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The small hive beetle (Aethina tumida) is an invasive pest from sub-Saharan Africa that has posed increasing threats to European honey bee (Apis mellifera) colonies in the United States over the past two decades. While control has been attempted, consistently effective management strategies still not been developed. This study sought to explore novel experimental methods to better understand and use A. tumida biology to target this pest. One aspect of A. tumida biology that has emerged as potential basis for improved control is olfactory manipulation, which could be used to disrupt beetles as they seek out A. mellifera colonies. Through olfactometry and electroantennography, key volatiles in A. tumida attraction and repulsion were tested and sensitivity of A. tumida to several attractants and repellents was quantified on behavioral and physiological levels. An additional source of attractive volatiles is the A. tumida fungal symbiont Kodamaea ohmeri, which ferments larval waste and is present throughout the A. tumida lifecycle, both externally and in the GI tract. This study explored the development of feeding and soil bioassays to test the effects of several insecticides on A. tumida larvae. Feeding and injection bioassays were also used to deliver a fungicide with the goal of repressing K. ohmeri, which was expected to detrimentally impact A. tumida health. The results of this work enhance our current knowledge or A. tumida biology and provide a useful basis for development of safe and selective management A. tumida management options for the future. / Doctor of Philosophy / The small hive beetle is an invasive European honey bee pest that poses a significant threat to apiaries in the United States. These beetles feed on hive products and brood, pollute the hive with fermenting waste, and, in severe infestations, cause colonies to abandon their hives. This project investigated previously unexplored control options that take small hive beetle biology into account. Small hive beetles have an exceptional sense of smell compared to other beetles, and this ability helps them to locate honey bee hives. Therefore, behavioral responses to attractants and repellants were tested through olfactometry, in which beetles were given a choice to travel toward or away from specific odors or odor blends. Responses to these odors on a physiological level were also quantified through electrical recordings of beetle antennae. Small hive beetles are also known to have a yeast-like symbiont, which is present throughout the small hive beetle lifecycle, both externally and internally. Feeding bioassasy for small hive beetle larvae, along with soil bioassays for the delivery of insecticides were also developed and used to test several compounds against small hive beetle larvae. Feeding bioassays were also used to deliver a fungicide to larval and adult beetles with the goal of repressing internal fungal activity The results of these studies help expand the knowledge of small hive beetle olfaction and provide a background for the development of novel control options to effectively manage this destructive pest.

Plum curculio, Conotrachelus nenuphar (Herbst), is an endemic pest of stone and pome crops of the eastern United States. Two morphologically identical strains of plum curculio have been described and documented in Virgina: a univoltine strain and a multivoltine strain. Because of the cryptic coloring and behaviours of the plum curculio adults, monitoring in orchards is difficult and often ineffective. RAPD-PCR assay was an effective method for separation of the geographical strains. Of the tested primers four, OPE 01, OPE 03, OPE 04, and OPE 07, gave 21 amplimers that are useful for distinguishing individuals from the univoltine and multivoltine populations. Gene targeted PCR revealed the presence of Wolbachia in both populations. Analysis of the wsp gene sequence showed the univoltine population of plum curculio is associated with a strain of Wolbachia in supergroup B, most closely related to a strain identified from Perithemis tenera (Say) (Odonata). The multivoltine populations of plum curculio are associated with strains of Wolbachia which are in supergroup A, and most closely related to Wolbachia strains associated with Dacus destillatoria, Bactrocera sp., and Callosobruchus chinensis Linn. Three different trap designs baited with grandisoic acid, plant volatiles, and a combination of pheromone and plant volatiles were tested. In 1999, significantly more plum curculios were captured with Tedders traps baited with grandisoic acid and unbaited control in traps baited with limonene, plum essence or ethyl isovalerate. In 2000, Circle traps baited with plum essence, sour cherry essence and grandisoic acid yielded no results. In 2001, branch mimic traps yielded no significant differences among three release rates of a blend of benzaldehyde, ethyl isovalerate, trans-2-hexenal, and limonene; although, there was a significant interaction between the pheromone and the host plant volatiles. Overall however, all three of the traps were not effective. / Master of Science

Female mice exhibit a hardwired preference to investigate pheromones released by male conspecifics. The neural pathways that convey pheromonal inputs to brain regions controlling motivated behaviors remain largely unknown. One brain region known to process pheromonal information conveyed via main- and accessory olfactory bulb inputs is the Medial Amygdala (Me), a limbic structure comprised of anterior (MeA) and posterior (MeP) subdivisions. Electrolytic lesions of the MeP blocked the normal preference of estrous female mice to investigate urinary odors emitted from breeding as opposed to castrated males whereas lesions of either the MeA or MeP significantly reduced females' display of the receptive lordosis posture in response to male mounts. Quantitative analysis of synaptic puncta in the efferent projection targets of these two amygdaloid subregions, visualized using fluorescent anterograde tract tracing techniques, revealed that the MeA and MeP differentially innervate several forebrain regions. The medial olfactory tubercle (mOT; a component of the ventral striatum) receives dense monosynaptic input from the MeA and responds selectively to breeding male (but not female) soiled bedding volatiles, indexed by augmented FOS expression. Using injections of the retrograde tracer, cholera toxin B (CTb), neurons were identified in the MeA and ventral tegmental area (VTA) that projected to the mOT in female mice and which also co-expressed FOS after exposure to breeding male, but not female, soiled bedding/urinary volatiles. This suggests that the MeA and VTA convey opposite-sex (male) pheromonal information to the mOT. Bilateral dopaminergic lesions of the anteromedial VS (a region which includes the mOT) eliminated females' preference for breeding male vs. female urinary pheromones, suggesting that dopaminergic modulation in the VS is necessary for the display of these behaviors. Lastly, bilateral silencing of mOT neuronal firing by the activation of the inhibitory DREADD receptor, hM4Di, induced by intraperitoneal injection of its ligand (CNO), also disrupted females' preference to investigate urinary odors from breeding males; this deficit was reversed when saline was administered instead of CNO. The Me, VTA, and mOT are essential segments of a neural reward circuit that motivates estrous female mice to seek out male pheromones, thereby facilitating mate recognition and reproductive success.

<p>Sensory neuron diversity is a common theme in the animal kingdom. It provides the cellular infrastructure that supports the accurate perception of the external world. Among all sensory systems, the olfactory system demonstrates an extreme in the extraordinarily diversified neuronal classes it holds. The system-wide cellular diversity is in sharp contrast with the individual specialization of olfactory receptor neurons (ORNs) per se. How the nervous system, particularly the olfactory system, uses limited genetic information to generate a huge variety of neurons with distinct properties remains elusive. </p><p>The adult Drosophila olfactory system is an excellent model to address this question due to its conserved organizational principles and reduced complexity. The fly olfactory appendages contain 50 ORN classes, each of which expresses a single receptor gene from a family of ~80 genes. Stereotyped clusters of 1-4 ORN classes define about 20 sensilla subtypes, belonging to 3 major morphological types. All cellular components within a sensillum are born by a single sensory organ precursor (SOP) via asymmetric divisions. The molecular mechanisms that determine SOP differentiation potentials to develop into distinct sensilla subtypes and the associated ORN classes are unknown.</p><p>From a genetic screen, we identified two mutant alleles in the rotund (rn) gene locus, which has a critical function in diversifying ORN classes. Rn is required in a subset of SOPs to confer novel sensilla subtype differentiation potentials from otherwise default ones within each sensilla type lineage. In rn mutants, ORNs in rn-positive sensilla subtypes are converted to lineage-specific default rn-negative fates, resulting in only half of the normal ORN diversity. This work is described in Chapter 2.</p><p>Based on an unbiased time-course transcriptome analysis, we discovered two critical downstream targets of Rn, Bric-à-brac (Bab) and Bar. In light of the knowledge about leg development, we found these genes, along with Apterous (Ap) and Dachshund (Dac), are part of the conserved proximal-distal (PD) gene network that play a crucial role in patterning the antennal precursor field prior to proneural gene-mediated SOP selection. Interactions between these PD genes under the influence of morphogen gradients separate the developing antennal disc into 7 concentric domains. Each ring is represented by a unique combination of the aforementioned transcription factors, coding the differentiation potentials for a limited number of sensilla subtypes. Genetic perturbations of the network lead to predictable changes in the ratios of different sensilla subtypes and corresponding ORN classes. In addition, using CRISPR/Cas9 technology, we were able to add tags to specific rn isoforms in the endogenous locus, and show positive regulation of Bab and negative regulation of Bar by the direct binding of Rn to the promoters in vivo. This work is presented in Chapter 3.</p><p>We proposed a three-step mechanism to explain ORN diversification, starting from pre-patterning of the precursor field by PD genes, followed by SOP selection by proneural genes, and ended with Notch-mediated neurogenesis. The final outcomes are greatly determined by the pre-patterning phase, which may be modified during evolution to compensate special olfactory needs by individual species. In our model, each step serves a single purpose, which displays context-dependent functions. By changing contexts, reassembly of the same logical steps may guide neuronal diversification in parallel systems with completely different identities. This step-wise mechanism seems to be a common strategy that is used by many other systems to generate neuronal diversity.</p> / Dissertation

O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos. / The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

There is little knowledge about olfactory learning in primates, even though primates are known to use olfaction in several behaviors including food selection and territorial defense. Therefore I assessed the olfactory learning and memory performance in five adult spider monkeys (Ateles geoffroyi) using a food-rewarded two-choice olfactory discrimination paradigm. The spider monkeys acquired the initial odor discrimination in 530-1102 trials and in a series of intramodal transfer tasks they needed 30-510 trials to reach the learning criterion. There was a significant negative correlation between the number of trials needed to reach the learning criterion and the number of transfer tasks completed. Thus, as a group, the animals displayed olfactory learning set formation. The number of trials that the spider monkeys needed in initial olfactory learning was comparable to that of other primate species tested previously but higher compared to that of other mammals such as dogs and rats. The learning speed of the spider monkeys in intramodal transfer tasks was similar to that of other mammals tested, suggesting that primates are less prepared to use olfactory cues in the initial solving of a problem but that once they learn the concept, their learning speed with novel odor discrimination problems is not generally slower than that of non-primate mammals. All spider monkeys tested reached the learning criterion in the memory tasks straight on the first testing day, that is: within 30 trials, suggesting similar long-term odor memory capabilities in spider monkeys and other mammals such as dogs, mice and rats.

Disorders of the autonomic nervous system constitute serious complications of spinal cord injury (SCI) and their treatment is usually highly prioritised by spinal patients. Among these, autonomic dysreflexia and impaired thermoregulation are potentially life threatening conditions and require effective management. Olfactory ensheathing cells (OECs), progenitor cells and polymeric scaffolds have been tested in animal models of SCI and some of them have been considered for clinical trials. However, evaluation of the effect of such interventions on autonomic functions has received only rudimentary attention and would require a more thorough experimental assessment before the methods are utilised in human patients. This thesis tested two potential therapeutic strategies for autonomic dysreflexia and examined disorders of thermoregulatory functions in a rat model of spinal cord transection. Magnitude and duration of autonomic dysreflexia were evaluated with radio telemetry in spinalised animals treated with (i) implants of OECs and olfactory neurosphere-derived cells seeded in poly(lactic co glycolic) porous scaffolds or with (ii) transplants of OECs alone. (iii) Effects of SCI and of OECs on the morphology of sympathetic preganglionic neurons (SPNs; which are involved in pathogenesis of autonomic dysreflexia) stained for NADPH diaphorase were examined. (iv) Doppler ultrasonography and infrared thermography were used to assess responses of tail blood flow and surface temperature to cold. Transplants of OECs alone, but not in combination with olfactory neurosphere-derived cells and polymeric scaffolds, resulted in significantly shortened episodes of autonomic dysreflexia. This may be attributed to the alterations to the morphology of SPNs adjacent to the lesion: a transient increase in the morphometric features of the SPNs was evoked by spinal cord transection and this was further altered by transplantation of OECs. The thesis also showed that local responses of tail blood flow and temperature to cold were not abolished by complete SCI suggesting that temperature homeostasis could still be maintained in response to cold. It is hypothesised that OECs facilitate improved recovery from autonomic dysreflexia through alteration of the morphology of SPNs. Furthermore, it is suggested that the role of the tail in heat conservation can be regulated by mechanisms that are independent of the descendent neural control from supraspinal centres.