Parasitic reproductive endosymbionts are emerging as formidable threats to insect biodiversity. Wolbachia are prevalent maternally inherited intra-cellular bacteria found in >50% of arthropod species. These symbiotic bacteria interact with their hosts in diverse ways, most often they alter host reproduction causing four conditions that all selectively favor infected females: feminization, male killing, parthenogenesis, and cytoplasmic incompatibility (CI). Furthermore, depending on strain-type and host genetic background, Wolbachia are known to affect insect behavior, expand or shift host thermal tolerance ranges, and confer anti-viral protection to their hosts. Because Wolbachia both reside in and are transmitted with host cell cytoplasm, mitochondria and other cytoplasmically inherited genetic elements become linked with the bacteria. Thus, by enhancing their own transmission, Wolbachia-induced phenotypes can lead to mitochondrial selective sweeps, which may have profound impacts on vulnerable and small insect populations. Elucidating the extent to which endosymbionts influence biological and ecological functions is pivotal to making management decisions regarding imperiled insect species. My dissertation investigates biological and ecological impacts of host-endosymbiont interactions by examining Wolbachia infections in three different host systems. First, I used the federally threatened butterfly species Speyeria zerene hippolyta to determine whether the general reproductive success of local populations was affected by the introduction of CI-inducing Wolbachia-infected butterflies through implemented species recovery programs. Next, by characterizing the Wolbachia infections of parasitoids associated with the Eurema butterfly clade, I analyzed whether host-parasitoid interactions provide a path for interspecies horizontal transmission. Finally, I conducted a laboratory experiment using an isogenic Drosophila melanogaster line to determine whether Wolbachia influence host temperature preference. Together, my research examines how the individual level effects of host-endosymbiont interactions can expand into populations, have broader impacts on insect communities, and potentially impede the conservation and management of insects in nature. In chapter one, I screened S. z. hippolyta samples from three extant populations for Wolbachia infection. To examine the impacts of Wolbachia on small populations, I analyzed and compared infected and uninfected S. z. hippolyta reproductive data and showed that, in a population composed of infected and uninfected S. z. hippolyta, uninfected butterflies had reduced reproductive success (GLMM z = -8.067, P < 0.0001). I then developed a single-population demographic theoretical model using these same reproductive data to simulate and analyze different potential dynamics of small populations resulting from population supplementation with uninfected, CI-Wolbachia infected, or combined uninfected and infected butterflies. Analysis of model simulations revealed that supplementation with CI-inducing butterflies significantly suppressed host-population size (ANOVA F5,593 = 3349, PWolbachia-infected individuals (Tukey's post-hoc test P < 0.0001). In addition, supplementation by multiple releases using a combination of 50 infected and 300 uninfected butterflies has a less severe suppression effect, reducing the population by 75.8%, but the reduction occurs 42.6% faster than with the single release of 50 Wolbachia-infected butterflies (Tukey's post-doc test P < 0.0001). Parasitoid-host interactions have emerged as probable ecological relationships to facilitate horizontal transmission of Wolbachia. In chapter two, I addressed horizontal transmission using Eurema butterflies and their associated parasitoids. From four locations in Northern Queensland, Australia, I collected a total of 404 Eurema hecabe butterfly larvae. Twenty-three parasitoids emerged from the larvae of which 21 were Diptera and two were Hymenoptera. I amplified COI loci fragments from each parasitoid for BLAST query searches and found that 20 individual Diptera parasitoids matched to the genus Exorista and one to the genus Senometopia. One of the Hymenoptera parasitoids matched to the genus Microoplitis and the other to the genus Cotesia. To characterize Wolbachia infections, I used Wolbachia Multi Locus Sequencing Technique (MLST) and discovered that all 20 Exorista parasitoids were infected with an identical Wolbachia strain (ST-41), which is the same strain infecting their Eurema hecabe butterfly hosts. Although, further experiments are necessary to definitively determine that ST-41 Wolbachia are incorporated into germline cells of the parasitoids, this is the first study to provide ecological evidence for inter-ordinal Wolbachia transmission between Lepidoptera and Diptera. Furthermore, this discovery exposes the risk of population augmentation programs that move insects, potentially facilitating the spread of Wolbachia between species within a community through the accidental introduction of new Wolbachia-infected parasitoids. Finally, both Wolbachia and their insect hosts are temperature sensitive organisms. Wolbachia's replication behavior in their hosts is positively-temperature dependent, while environmental variation can have profound effects on insect's immune function, fitness, and fecundity. In chapter three, I conducted a laboratory experiment using a thermal gradient choice assay and an isogenic Drosophila melanogaster line with four different Wolbachia infection statuses -- uninfected, wMel, wMelCS, and wMelPop - to assess whether a relationship existed between Wolbachia infection and host temperature preference. Results from my laboratory experiment revealed that Wolbachia-infected flies preferred cooler temperatures compared to uninfected flies. Moreover, D. melanogaster temperature preferences varied depending on the Wolbachia strain variant with which they were infected; flies infected with the wMel strain had temperature preferences 2°C cooler compared to uninfected flies; flies infected with either wMelCS or wMelPop strains had preferred temperatures 8°C cooler compared to uninfected flies. Wolbachia-associated temperature preference variation within a species can lead to conspecifics occupying different microclimates, genetically adapting to different sets of specific environmental conditions, and may eventually result in ecological and reproductive isolation. While, reproduction isolation is recognized as one of the first stages in speciation, in small populations of endangered and threatened species, the inability to reproduce between conspecifics can drive species to extirpation or extinction. Collectively, the three chapters of my dissertation set precedent for future integration of host-endosymbiont research prior to implementing population supplementation or translocation programs for the conservation of imperiled insects.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb. 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Georgia Davis. Vita Includes bibliographical references.

Mosquito-borne diseases account for hundreds of thousands of deaths each year, highlighting the need for successful intervention methods, which can be targeted at either the pathogen, mosquito vector, or human host. This thesis aims to contribute to better intervention methods focused against malaria and dengue by either (i) improving available research tools, (ii) enhancing the understanding of a promising intervention method or (iii) designing new intervention candidates. Firstly, a superior method for studying in vitro malaria infection of the liver is shown, with implications for vaccine and drug interventions. Secondly, the biology of Wolbachia infection in Anopheles gambiae mosquitoes in the context of the target of rapamycin signalling cascade is investigated in an attempt to improve our understanding of its malaria inhibitory phenotype and inability to stably infect An. gambiae mosquitoes. Finally, an algorithm is developed for the design of a hypothesis driven conservation-based vaccine against viral mosquito diseases with a particular focus on dengue.

Phlebotomine sand flies are proven vectors of many pathogens including parasites of genus Leishmania. Leishmania develop in sand fly midgut which is colonized also by many others microorganisms, creating rich community known as gut microbiota. The presence and composition of gut microbiota affect sand fly mortality, but also development of transmitted pathogens. In contrast to mosquitoes, sand fly gut microbiota is not well studied. This thesis focuses on bacteria of the genera Asaia and Wolbachia and their potential impact on Leishmania in sand fly midgut. Thesis reports the first finding of Asaia sp. and Wolbachia sp. in sand flies from Balkan penninsula - hotspot for visceral leishmaniasis and phleboviruses. In 273 individuals from subgenera Larroussius were Asaia sp. and Wolbachia sp. detected with infection prevalence 2,5 % and 8,4 %, respectively. In addition, laboratory-reared sand flies were tested for presence of these bacteria: from twelve studied colonies, only Phlebotomus perniciosus was infected by Wolbachia sp. Then, we focussed on elimination of Wolbachia sp. from this laboratory colony with the aim to use Wolbachia-negative sand flies in future experiments with Leishmania. The final part of the thesis was dedicated to bacteria of the genus Asaia (specifically A. krungthepensis),...

Wolbachia are maternally transmitted endosymbionts that infect a large proportion of arthropods and reduce vector competency of mosquitoes carrying deadly diseases such as Dengue, Zika, West Nile Virus, and Chikungunya. Wolbachia preferentially infect specific host cells, a phenomenon termed tissue tropism. Wolbachia tissue tropism has consequences in proper vertical and horizontal transmission. Although Wolbachia tropism to multiple Drosophila gonadal cell types has been identified, the mechanisms of tropism during development have not been characterized, in part because Wolbachia infected cell types are challenging to study during morphogenesis. Here we describe a novel Wolbachia tropism to polar cells (PCs) of the Drosophila ovary, a developmentally well characterized system. We show that Wolbachia intracellular accumulation is triggered by specific events of PC morphogenesis including differentiation from progenitors, and during stage 8 to 9 transition. Using genetic tools, we also demonstrate that induction of ectopic PC fate is sufficient to drive Wolbachia tropism. These findings implicate a tight coordination of host developmental events with Wolbachia tropism. Wolbachia tropism to multiple host cell types suggest that host pathways common to these would be conducive to intracellular Wolbachia growth. Indeed, we found that Wnt signaling, is active in gonadal cell types with Wolbachia tropism. Wnt signaling, first characterized in embryonic development and patterning, has novel functions in immunity and intracellular pathogen survival. Using RNAi mediated gene knockdowns, we studied the effect of Wnt signaling on Wolbachia in various infected cell types, including the testis stem cell niche, PCs and germline. Reduction of Wnt signaling caused a decrease in Wolbachia density and increased signaling led to higher density suggesting the reliance of Wolbachia on host Wnt signaling for its tropism. Moreover, expression of ectopic Wnt signaling was sufficient to drive Wolbachia tropism to previously uninfected tissues such as the D. melanogaster female germline stem cell niche. Finally, small molecule Wnt signaling agonists were sufficient to drive high Wolbachia titers in mosquito cell lines. These findings describe the effect of host signaling on Wolbachia tropism and provide an approach to affect Wolbachia levels in disease-causing vectors, thereby contributing to Wolbachia based vector control strategies.

West Nile virus (WNV) is a geographically endemic mosquito-borne flavivirus that has spread across the United States infecting birds, mosquitos, humans, horses and other mammals. The wide spread nature of this virus is due to the ability of the mosquito vector to persist in broad, ecological diverse environments across the United States. In this study, mosquito populations in North Texas region were sampled for detection of Wolbachia, blood meal source, and WNV. The ultimate goal of this study was to examine the potential of a biocontrol agent, Wolbachia sp. that colonizes the hindgut of various insects, including mosquitos, as a natural means to interrupt virus transmission from mosquitos to other hosts, including humans. In Australia, Wolbachia sp. from fruit flies (Drosophila melanogaster) have been successfully used to block transmission of a similar pathogenic virus from mosquitos responsible for transmission of Dengue fever. Here, mosquitoes were collected using CDC style Gravid Traps in Denton, Texas, from October 2012 through September 2014. Collected mosquitoes were identified, sexed, and categorized as to the amount of host blood in their alimentary system using a Zeiss Axio Zoom microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). Culex quinquefaciatus was the dominant blood engorged species collected. Smaller populations of Culex tarsalis and Aedes albopictus, another known vector for WNV were also collected. Mosquito larva were also collected from the UNT water research field station and reared to adults. Cx. tarsalis was the dominant mosquito taken from this habitat. Samples of Cx. quinquefasciatus, Cx. tarsalis and A. albopictus were analyzed for Wolbachia sp. and to identify host blood in the mosquito alimentary system. Total DNA extraction from the pool of mosquito samples was by both commercially available DNA extraction kits (Qiagen, Valencia, CA) and salt extraction technique. Polymerase chain reaction (PCR) was used to amplify and identify Wolbachia sp. 16SrDNA and mitochondrial DNA from vertebrate blood. The maternally inherited endosymbiont, Wolbachia, were found to be uniformly distributed across the mosquitoes sampled in this study. Blood meal analysis by PCR showed that Cx. quinquefaciatus fed more on birds than on mammalian blood sources based on the previously developed primers used in this study.

Embora muitos aspectos sobre a história de vida dos isópodos terrestres na América do Sul tenham sido investigados, alguns ainda são desconhecidos. Entre estes, está a relação simbiótica entre os isópodos terrestres e Wolbachia pipientis, uma espécie de alpha-proteobactéria, considerada um parasito intracelular obrigatório que infecta espécies de nematóides e praticamente todos os grupos de artrópodos, incluindo os isópodos terrestres (Oniscidea). Apesar de contestada, a filogenia mais aceita inclui as linhagens de Wolbachia em oito supergrupos (A-H), a partir da qual a transmissão horizontal (entre taxons) é corroborada. Wolbachia está presente em vários tecidos do hospedeiro, principalmente em tecidos reprodutivos e propaga-se por via materna para a prole, utilizando como estratégia de disseminação, a produção de fêmeas ao longo das gerações. Esse aumento na proporção de fêmeas surge como consequência dos fenótipos induzidos gerados pela interação Wolbachia-hospedeiro. A cotransmissão de Wolbachia e outros elementos presentes no citoplasma (p. ex. mitocôndrias) gera um desequilíbrio de ligação entre os mesmos. Este fenômeno juntamente com a taxa de transmissão vertical, competição entre linhagens e ancestralidade da invasão são fatores que podem contribuir para alterações na estruturação do mtDNA nas populações hospedeiras. Todas essas características fazem de Wolbachia um fator que pode estar envolvido no processo de especiação. O presente trabalho descreve o primeiro registro de Wolbachia em espécies de isópodos terrestres endêmicas da América do Sul, mais especificamente em duas espécies do gênero Balloniscus (Crustacea, Oniscidea). No total, foram observadas 16 novas linhagens em populações distribuídas ao longo da Planície Costeira do Rio Grande do Sul. A partir de uma abordagem populacional, observou-se variação nas prevalências intra e interespecíficas. Em média, as prevalências foram mais altas em populações de B. glaber do que em relação a B. sellowii. Todas as linhagens encontradas situaram-se dentro do Supergrupo B, no qual situam-se também as demais linhagens presentes em isópodos terrestres. Entretanto, as topologias geradas sugerem rotas de transmissão horizontais alternativas para as linhagens de Wolbachia encontradas em ambas as espécies de Balloniscus. A partir da idéia de impacto de Wolbachia sobre a estruturação do mtDNA, foi realizado um estudo genético populacional ao longo da Planície Costeira do Rio Grande do Sul baseado no gene COI. Apesar dos fracos indícios do impacto de Wolbachia sobre a estruturação do mtDNA, outros resultados foram alcançados. A história geológica de formação da Planície Costeira do Rio Grande do Sul parece ser diretamente associada aos agrupamentos (clados) observados. Os clados I, II e III incluiram populações de B. sellowii, enquanto que o clado IV foi composto exclusivamente por populações de B. glaber. As análises filogenéticas e a análise da variação genética entre as duas espécies sugeriram que existe um grande complexo composto pelas populações de B. sellowii e, possivelmente B. glaber seria uma espécie derivada de B. sellowii. Além disso, as análises sugerem ainda a população de São Lourenço do Sul (B. sellowii) pode se tratar ou de uma linhagem muito basal dentro do complexo B. sellowii ou de uma nova espécie. Essa populaçào apresentou os níveis de diversidade mais elevados, sendo lançada a hipótese de que Wolbachia poderia estar envolvida no polimorfismo observado. / Although many aspects of the life history of South American terrestrial isopods have been investigated, some of them are still unknown. Among them is the symbiotic interaction between terrestrial isopods and Wolbachia pipientis, an alpha-proteobacteria, considered an obliglate intracellular parasite which infects nematodes species and almost all arthropods groups, including terrestrial isopods (Oniscidea). Despite some disagreements, the most accepted Wolbachia phylogeny is comprised by eight supergroups (A-H), from which the horizontal transmission is corroborated. Wolbachia, is found in several tissues, mainly in reproductive ones; it is maternally disseminated to the progeny using as strategy, the production of females over the generations. This increase in female proportion arises as a consequence of induced phenotypes generated by the interaction between Wolbachia and its host. The cotransmission between Wolbachia and other cytoplasm elements (e.g mitochondria) creates linkage disequilibrium between them. This phenomenon together with the vertical transmission, competition between lineages and ancestry of the invasion are factors that may contribute to changes in the mtDNA structure over host populations. All these characteristics make Wolbachia a factor that may be involved in the process of speciation. This study shows the first record of Wolbachia in endemic South American terrestrial isopods, specifically in two species of the genus Balloniscus (Crustacea, Oniscidea). Overall, 16 new strains were observed in populations distributed along the coastal plain of Rio Grande do Sul. Based on a population approach, we observed variation in intra and interspecific prevalences. On average, the prevalence was higher in B. glaber than in B. sellowii populations. All strains were grouped in supergroup B, in which all Wolbachia strains found in terrestrial isopods are situated. However, the topologies indicated alternative routes for horizontal transmission of Wolbachia found in both Balloniscus species. From the idea of Wolbachia impact on the mtDNA structuring, we conducted a genetic study population along the coastal plain of Rio Grande do Sul based on COI gene. Despite the weak evidence of the impact of Wolbachia on the mtDNA structuring, other results have been achieved. The geological history of formation of the Coastal plain of Rio Grande do Sul seems directly involved on the grouping (clades) observed. The clades I, II and III included populations of B. sellowii, while clade IV was composed exclusively of populations of B. glaber. The phylogenetic and genetic variation analysis between the two species suggests that there is a large complex formed by the populations of B. sellowii and, possibly B. glaber may be a species derived from a B. sellowii. Furthermore, our analysis also suggests that the population of São Lourenço do Sul (B. sellowii) may be very basal lineage within the B. sellowii complex or a new species. This population showed the highest levels of diversity, and we hypothesized that Wolbachia could be involved in the observed polymorphism.

The phenomenon of repeated evolution runs counter to expectations about the role of contingency in adaptation. However, many examples of independently acquired similar traits show that evolution sometimes does follow the same path. Factors influencing the probability of such an event include selection, trait complexity and relatedness. Previous investigations of repeated adaptation have primarily focused on low-complexity traits subject to strong selection. Studies of systems with varying levels of trait complexity, selection, and relatedness are needed to evaluate the relative contributions of these factors. The series of studies reported here 1) establishes a system for inquiry into the role of parallel adaptation among hosts and parasites and 2) provides an assessment of the role of parallel genetic change in the evolution of a complex trait. In Chapter 2, I show that all-female broods in a line of Drosophila borealis are caused by infection with a male-killing strain of Wolbachia that is very closely-related to another male-killing strain infecting a geographically and evolutionarily distant species of Drosophila. This host-parasite system, together with two other known male-killing Wolbachia strains infecting Drosophila provides a framework for investigating the role of parallel evolution in the independent acquisition of the male-killing trait among Wolbachia, as well as in the adaptation of divergent hosts to similar male-killing parasites. In Chapters 3-5, I investigate the role of parallel genetic change in a complex trait in two species of Drosophila by searching for evidence of adaptation in the Drosophila americana homologs of genes thought to underlie adaptation to climate in Drosophila melanogaster. In Chapter 3, I investigate the D. americana homolog of Alcohol dehydrogenase (Adh). In contrast with D. melanogaster, which segregates functionally distinct variants in Adh that represent local adaptation to climate, D. americana segregates little variation. This is surprising, especially because Adh of D. americana is found near a polymorphic chromosomal rearrangement that does segregate geographically-structured alleles across the species' range. In Chapter 4, I report similarities at the Phosphoglucomutase (Pgm) locus in the two species, including a shared excess of nonsynonymous variants and the presence of clinal alleles. However, while variation at Pgm of D. melanogaster is proposed to underlie local adaptation, variation at Pgm of D. americana appears to be predominantly neutral. In Chapter 5, I investigate the role of positive selection in sequence evolution in the D. americana homologs of a group of genes thought to underlie local adaptation to climate in D. melanogaster. The two species share a large geographic range and exhibit levels of sequence variation that indicate a similar effective population size, but D. melanogaster appears to undergo more frequent fixation of advantageous alleles. Approximately half of all amino acid divergence in D. melanogaster is attributable to positive selection, but I find no signs of positive selection in the investigated genes in D. americana. Overall, the results reveal little or no parallel evolution at the single genes analyzed. This lack of parallel evolution is likely a result of the high complexity of adaptation to climate as well as contingency.

Symbiotic associations between animals and microbes are widespread in nature, yet the factors controlling the abundance and distributions of particular symbionts are mostly unknown. Vertically transmitted (VT) symbionts can invade host populations by providing net benefits to hosts. While most beneficial symbiotic interactions that have been characterized are nutritional, other major pathways likely exist that facilitate symbiont transmission within host populations. Natural enemies, for example, are important selective forces in shaping the life history of many invertebrates and hosts may benefit from collaborations with microbes to aid in their defense.In this dissertation I have addressed the role of VT, facultative (= secondary) symbionts (SS) of Acyrthosiphon pisum in mediating interactions with an important natural enemy, the parasitic wasp, Aphidius ervi. I found that, in a common genetic background, two A. pisum SS (R- and T-type SS) confer resistance to A. ervi, by causing mortality to developing wasp larvae. Defensive mutualisms with microbes provide a mechanism for the spread and persistence of VT symbionts.A. pisum superinfected with both R- and T-type SS were found to be more resistant to parasitism that those singly infected with either SS. Despite this added benefit to resistance, R + T-type superinfections were rare in a survey of A. pisum symbionts, likely attributable to severe fecundity costs. R-type densities increased dramatically in superinfected hosts and over-proliferation of SS may result in poor aphid performance. Thus, interactions among the symbionts themselves also likely play a critical role in determining the distributions of symbionts in nature.I also found that four T-type isolates from A. pisum, and one from another aphid species, all conferred resistance to parasitism in the same A. pisum host background. The levels of resistance varied greatly among isolates, including one that conferred nearly complete resistance. A single T-type isolate was also found to confer similar levels of resistance in five A. pisum backgrounds. These results indicate that SS-mediated resistance is a general phenomenon in A. pisum and that the SS isolate is more important in determining the level of resistance than is the aphid genotype or interaction between isolate and aphid genotype.

Reciprocal and homologous crosses were carried out using pairs or groups of twenty males and females for three biotypes of the whitefly Bemisia tabaci complex. Crosses were undertaken for the A biotype-Arizona (AzA), the B biotype-Africa (AzB), and the monophagous, Jatropha (Jat) biotype-Puerto Rico. The maternal haplotype pedigree of parents and selected offspring (F₁, F₂) was determined using the mitochondria cytochome oxidase I sequence. All reciprocal crosses yielded viable female offspring, indicating reproductively compatibility, except for AzB♀ x AzA♂, or AzB♀ x Jat♂ crosses, which yielded females unidirectionally. As an unidirectional pattern was reminiscent of cytoplasmic-mediated incompatibility (CI), the possibility was investigated that the phenotype might be caused by CI-bacteria, instead of a germ line barrier. Using the 16S rRNA sequence three prospective CI-bacterial species were identified in whitefly colonies. A Cardinium spp. (Bacteroidetes) was present in the A biotype (isolates AzA, CulA, RivA, SalA), whereas the B biotype (isolates AzB, FlB1, FlB2) was infected with Rickettsia bellii (Proteobacteria), and a Wolbachia spp. (Proteobacteria) was associated with the Jat biotype. The unidirectional incompatible phenotypes were consistent with CI-bacterial infection of AzA (Cardinium) and Jat (Wolbachia), but no such association was apparent for B biotype-Rickettsia infections. The bidirectional compatibility for Jat x AzA suggested a CI-bacteria-mediated reciprocal rescue. However, that Wolbachia-infected Jat♀ and Cardinium-infected AzA♂ crosses yielded fewer females, compared to AzA-Cardinium♀ and Jat- Wolbachia♂ crosses, suggested that Cardinium could better counter Wolbachia-induced female mortality than Wolbachia. This suggested the possibility that these phylogenetically divergent bacteria might utilize similar CI-mechanisms. In this study, the suspect CI-bacteria were strongly associated with complete or partial obstruction of gene flow in certain crosses, and with sex bias in the AzA x Jat crosses. This is the first evidence that female offspring can be produced between phylogeographically divergent, and polyphagous and monophagous B. tabaci, for which gene flow barriers are widely reported, suggesting that hybridization is utilized as a means of diversification in B. tabaci. The inability to rid colonies of CI-bacteria has necessitated introgression experiments to investigate direct CI-causality over CI-association.