventilation "broke through", and in these periods the external nares were closed and buccal pressure was held independent of atmospheric pressure. Reduction of the stimulation frequency by a factor of ten after the initiation of apnoea, simulating adaptation of the sensory nerves, proved as effective in maintaining apnoea as continued stimulation at the original frequency. / Science, Faculty of / Zoology, Department of / Graduate

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Quantitative relationships between blood flow velocity, vessel diameter, width of the peripheral plasma layer and induction of thrombus formation were studied in small blood vessels of the retrolingual membrane and intestinal mesentery of the frog, Rana Pipiens, and of the cheek pouch of the golden hamster, Mesocricetus auratus. Blood flow velocity was measured by a modification of the technique described by Hugues (Arch. Int. de Physiol. 61: 565, 1953). Internal vessel diameters and widths of the total peripheral plasma layer were measured with an ocular micrometer. Thrombus thresholds were determined by graded electrical stimulation. Determinations were made on 202 vessels in retrolingual membranes of frogs prepared by single pithing. In 95 arterioles (diameters from 10 to 40 microns), the mean flow velocity was 2.84 mm/sec. and the mean volume flow rate, 1.68xl0-3 cu mm/sec. The mean width of the total peripheral plasma layer was 3.6 microns and its ratio to mean vessel diameter was 1:6. In 107 venules (diameters from 10 to 50 microns), the mean flow rate was 1.20 mm/sec. The mean volume flow rate was 1.02x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 4.1 microns and its ratio to mean vessel diameter was 1:7.4. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.3 and 1:1.6 respectively. Measurements were made on 100 mesenteric vessels of frogs prepared by single pithing. In 50 arterioles (diameters from 15 to 45 microns), the mean velocity was 3.77 mm/sec. The mean volume flow was 3.05xlo-3 cu mm/sec. The mean width of the total peripheral plasma layer was 2. 7 microns and its ratio to mean vessel diameter was 1:11.5. In 50 venules (diameters from 20 to 50 microns), the mean velocity was 1.55 mm/sec. and the mean volume flow rate, 1.76x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 6.7 microns and its ratio to mean vessel diameter was 1:5.3. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.3 and 1:1.7 respectively. A total of 109 mesenteric vessels were studied in frogs anesthetized with urethane. In 55 arterioles (diameters from 15 to 45 microns), the mean velocity was 3.28 mm/sec. The mean volume flow was 2.87x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 3.2 microns and its ratio to mean vessel diameter was 1:9.3. In 54 venules (diameters from 20 to 50 microns), the mean flow rate was 1.61 mm/sec. and the mean volume flow, 1.83x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 5.6 microns and its ratio to mean vessel diameter was 1:5.9. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.0 and 1:1.5 respectively. In these three series of studies in the frog, no relationship was clearly apparent between velocity and either vessel diameter or width of the peripheral plasma layer in arterioles. Suggestions of a direct relationship between velocity and peripheral plasma layer in veins, however, were evident. In all cases, and in both arterioles and venules, vessel diameter and peripheral plasma layer were clearly and directly related. The effects of flow velocity changes on width of the total peripheral plasma layer in individual vessels were studied in 26 arterioles of the hamster cheek pouch. Blood flow was varied by means of a cuff described by Copley (Biorheology 1: 3, 1962). A direct relationship between velocity and width of the peripheral plasma layer was clearly demonstrated. Thrombus thresholds were determined in mesenteric vessels of the frog. In 103 arterioles (diameters from 20 to 140 microns), the mean strength of stimulus necessary to produce a platelet thrombus was 24.8 volts with an amperage of 0.18 milliamperes. In 100 venules (diameters from 20 to 140 microns), the mean strength of stimulus necessary was 20.7 volts with an amperage of 0.12 milliamperes. Possible relationships of thrombus thresholds to flow velocity in mesenteric arterioles and venules are discussed. / 2031-01-01

A primary goal of population genetics is to identify the role of microevolutionary forces in producing observed patterns of molecular and phenotypic variation. I conducted four studies in the northern leopard frog, Rana pipiens, to determine just how mutation, migration, genetic drift, and selection influenced, genetic structure of mitochondrial DNA (mtDNA), nuclear DNA, and a single locus polymorphism that determines dorsal coloration. In the first study, I surveyed the literature concerning color and pattern polymorphisms in anurans. I conclude that anuran polymorphisms remain a rich but largely unexploited system for studying the evolution of phenotypic variation in nature. In the second study, I compared mitochondrial DNA variation from 35 populations distributed across the species' range. A phylogenetic analysis indicated R. pipiens is split into two deeply divergent mtDNA groups, a western group and an eastern group. Phylogeographic and demographic analyses indicated that although restricted gene flow with isolation by distance explained the majority of the processes influencing current genetic structure, population bottlenecks and expansions also played an important role. In the third study, I investigated mtDNA and microsatellite variation in Pacific Northwest populations of R. pipiens, where a recent range contraction had occurred. I found that peripheral populations had reduced levels of genetic variation compared to more interior populations. Moreover, I found that historic samples from peripheral population already had reduced levels of genetic variation. Therefore, low diversity in the remnant populations could not be ascribed to the recent range contraction. In the fourth study, I compared genetic structure from a suite of putatively neutral molecular markers with that derived from the color polymorphism locus. Genetic structure at the color locus, assessed both spatially and temporally, was indistinguishable from structure at neutral loci. This study exemplifies the importance of investigating for evidence of selective maintenance before studies attempt to measure the selective mechanisms maintaining a polymorphism. Overall, my research helps to elucidate how biogeographic and microevolutionary forces influence a wide-spread North American species, R. pipiens. / Graduation date: 2004

Emerging infectious diseases, pollution and climate change are associated with amphibian extinction events worldwide; however, direct causation is often obscured by the interactions of these stressors. Elucidating the possible synergies between metal contamination and disease is, therefore, critical in advancing our knowledge of the co-evolutionary mechanisms in host-pathogen systems and helping with the ability to better forecast the spread of diseases in metal-stressed environments. Additionally, increasing ecotoxicological research has improved our understanding of the complex influence trace metals may have on the physico-chemical nature of aquatic systems; however, the discrepancy in concentration-response within the toxicological literature makes it difficult to accurately define the range of toxicity, often due to the variability in media used in experimentation. The first chapter of this thesis reports an evaluation of copper, nickel and copper/nickel concentrations on several Northern Leopard Frog (Lithobates [Rana] pipiens) larvae life history traits within field collected smelting effluent water. Overall, results indicated that copper had a stronger negative impact on survival than nickel. However, tadpoles exposed to copper displayed increased growth and developmental patterns while those exposed to nickel demonstrated opposing life history traits. These results indicate that tadpoles are displaying different fitness strategies, in terms of survival and life history, in the presence of increased copper and/or nickel stress.

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.

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.

<p>Perfluoroalkyl substances are synthetic organic chemicals of environmental concern because they have been associated with adverse effects in both human epidemiological studies and standard laboratory animals. In the environment, PFAS occur as mixtures, especially in areas with a history of PFAS application, such as aqueous film forming foam (AFFF) sites. Among the PFAS, perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) are the most common, and occur at the highest concentrations. Thus, amphibian populations at or near AFFF sites are at risk of exposure to known bioaccumulative and persistent chemicals, likely compromising the physiology and body condition of the animals. Here, we exposed northern leopard frogs to environmentally relevant concentrations of 0.5 and 1 ppb PFOS and PFHxS, alone or as a mixture comprised of 0.5 ppb PFOS and 0.5 ppb PFHxS. Univariate analyses showed that in the larval stages, tadpoles exposed to PFAS had significantly reduced scaled mass indexes (SMI’s) relative to the control, and only the organisms exposed to PFHxS 0.5 ppb were significantly larger. Sex did not significantly influence toxicity in the later stages (GS 42 & 46), indicating no sex-related effects. Altered body condition (i.e., fat stores) in the larval stages indicate potential effects to energy balance. There is a need to assess fitness-related effects as amphibians’ transition into the terrestrial environment, and include endpoints such as: reproductive, developmental, immunological, mating, feeding, competition, and survival. Early developmental effects in the larval stages also suggests that earlier developmental endpoints may be of interest. Establishing ecological risk assessments for PFAS are necessary, as they are toxic, persistent, and bioaccumulative.</p>

Despite a thorough understanding of the proximate mechanisms that drive genetic diversity, we are still very poor at predicting the genetic diversity of natural populations. Understanding patterns of genetic diversity is important for many reasons, including predicting species' adaptation to climate change and predicting the spread of invasive species, but it is particularly important for species that are declining. This dissertation attempts to explain patterns in genetic diversity at multiple spatial scales across the range of the Northern Leopard Frog, Rana pipiens, which is declining across large portions of its range. Genetic diversity is often lower in edge populations than in central populations. Genetic diversity may be reduced in edge populations per se, or populations that occur at the edge of the species' range may have low diversity because they have recently expanded into new habitat and thus show signs of founder effects. In Chapter 2, we tested several alternative hypotheses to explain genetic diversity across the species' range, and to explain why some edge populations may not show reduced genetic diversity. We found that genetic diversity was reduced in edge populations relative to central populations, but was not reduced in populations in previously glaciated areas relative to previously unglaciated areas; therefore position at range edge had a stronger effect in reducing diversity than recent colonization of new habitat. We found that genetic diversity declined linearly towards the range edge in one of two transects from range center to range edge. We concluded that genetic diversity in this species is generally reduced by position at the range edge, but that this effect may differ among edges. In Chapter 3, we tested the hypothesis that eastern and western populations were genetically distinct. We found two distinct clades that introgress in some markers but are distinct and defined by narrow boundaries in the eastern Great Lakes region in others. We concluded that genetic diversity in the Mississippi River region was elevated by the introgression of descendants from two Pleistocene refugia. In Chapter 4, we analyzed genetic diversity within populations throughout Arizona to assess potential source populations for reintroductions. We also analyzed mitochondrial DNA to determine whether any populations contained genetic material not native to the region. Populations in one area had high genetic diversity and high gene flow among populations, but also contained evidence of introduction of eastern frogs. We conclude that supplementing genetic diversity in other populations with translocations from this area is not recommended.

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