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G protein-coupled receptors (GPCRs) are composed of seven transmembrane domains and play an essential role in regulating physiological functions and mediating responses to environmental stimuli, biogenic amines, neurotransmitters, peptides, lipids, and hormones. The calcitonin-like diuretic hormone 31 (DH31) is known to elicit natriuresis from the Malpighian tubules (MTs) of mosquitoes Anopheles gambiae and Aedes aegypti upon blood feeding. However, the contribution of DH31 cognate receptor, calcitonin receptor 1 (GPRCAL1), has not been evaluated with respect to postprandial fluid regulation or myostimulatory activity in blood feeding insects. Thus, this dissertation has investigated potential roles of AedaeGPRCAL1 in the regulation of fluid homeostasis and hindgut muscle contraction in female A. aegypti mosquito. The full length cDNA encoding AedaeGPRCAL1 was cloned and sequenced. The receptor expression in the MTs and hindgut from female mosquito was analyzed by western blot and immunohistochemistry using anti-AedaeGPRCAL1 affinity purified antibodies, and subsequently its role in fluid transport and hindgut contraction was evaluated by RNA interference (RNAi). The mosquitoes that underwent knock-down of the AedaeGPRcal1 exhibited up to 57% lower rate of MT fluid secretion in presence of Aedae-DH31 in the in vitro assay and a ~30% reduction in the fluid excreted from live females upon blood feeding. The receptor was immunolocalized in principal cells, predominantly towards the distal end of MTs. Analyses of receptor signal probability indicate the receptor is expressed in a gradient-like fashion along the length of the MTs. A striking discovery was the fact that not all principal cells express the receptor, contrary to previous belief. Immunolocalization revealed the AedaeGPRCAL1 is expressed in hindgut circular and longitudinal muscles. The application of DH31 increased the frequency of hindgut contractions in all female mosquitoes, those injected with AedaeGPRcal1 dsRNA and controls, as compared to their basal contraction rate, but the percent change in frequency of hindgut contraction from AedaeGPRcal1 knock-down females was about 2-fold lower than the controls after application of Aedae-DH31. To my knowledge, this is first evidence of RNAi-induced phenotypes in any invertebrate that allowed the quantification of the contribution of single family B GPCR to fluid loss and muscle contractility.

Master of Science / Animal Sciences and Industry / Teresa L. Douthit / Abrupt dietary changes increase a horse’s risk for developing gastrointestinal diseases, such as colic or laminitis. Understanding the impact of various feeds and feeding practices on feeding behavior and gastrointestinal function creates a whole-animal perspective that allows for a more holistic interpretation of the effects of abrupt dietary changes on the hindgut environment. Unfortunately, few reports exist that have examined the effects of abrupt dietary changes in the horse. This study was designed to determine the effects of various abrupt dietary changes on the hindgut environment. In 4 sequential experiments, horses were exposed to an abrupt change from a baseline ration to a complete pelleted diet, an abrupt change from a baseline ration to a 100% grass hay diet, an abrupt change from a prairie hay ration to an alfalfa hay ration, and an abrupt change from a baseline ration to a large concentrate meal. These dietary challenges were chosen to mimic real-world scenarios that horse owners are likely to encounter. These experiments were arranged into a longitudinal trial in which the effects of the abrupt dietary change on cecal and fecal pH, total lactate and volatile fatty acid (VFA) concentrations, cecal lactate-utilizing bacterial populations, and fecal dry matter (DM) were compared to values obtained while horses were consuming the baseline diet. In the first experiment, decreased cecal (P ˂ 0.0001) and fecal (P ˂ 0.0001) pH values combined with increased cecal total lactate (P ˂ 0.001) and fecal VFA concentrations (P ˂ 0.0001) indicate that the abrupt change to a complete pelleted diet disrupted the stability of the hindgut environment. Because cecal pH values were below 6.0, this dietary challenge may be significant enough to elicit subclinical fermentative acidosis and, thereby, increase colic risk. The dietary change to grass hay had little impact on the hindgut environment, as pH, total lactate, and VFA concentrations remained stable (P ≥ 0.05). In general, horses may well tolerate an abrupt increase in the fibrous component of the diet and the elimination of concentrate, a dietary shift that presents a more natural diet to the horse. The abrupt change to alfalfa hay elicited alterations in cecal pH (P ˂ 0.01), total lactate (P ˂ 0.0001) and VFA concentrations (P ˂ 0.05), and lactate-utilizing bacterial populations; however, fecal parameters varied little in response to the dietary change (P ≥ 0.05), indicating that the distal hindgut may be more tolerant to abrupt changes in forage sources than the cecal environment. Here, the potentially adverse shifts in cecal parameters indicate that an abrupt change in hay type and quality alters the fermentative environment of the proximal hindgut and may increase a horse’s risk for gastrointestinal disease. Similarly, the abrupt introduction of a large concentrate meal elicited a decrease in cecal pH (P ˂ 0.005) along with increases in total lactate (P ˂ 0.001) and VFA concentrations (P ˂ 0.05) in the cecum that were consistent with previously reported experiments in which horses were presented with large increases in dietary concentrates. Notable shifts in lactate-utilizing bacterial growth curves were also observed. Overall, these results provide evidence of environmental alterations in the equine hindgut that support epidemiological reports that associate abrupt changes in the amount and type of concentrate, hay type and quality, and forage:concentrate ratio with increased risk for gastrointestinal disease in horses.

Background: Rectal hyposensitivity (RH) is associated with functional hindgut disorders. It is hypothesized to involve afferent pathway dysfunction. However, little is known regarding its clinical impact. Aims: To assess whether RH is: • clinically important and associated with specific symptoms; • secondary to afferent neuronal dysfunction; and • primarily a pelvic abnormality. Methods: Epidemiological studies were conducted: (1) a case-controlled study stratified by sensory status, assessing symptoms of constipation and incontinence, health status and quality of life; (2) an observational study exploring RH in faecal incontinence in men; (3) an observational study examining the impact of RH on defaecatory urge. Pathophysiological studies were also conducted: (1) transmission of visceral sensory information was evaluated using rectal evoked potentials;(2) somatic sensory function and visceral efferent function were examined in patients with and without RH. Results: RH is associated with constipation. Patients with RH have more severe symptoms and worse health status and quality of life. Constipated patients report altered defaecatory urge compared to controls, most notably in those with RH. RH is associated with concurrent constipation and evacuatory dysfunction in males with incontinence. Patients with RH have delayed evoked potential latencies, without alteration of cortical activation. A proportion have elevated somatic sensory thresholds although efferent function is similar between groups. Conclusions: 1. In patients with constipation, those with RH have a worse clinical phenotype, with poorer health status and quality of life. Patients with constipation, (particularly those with RH), have alteration of defaecatory urge. 2. RH and constipation may contribute to incontinence in males where sphincter dysfunction is less important. 3. RH is associated with delayed afferent transmission indicating primary afferent pathway dysfunction. In a proportion, reflecting a possible generalised sensory neuropathy. These studies confirm that intact rectal sensation is fundamental to normal hindgut function. Impaired visceral sensation is thus an important therapeutic target.

Made available in DSpace on 2017-07-10T14:17:03Z (GMT). No. of bitstreams: 1 sostenez.pdf: 5444547 bytes, checksum: 0e57c73e9d81ba62bd8cce82698d43f6 (MD5) Previous issue date: 2013-10-17 / Bombyx mori nucleopolyhedrovirus (BmNPV) is an entomopathogenic and poliorganotrophic virus that infects Bombyx mori. BmNPV consists of a double strand DNA with two distinct phenotypes: the derived polyhedral virus (PDV), responsible for the primary infection in the insect s midgut; and the budded virus (BV), which disperses the infection in the hemocele, causing secondary or systemic infection. It is extremely virulent and when it infects silkworms, causes serious damages to the insect, usually leading to its death or harming the silk production, affecting all the productive chain, leading to economic losses. For a good silk production, a determinant factor is the functioning of the food duct, which is divided in foregut, midgut and hindgut, being the hindgut the place for water and mineral salts absorption and for the end of the digestive process. Several tissues have been established as virus targets, however, others have shown to be resilient to BmNPV. Given the importance of the hindgut, the present paper aimed to verify the susceptibility of its components, ileum, colon and rectum to an geographic isolated of BmNPV in Paraná, Brasil, the BmMNPV. In different post-inoculation days (dpi), the hindgut segment of 5th instar silkworms was dissected and processed for analysis in light microscopy, using conventional dyes and cytochemistry for viral detection and electronic scanning microscopy for the morphological details. The results revealed that the ileum, colon and rectum of the B. mori are constituted by simple epithelium, with alterations in cell morphology, covered by an intima in its luminal side, and that its organization is similar to that of other described insects. The cytological analysis of the ileum, colon and fore rectum revealed that its epithelial cells are not susceptible to BmMNPV in neither of the times analyzed. However, the posterior hindgut or anal duct showed itself to be susceptible to the virus after the 5º dpi, developing all the classic signs described for the infection with AlphaBV, as the presence of viroplasm, nuclear hypertrophy, polyhedra in formation and mature ones. At the end of the infectious cycle, occurs cell lysis with the liberation of viral polyhedra in the intestinal lumen and, consequently, to the external medium, coinciding with the death of the insect. Even with no infection in the other regions of the hindgut, the surrounding tissues have shown infected, affecting the normal functioning of this intestinal region, verified through changes in fecal pellet, which was less compact and changed format. These results will contribute in the establishment of the BmMNPV infectious cycle. Furthermore, the basic knowledge of viral behavior is important for the development of infection control, prevention and previous identification methods of this disease in the field, once it also makes possible the removal of infected silkworms, diminishing the horizontal transmission of the virus in the creation rooms, in a way to reduce the loss of cocoons to be used in the confection of silk yarn / Bombyx mori nucleopolyhedrovirus multiple (BmMNPV) é um vírus entomopatogênico e poliorganotrófico, constituído por DNA fita dupla e apresenta dois fenótipos distintos: o vírus poliédrico derivado, responsável pela infecção primária no intestino médio do inseto; e o vírus broto, que se dispersa na hemocele causando a infecção secundária ou sistêmica. É extremamente virulento e quando infecta lagartas do bicho-da-seda, causa sérios danos ao inseto, geralmente levando-o à morte ou prejudicando a produção da seda, comprometendo toda a sua cadeia produtiva e gerando prejuízos econômicos. Para uma boa produção de seda, um fator determinante é o funcionamento do canal alimentar, que nos insetos é dividido em anterior, médio e posterior, sendo o posterior, local de absorção de água e sais minerais e da finalização do processo digestório. Vários tecidos já foram estabelecidos como alvos do BmMNPV, entretanto outros se mostraram resistentes. Dada a importância do intestino posterior, o presente trabalho objetivou verificar a susceptibilidade de seus componentes, íleo, cólon e reto de lagartas de B. mori do 5° instar ao BmMNPV isolado geográfico do Paraná. Em diferentes dias pós-inoculação (dpi), o intestino posterior foi dissecado e processado para análise em microscopia de luz, utilizando colorações convencionais e citoquímica para detecção viral, e microscopia eletrônica de varredura, para os detalhes morfológicos. Os resultados revelaram que o íleo, cólon e reto de B. mori apresentou morfologia semelhante a de outros lepidópteros. A análise citopatológica do íleo, cólon e o reto anterior revelou que suas células epiteliais não são susceptíveis ao BmMNPV, em nenhum dos tempos analisados. Já o reto posterior ou canal anal, mostrou-se susceptível ao vírus a partir do 5º dpi, desenvolvendo todos os sinais clássicos descritos para a infecção com o AlphaBV, como presença de viroplasma, hipertrofia nuclear, poliedros em formação e maduros. No final do ciclo infeccioso, ocorre a lise celular com a liberação dos poliedros virais para luz intestinal e, consequentemente, para o meio externo, coincidindo com a morte do inseto. Mesmo não havendo infecção nas demais regiões do intestino posterior, os tecidos circunvizinhos se mostraram infectados, o que possivelmente afetou o funcionamento normal desta região, sendo visíveis as modificações na formação do pellet fecal, que se mostrou menos compacto e com alterações no formato. Os resultados obtidos irão contribuir no estabelecimento do ciclo infeccioso deste patógeno. Além disso, o conhecimento básico do comportamento viral é importante para o desenvolvimento de métodos de controle da infecção, prevenção e identificação prévia desta doença no campo, pois, possibilita ainda, a retirada de lagartas infectadas, diminuindo a transmissão horizontal do vírus nos barracões de criação, de forma a reduzir a perda de casulos, a serem utilizados na confecção dos fios de seda

Serotonin (5-hydroxytryptamine, 5-HT) has long been associated with the vertebrate gut and is an important neuromodulator of crustacean foregut. This dissertation presents evidence that 5-HT initiated peristalsis in crayfish hindgut and enhanced the power of contractions in caudal regions of the hindgut. 5-HT receptor immunoreactivity studies showed that the two identified crustacean 5-HT receptors, 5-HT1α and 5-HT2β are present on the hindgut in different and distinctive patterns. 5-HT immunoreactivity (5-HT-ir) studies revealed that the fibers from central neurons found on the hindgut showed a broad range of 5-HT-ir intensity, which led to the hypothesis that they borrowed 5-HT. This hypothesis was tested by first determining that the HGNs can take up 5-HT through a serotonin transporter and that uptake can be blocked by a serotonin reuptake inhibitor. Second, synthesis was tested by superfusing tryptophan and using 5-HT-ir to determine the presence of 5-HT. No constitutive 5-HT synthesis occurred under these conditions. Superfusion of the intermediate product of 5-HT synthesis, 5-hydroxytryptophan (5-HTP), did lead to 5-HT-ir. The HGNs can take up 5-HT but have only one of the synthetic enzymes. The lack of nearby sources for 5-HT led to the hypothesis that hormonally supplied 5-HT may be the source for 5-HT in the HGNs. High performance liquid chromatography measurements of 5-HT and 5-HTP levels in tissue following injection of 5-HT into the hemolymph revealed that levels of 5-HT significantly increased in the terminal ganglion and hindgut, where the HGNs cell bodies and projections are respectively located. All other areas of the central nervous system, with the exception of the brain, also showed a significant increase in 5-HT levels. Injection of tryptophan produced a significant increase in 5-HTP levels in the brain. Quantitative 5-HT-ir indicated that feeding increased the intensity of 5-HT-ir in the HGNs. Feeding was determined to be a relevant stimulus to examine facultative synthesis of 5-HT. The enzyme that converts 5-HT to 5-HTP was blocked and 48 hrs after feeding 5-HTP-ir was used to indicate that facultative synthesis did not occur. At the same time, 5-HT-ir was used to indicate that uptake of 5-HT by the HGNs more likely occurred.

The overall aim of this study was to investigate the potential influence of chitosan, a biodegradable and antimicrobial compound, on termite hindgut symbionts. For this purpose, a morphological quantifying technique was conducted on the protist community’s hindgut after feeding termites on chitosan-treated wood. The aim was to characterize the diversity of protist species in the economically important dark southern subterranean termite, Reticulitermes virginicus. A molecular phylogenetic analysis of the V3 and V4 hyper-variable regions of 16S ribosomal RNA (rRNA) gene of the bacterial community in the hindgut of R. virginicus was performed on termites exposed to chitosan treatment. Light microscopy visualization of protist species residing in the hindgut of workers showed presence of ten protist species both in the control sample and in termites fed a low concentration of chitosan. In this study, the coexistence of two species of the genus Trichonympha (T. agilis and T. burlesquei) is reported for the first time in R. virginicus. Monocercomonas sp. and Trichomitus trypanoides were the only two protists found in termites exposed to wood treated with higher chitosan concentration solutions and the absence of wood fragments in their food vacuoles was clear. This feature indicates that these two protists may not be involved in the digestion of the wood fragments impregnated with chitosan. The results of this study indicated that the potential effect of chitosan caused elimination of the protist species in termite hindguts. The genomic DNA of bacterial hindgut community of R. virginicus were profiled using sequences which amplified theV3-V4 sub-regions of 16S rRNA gene. Sequences were analyzed using a taxonomic analysis tool, Quantitative Insights Into Microbial Ecology (OIIME 2), in order to infer the effect of chitosan on the composition of the bacterial fauna in the hindgut. The richness and evenness results indicated that the most diversity was observed in the bacteria from termites not being exposed (UNX) to treatment compared to other treatment groups. On the other hand, the lowest richness and evenness were determined for chitosan-treated wood (CTE) and starved termites (STV). Of 28 bacterial phyla, Bacteroidetes, Firmicutes, Elusimicrobia, and Proteobacteria were the most dominant phyla across all the treatment groups. The results suggest that chitosan treated wood led to the microbial community shifts in R. virginicus. In addition, lack of a nutrition source and other changes in termite’s food affect the termite hindgut bacterial diversity.

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