O objetivo geral desta pesquisa foi avaliar a fisiologia reprodutiva de Nezara viridula (L, 1758) (Hemiptera: Pentatomidae) e os fatores que influenciam o seu processo reprodutivo, como a frequência de cópula, a associação a bactérias simbiontes e a utilização de recursos nutricionais. Análises da composição bioquímica da hemolinfa durante o processo de maturação reprodutiva das fêmeas indicaram que a concentração de proteína total aumentou gradativamente durante o período de maturação dos ovários, sendo que as proteínas ligadas ao desenvolvimento de oócitos, as vitelogeninas, tornaram-se disponíveis na hemolinfa a partir do décimo dia de idade, período que corresponde à fase de pré-cópula. O desenvolvimento e a maturação de oócitos ocorreram de forma gradativa em função do aumento das proteínas disponíveis na hemolinfa das fêmeas. A cópula não foi essencial para o desenvolvimento dos ovários, indicando a inexistência de estímulos fisiológicos associados à distensão da espermateca ou à transferência de moléculas associadas ao fluído seminal de machos. Entretanto, a freqüência com que as fêmeas copularam afetou a capacidade reprodutiva de N. viridula, sendo as fêmeas que copularam por duas vezes as mais fecundas em relação àquelas que copularam múltiplas vezes. Este fato também indica a existência de custos fisiológicos associados à cópula para fêmeas, sendo ainda evidente a inexistência de relação positiva entre os possíveis benefícios da transferência de nutrientes pelo macho a fêmea e o número de cópulas realizadas. Análises relacionadas à utilização de recursos nutricionais indicaram que o desenvolvimento do ovário de N. viridula é basicamente dependente de nutrientes adquiridos na fase adulta, sendo que a fecundidade de fêmeas oriundas de ninfas criadas em dieta de valor nutricional reduzido, foi recuperada quando adultos foram alimentados em dieta de valor nutricional adequado. Foi verificado ainda uma rica diversidade de bactérias associadas ao aparelho reprodutor masculino de N. viridula, com predominância da Enterobacteriaceae Klebsiella sp, a qual foi anteriormente relatada associada ao intestino de N. viridula. Fêmeas copuladas com machos infectados ou não por estas bactérias não apresentaram qualquer efeito em sua capacidade reprodutiva. / The objective of this research was to evaluate the reproductive physiology of Nezara viridula (L., 1758) (Hemiptera: Pentatomidae) and the factors that influence their reproductive process, such as mating frequency, association to symbiotic bacteria and the use of food resources. Analysis on biochemical composition of haemolymph during the reproductive maturation process of females indicated that the total protein concentration increased gradually during ovary maturation the proteins related to oocyte development, the vitellogenins, became available in the hemolymph from the tenth day of age, corresponding to the pre-copulation. Oocyte development and maturation occurred gradually due to the increase of protein available in hemolymph of females. Copulation was not requered for ovary development, indicating the absence of physiological stimuli associated with spermatheca distension or molecules with on male seminal fluids. However, the frequency females mated affected the reproductive capacity of N. viridula, to females that mated twice were the most fecund if compared to those mated multiple times. This also indicates that there are physiological cost associated with mating frequency, and that there is no positive relationship between the potential benefits from the transfer of nutrients from male to female and the number of mates performed. Analysis related to the use of food resources indicated that ovary development of N. viridula is basically dependent on nutrients acquired during adulthood. Fecundity of females obtained from nymphs reared on a low nutritional value diet was recovered when adults were fed on an adequate-nutritional diet. We further verified a rich diversity of bacteria associated with the male reproductive system of N. viridula, with predominance of the Enterobacteriaceae Klebsiella sp, which was previously reported associated with the gut of N. viridula. Females copulated by males infected or not by these bacteria did not show any effect on their reproductive capacity.

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / The three experiments described below offer support of immune function by the swine gastrointestinal epithelium. Experiment one evaluated mediators that regulate the movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (interleukin 8; IL8), dendritic cells (CC chemokine ligand 20; CCL20) and epithelial remodeling (osteopontin; OPN) in pigs challenged with Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The proximal ileum had greater IL8 expression than the distal ileum (P < 0.05), and ST increased CCL20 (P < 0.05). In vitro, MIF, IL8, CCL20 and OPN mRNA expression induced by lipopolysaccharide (LPS), ST or SC using pig jejunal epithelial cells (IPEC-J2) resulted in increased IL8 secretion, and increased IL8 and CCL20 mRNA by ST and SC (P < 0.05). Experiment two evaluated how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from ST or SC in their ability to regulate, stimulate, or modify IL8, CCL20, and tumor necrosis factor α (TNFα) in IPEC-J2 cells. ST stimulated an increase in IL8 secretion, with increases in IL8 mRNA (P < 0.05). BL increased IL8 mRNA (P < 0.0001). CCL20 mRNA was upregulated by ST (P < 0.05) and BL (P < 0.05). Only ST increased TNFα mRNA (P < 0.05). Another objective evaluated whether pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. IL8 secretion was increased by ST (P < 0.0001), and reduced by LR (P < 0.05). Only the BL/ST co-treated wells blunted basolateral IL8 secretion (P < 0.0001). Experiment three characterized the swine CCL20 mRNA sequence and evaluated tissue expression. Cloning of CCL20 from the porcine jejunum predicted a 97 amino acid peptide. All healthy tissues expressed CCL20 mRNA. In animals challenged with Salmonella spp., SC increased spleen and liver CCL20 expression. The data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected proinflammatory mediators; Salmonella spp. elicited differing patterns of activation in vitro and in vivo; IPEC-J2 cells increased IL-8 secretion in response to ST and BL, but not LR, while ST stimulated secretion was inhibited basolaterally by BL pre-exposure; and numerous porcine tissues are prominent sources CCL20.

Doctor of Philosophy / Department of Anatomy and Physiology / Michael J. Kenney / Important bidirectional interactions exist between the central nervous system and the immune system. Neural-immune interactions provide a regulatory system in the body and disturbances in these interactions may lead to disease. Although the sympathetic nervous system is thought to play a key role in mediating neural-immune interactions, central neural mechanisms mediating sympathetic-immune interactions and the effect of centrally-induced alterations in sympathetic nerve discharge on immune function is not known. We tested the hypothesis that central activation of sympathetic neural circuits alters splenic cytokine gene expression. In a separate study, we tested the hypothesis that hypothermia-induced changes in visceral sympathetic nerve discharge (SND) would be attenuated in middle-aged and aged compared with young rats. Previous studies have demonstrated that skin sympathoexcitatory responses to skin cooling are attenuated in aged compared with young subjects, suggesting that advancing age influences sympathetic nerve responsiveness to cooling. The effect of age on sympathetic nerves innervating other targets organs during acute cooling remains unknown. Central activation of splenic SND was produced using three different experimental interventions: increased core body temperature produced by acute heating, intracerebroventricular injection of angiotensin II (ANGII), and decreased core body temperature produced by acute cooling. Changes in gene expression profiles were analyzed using inflammatory cytokine-specific gene-array and further validated using real-time RT-PCR analysis. The following observations were made. 1)Splenic SNDincreased in response to each experimental intervention except in acute cooled young rats where there was a decrease in splenic SND. 2) Splenic cytokine gene expression of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-2) and chemokines (GRO1, CXCL2, CCCL2 and, CXCL10) was increased in response to each experimental intervention. 3) Expression of splenic cytokine genes was reduced after splenic-denervation except in acute cooled rats. 4) Progressive hypothermia reduced splenic, renal, and adrenal SND in rats and was generally attenuated in middle-aged and aged rats. These results demonstrate the functional significance of changes in sympathetic nerve activity on splenic immune cell activation and the effect of age on SND responses to core body cooling.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The goal of this project was to define mechanisms responsible for Na+ transport in two hormonally-sensitive epithelium, the bovine mammary gland and porcine vas deferens. Glucocorticoid stimulation in these epithelia results in a significant increase in amiloride-sensitive ion transport, suggesting regulation of the epithelial Na+ channel, ENaC. ENaC has typically been described as a heteromultimeric ion channel with at least three different types of subunits, the most common being , β, and γ. Glucocorticoid-induced regulation of these subunits at the transcriptional level appears to be very different in the porcine vas deferens as compared to the bovine mammary gland. The aims of the study in mammary epithelium were to elucidate the mechanisms by which apical electrolytes and cytokines compromise barrier function in mammary epithelium. The long term goal is to better understand and manage the interaction between ionic composition of milk and breakdown of the gland epithelium that occurs during mastitis. Our results suggest a causal link between changes in milk electrical conductivity and epithelial barrier breakdown that has not been appreciated previously. Results will provide benefits to dairy farmers by characterizing steps that might prevent the development of mastitis or hasten recovery. The aims of the study using porcine vas deferens epithelial cells include determining the time course, concentration- and structure-dependency for regulation of amiloride-sensitive ion flux by corticosteroids. Corticosteroids caused a concentration-dependent increase in amiloride-sensitive Isc with a rank order of potency of dexamethasone>prednisolone>cortisol. Hill analysis indicates steep concentration dependency. The corticosteroid-induced, amiloride-sensitive current is Na+ absorption as indicated by radiotracer flux measurements. Studies employing selective antagonists (spironolactone, mifepristone) define glucocorticoid receptor mediation. These results suggest that vas deferens epithelia are exquisitely sensitive to corticosteroid exposure. Observed changes in epithelial function in response to corticosteroid exposure would rapidly and chronically affect the luminal environment to which sperm are exposed. Thus, physiological and pharmacological corticosteroid exposure is expected to affect male fertility.

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A reprodução é custosa para ambos os sexos, mas a magnitude dos gastos e sua relação com o sucesso reprodutivo diferem entre os gêneros. Os custos reprodutivos são divididos em duas categorias: custos de sobrevivência e custos energéticos. Crotalus durissus possui um ciclo reprodutivo sazonal com cópula ocorrendo no outono e a parturição no final no verão. Os machos competem por fêmeas receptivas. A inferência de custos reprodutivos associados à sobrevivência foi realizada por meio de levantamentos das taxas de atividade entre machos e fêmeas. A variação da quantidade de gordura abdominal e dos substratos energéticos do fígado e dos rins foi avaliada para mensurar o custo energético. Os resultados sugerem que para fêmeas a reprodução exige um alto investimento energético evidenciado pelos maiores níveis de gordura abdominal e de lipídios no fígado durante a fase vitelogênica e o padrão de atividade diferenciada entre machos e fêmeas sugere que a estação reprodutiva embute um custo de sobrevivência maior para os machos. / Reproduction is costly for both sexes, but the magnitude of spending and its relation to reproductive success differ between genders. Reproductive costs are divided into two categories: survival costs and energy costs. Crotalus durissus has a seasonal reproductive cycle with mating occurring in the fall and parturition in late summer. The males compete for receptive females. The inference of survival costs was accomplished through surveys of activity rates between males and females. The variation of the amount of abdominal fat and energy substrates in liver and kidneys was evaluated to measure the energy cost. The results suggest that for females reproduction requires a high energy investment - evidenced by the higher levels of abdominal fat and lipids in the liver during vitellogenic phase and activity patterns differentiated between males and females suggests that the reproductive season embeds a higher cost of survival for males.

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No description available.

In this dissertation, I explored the effects of developmental hypoxia on heart contractility in three separate species of ectotherms: the common snapping turtle (Chelydra serpentina), the American alligator (Alligator mississippiensis), and the zebrafish (Danio rerio). I began with the common snapping turtle and tested whether the utilization of the sarcoplasmic reticulum was altered in response to developmental hypoxia. In the next two chapters, developmental hypoxia of the American alligator was explored studying how the cardiac tissue was affected, specifically in physiological stressors, sarcoplasmic reticulum utilization and sensitivity to pharmacological increases in contractility. The last chapter explored how zebrafish heart contractility was altered in response to chronic hypoxia from egg to adult. Findings from these chapters suggest that while developmental hypoxia did alter cardiac contractility, it did not alter the response of the heart to physiological stressors such as increased heart rate or under hypoxia. Overall, these findings contribute to increasing the current understanding of how developmental hypoxia alters the cardiovascular system but with an emphasis on the cardiac tissue level.

Achievement of endothernic capacity is vital for independence from ambient temperature changes, sustained activity, optimal biochemical reactions and optimization of parental care. During early avian development, the core tenets of transition from ectothermy to endothermy are development of metabolic capacity (oxygen consumption, mitochondrial bioenergetics), enhanced cardiovascular function (heart rate and cardiac output), pulmonary ventilation and thermogenic capacity. Thyroid hormones, particularly T3, are key metabolic regulators of basal metabolism, thermogenesis, pulmonary ventilation and mitochondrial respiration. Thyroid hormone fluctuation patterns during both precocial and altricial avian endothermic transition suggest a prominent role in maturation of endothermy, cardiovascular, respiratory and skeletal muscle physiology. This body of work explores effects of T3 manipulations in two avian species: the precocial Pekin duck and the altricial Red-winged Blackbird. Increased plasma T3 during late incubation resulted in increased cardiac mass, elevated resting and intrinsic heart rate, intrinsic mean arterial pressure, increased cholinergic tone and blunted alpha-adrenergic tone in the precocial Pekin duck. In both Pekin duck and Red-winged blackbird, plasma T3 levels correlated with changes in the trajectory of endothermic ontogeny, systemic oxygen consumption, thermogenesis, maturation of pulmonary ventilatory function, altered growth and effects on skeletal and cardiac mitochondrial bioenergetics. These observations support the role of thyroid hormones as metabolic and developmental regulators at the time of attainment of endothermy during the perinatal period in precocial and altricial avian species. Insights into the role of thyroid hormone as a metabolic and development regulator at the time of avian endothermic attainment provide a more thorough understanding of metabolic and physical transitions a hatchling bird must undergo to reach the adult endothermic phenotype. Such insights also deepen understanding of the complex role thyroid hormones play in homeostasis and offer implications about the evolutionary history of endothermic capacity.