PROTEOMIC ANALYSIS OF WOLBACHIA SYMBIOSIS WITHIN THE DROSOPHILA OVARY

Wolbachia pipientis are bacterial endosymbionts carried by millions of invertebrate species, including ~40% of insect species and some filarial nematodes. In insects, basic Wolbachia research has potential applications in controlling vector borne disease. Conversely, Wolbachia of filarial nematodes are causative agents of neglected tropical diseases such as lymphatic filariasis and African river blindness. However, remarkably little is known about how Wolbachia interact with their hosts at the molecular level. Understanding this is important to inform the basis for symbiosis and help prevent human disease. I used a high-throughput proteomics approach to study how Drosophila host cells are modified by Wolbachia infection. This analysis identified 23 Drosophila proteins that significantly changed in amount as a result of Wolbachia infection. A subset of differentially abundant host proteins were consistent with Wolbachia-associated phenotypes reported previously. This study also provides the first ever discovery-based evidence for a Wolbachia-associated change in maternal germline histone loads, which has possible implications in Rescue of a common Wolbachia-induced reproductive manipulation known as Cytoplasmic Incompatibility.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.

Specificity, stability and comparative physiology of coral-Symbiodinium mutualisms: Evaluating the potential for acclimation and/or adaptation in reef corals

The relationship between reef corals and endosymbiotic dinoflagellates is fundamental to the existence of coral reefs. To evaluate the fidelity of coral-Symbiodinium mutualisms, corals maintained in aquaria for years were analyzed by denaturant gradient gel electrophoresis (DGGE). Comparing Symbiodinium populations of captive aquarium colonies with known associations in nature is a practical way of examining partner flexibility. The finding of "normal" symbiont populations in corals existing under highly variable conditions supports the premise that most coral colonies possess stable associations. High sensitivity real-time PCR (rtPCR) was used to evaluate background populations of the putatively stress-tolerant Symbiodinium D in reef corals of the Caribbean. Analyses of samples collected during periods of environmental stability indicate the ability of Symbiodinium D to associate with a wider diversity of host taxa than previously recognized. To gain a broader perspective with regard to the ecology of Symbiodinium D1a, rtPCR and DGGE were used to evaluate the symbiont populations of reef corals from Barbados before and after the 2005 mass coral bleaching. Background populations were observed in 56% of the host genera prior to observations of bleaching. These findings indicate that 'stress', not 'bleaching', caused the displacement of 'natural' symbiont population and the opportunistic proliferation of D1a in many host taxa. Of the 12 host taxa monitored before and after the bleaching event, there was a 40% increase in colonies hosting Symbiodinium D1a. Together, these studies elucidate the mechanism responsible for recent observations reporting the emergence of Symbiodinium D following thermal disturbances. These observations are now most easily explained as the disproportionate growth of existing in hospite symbiont populations, rather than novel symbiont acquisition subsequent to bleaching. To evaluate the comparative "fitness" of corals able to host multiple symbiont types, rates of calcification were measured in P. verrucosa hosting either Symbiodinium C1b-c or D1 at elevated temperature. Rates of calcification decreased significantly for both host-symbiont combinations, but differences attributable to symbiont composition were not detected. This research improves our knowledge of the symbiosis biology and ecology of reef corals and contributes information necessary to most accurately predict the response of these ecosystems to global climate changes.

Hindutva meets globalization: The impact on Hindu urban media women

This study examines the impact of globalization and religious nationalism on the personal and professional lives of urban Hindu middle class media women. The research demonstrates how newly strengthened forces of globalization and Hindutva shape Indian womanhood. The research rests on various data that reveal how Indian women interpret and negotiate constructed identities. The study seeks to give voice to the objectified by scrutinizing and challenging the stereotypical modern faces of Indian womanhood seen in the narratives of globalization and Hindutva. Feminist open-ended interviewing was conducted in English and Hindi in New Delhi, the capital of India, with 23 Hindu women, employed by electronic and print media corporations. Accumulated data were analyzed and interpreted using feminist critical discourse analysis. Findings from the study indicate that while the Indian middle class women have embraced professional opportunities presented by globalization, they remain circumscribed by mutating gender politics. The research also finds that as academic and professional progress empower the women within their homes, their public lives have become fraught with increasing gender violence and decreasing recourse to justice. Therefore, women accept the power stratification of their lives as being dependent on spatial and temporal distinctions, and have learnt to engage and strategize with the public environment for physical safety and personal-professional progress. While the media women see systemic masculine domination as being symbiotic with tenets of religious nationalism, they exhibit an unquestioned embracing of capitalism/globalization as the means of empowerment. My research also strongly indicates the importance of the media’s role in shaping gender dynamics in a global context. In conclusion, my research shows the mediawomen’s immense agency in pursuing academic and professional careers while being aware of deeply ingrained gender roles through their strong commitment towards their families. The findings of this study contribute to the literature on Third World nationalism, urban globalization and understandings of reworked-renewed masculine domination. Finally, the study also engages with recent scholarship on the Indian middle class (See Nanda 2010; Shenoy 2009; Lukose 2005; and Radhakrishnan 2006) while simultaneously addressing the notions of privilege and disengagement levied at the middle class woman, a symbiosis of idealization and imprisonment.

Acclimatization of the tropical reef coral Acropora millepora to hyperthermal stress

The demise of reef-building corals potentially lies on the horizon, given ongoing climate change amid other anthropogenic environmental stressors. If corals cannot acclimatize or adapt to changing conditions, dramatic declines in the extent and health of the living reefs are expected within the next half century. The primary and proximal global threat to corals is climate change. Reef-building corals are dependent upon a nutritional symbiosis with photosynthetic dinoflagellates belonging to the group Symbiodinium. . The symbiosis between the cnidarian host and algal partner is a stress-sensitive relationship; temperatures just 1°C above normal thermal maxima can result in the breakdown of the symbiosis, resulting in coral bleaching (the loss of Symbiodinium and/or associated photopigments) and ultimately, colony death. As ocean temperatures continue to rise, corals will either acclimatize or adapt to changing conditions, or will perish. By experimentally preconditioning the coral Acropora millepora via sublethal heat treatment, the coral acquired thermal tolerance, resisting bleaching during subsequent hyperthermal stress. The complex nature of the coral holobiont translates to multiple possible explanations for acclimatization: acquired thermal tolerance could potentially originate from the host itself, the Symbiodinium, or from the bacterial community associated with the coral. By examining the type of in hospite Symbiodinium and the bacterial community prior acclimation and after thermal challenge, it is shown that short-term acclimatization is not due to a distinct change in the dinoflagellate or prokaryote community. Though the microbial partnerships remain without considerable flux in preconditioned corals, the host transcriptome is dynamic. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments, showing a modulated transcriptomic response to stress. Additionally several genes were upregulated in association with thermal tolerance, including antiapoptotic genes, lectins, and oxidative stress response genes. Upstream of two of these thermal tolerance genes, inhibitor of NFκB and mannose-binding lectin, DNA polymorphisms were identified which vary significantly between the northern and southern Great Barrier Reef. The impact of these mutations in putative promoter regions remains to be seen, but variation across thermally-disparate geography serves to generate hypotheses regarding the role of regulatory element evolution in a coral adaptation context.

Understanding the role of Dimethylsulfoniopropionate as a potential chemotactic cue for coral-associated bacteria

Most reef-building corals are known to engage in non-pathogenic symbiosis not only with unicellular dinoflagellates from the genus Symbiodinium, but also with other microscopic organisms such as bacteria, fungi, and viruses. The functional details of these highly complex associations remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and their coral host. Studies have shown that certain bacterial orders associate with specific certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enable both parties to find one another and thus generate the symbiosis. The production of these cues by the symbionts may be the result of environmental stimuli such as elevated ocean temperatures, increased water acidity, and even predation. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP during times of stress. Marine bacteria utilize DMSP as a source of sulfur and carbon. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. This would enable them to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. To test the hypothesis that coral-produced DMSP plays a role in attracting symbiotic bacteria, this study utilized the advent of high-throughput sequencing paired with chemotactic assays to determine the response of coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Chemotaxis assays revealed that some isolates responded positively towards the DMSP compound. This finding adds to existing evidence suggesting that coral-associated pathogens utilize chemotaxis as a host colonization and detection mechanism. Thus the symbiotic bacteria that make up the coral microbiome may also employ this process. Furthermore this study demonstrates that bacterial motility may be a strong contributing factor in the response to the chemotactic cue. Swarming motility may be better suited for bacteria that need to respond to a chemical gradient on the surface of the coral. Therefore the isolates that were able to swarm seemed to respond more strongly to the DMSP.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Buckwheat as a Cover Crop in Florida: Mycorrhizal Status, Soil Analysis, and Economic Assessment

This thesis analyses buckwheat as a cover crop in Florida. The study was designed to demonstrate: soil enrichment with nutrients, mycorrhizal arbuscular fungi interactions, growth in different soil types, temperature limitations in Florida, and economic benefits for farmers. Buckwheat was planted at the FIU organic garden (Miami, FL) in early November and harvested in middle December. After incorporation of buckwheat residues, soil analyses indicated the ability of buckwheat to enrich soil with major nutrients, in particular, phosphorus. Symbiosis with arbuscular mycorrhizal fungi increased inorganic phosphorus uptake and plant growth. Regression analysis on aboveground buckwheat biomass weight and soil characteristics showed that high soil pH was the major limiting factor that affected buckwheat growth. Spatial analysis illustrated that buckwheat could be planted in South Florida throughout the year but might not be planted in North and Central Florida in winter. An economic assessment proved buckwheat to be a profitable cover crop.

The Effects of Arbuscular Mycorrhizal Fungi on four Legume Hosts in South Florida Pine Rockland Soils

This study addressed the effects of salinity and pot size on the interaction between leguminous plant hosts and arbuscular mycorrhizal fungi in four pine rockland soils using a shade house trap-plant experiment. Little is known about the belowground diversity of pine rocklands and the interactions between aboveground and belowground biota – an increased understanding of these interactions could lead to improved land management decisions, conservation and restoration efforts. Following twelve weeks of growth, plants were measured for root and shoot dry biomass and percent colonization by arbuscular mycorrhizal fungi. Overall, arbuscular mycorrhizal fungi had positive fitness effects on the four legume species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima and Abrus precatorius), improving their growth rate, shoot and root biomass; pot size influenced plant-fungal interactions; and percent colonization by arbuscular mycorrhizal fungi was influenced by soil type as well as salinity.