Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.

Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

Drosophila Adaptation to Viral Infection through Defensive Symbiont Evolution

Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. Here we show that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit.

Dissecting signaling pathways that control glia migration in the developing Drosophila retina

The function of a complex nervous system relies on an intricate interaction between neurons and glial cells. However, as glial cells are generally born distant from the place where they settle, molecular cues are important to direct their migration. Glial cell migration is important in both normal development and disease, thus current research in the laboratory has been focused on dissecting regulatory events underlying that crucial process. With this purpose, the Drosophila eye imaginal disc has been used as a model. In response to neuronal photoreceptor differentiation, glial cells migrate from the CNS into the eye disc where they act to correctly wrap axons. To ensure proper development, attractive and repulsive signals must coordinate glial cell migration. Importantly, one of these signals is Bnl, a Fibroblast Growth Factor (FGF) ligand expressed by retinal progenitor cells that was suggested to act as a non-autonomous negative regulator of excessive glial cell migration (overmigration) by binding and activating the Btl receptor expressed by glial cells. Through the experimental results described in chapter 3 we gained a detailed insight into the function of bnl in eye disc growth, photoreceptor development, and glia migration. Interestingly, we did not find a direct correlation between the defects on the ongoing photoreceptors and the glia overmigration phenotype; however, bnl knockdown caused apoptosis of eye progenitor cells what was strongly correlated with glia migration defects. Glia overmigration due to Bnl down-regulation in eye progenitor cells was rescued by inhibiting the pro-apoptotic genes or caspases activity, as well as, by depleting JNK or Dp53 function in retinal progenitor cells. Thus, we suggest a cross-talk between those developmental signals in the control of glia migration at a distance. Importantly, these results suggest that Bnl does not control glial migration in the eye disc exclusively through its ability to bind and activate its receptor Btl in glial cells. We also discuss possible biological roles for the glia overmigration in the bnl knockdown background. Previous results in the lab showed an interaction between dMyc, a master regulator of tissue growth, and Dpp, a Transforming Growth Factor-β important for retinal patterning and for accurate glia migration into the eye disc. Thus, we became interested in understanding putative relationships between Bnl and dMyc. In chapter 4, we show that they positively cooperate in order to ensure proper development of the eye disc. This work highlights the importance of the FGF signaling in eye disc development and reveals a signaling network where a range of extra- and intra-cellular signals cooperate to non-autonomously control glial cell migration. Therefore, such inter-relations could be important in other Drosophila cellular contexts, as well as in vertebrate tissue development.

Filling in gaps of Drosophila melanogaster urate degradation metabolic pathway using metabolomics approaches: towards the core metabolome of the fruit fly

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound.

Defining the role of NatC dependent N-terminal acetylation in Drosophila fertility

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Bioecologia, monitoramento e controle de Drosophila suzukii na cultura do morangueiro.

2015

Bioecologia, monitoramento e controle de Drosophila suzukii na cultura do morangueiro.

O Brasil ocupa a terceira posição no ranking mundial de produção de frutas. A produção é diversificada devido às condições climáticas do País, que permitem produzir frutas tropicais, subtropicais e temperadas. A maior parte da produção tem como destino o mercado interno e apenas 2,5% destinam-se à exportação. Com a globalização da economia e as dificuldades de monitoramento das fronteiras brasileiras, a produção agrícola está vulnerável a diversos problemas fitossanitários. A fruticultura tem sido um dos setores mais afetados pelo registro de novas pragas, que aumentam os custos de produção e comprometem a qualidade das frutas devido às injurias causadas, e pelo maior risco da presença de resíduos de inseticidas utilizados para o seu controle, além de dificultar as exportações devido às barreiras quarentenárias. Dentre essas pragas, as moscas-das-frutas têm sido uma ameaça constante. Historicamente, a primeira espécie registrada no País foi a mosca do mediterrâneo, Ceratitis capitata, em 1901. Quase 100 anos depois, em 1996, foi registrada a ocorrência da mosca da carambola, Bactrocera carambolae, no Estado do Amapá, e em 1999 foi detectado Zaprionus indianus, em São Paulo. Diferentemente das duas primeiras espécies, que são tefritídeos, Z. indianus é um drosofilídeo, mas causa os mesmos danos nos frutos. Em 2013, no Rio Grande do Sul, foi confirmada a presença da drosófila da asa manchada Drosophila suzukii, considerada uma das principais pragas das chamadas ?pequenas frutas?, que incluem morango, mirtilo, amora-preta, framboesa e cereja, mas a drosófila da asa manchada também ataca outras frutíferas, como videira, nectarineira, pessegueiro, entre outros. No Brasil, já foram constatadas perdas em morango e teme-se que o inseto possa se adaptar às nossas condições e causar perdas econômicas significativas em outros cultivos. Dada a tradição da Embrapa Clima Temperado e da Embrapa Uva e Vinho em pesquisas sobre morangueiro, este documento tem o objetivo de disponibilizar informações sobre a identificação, a bioecologia e as estratégias para o monitoramento e controle da drosófila da asa manchada, baseadas nos trabalhos realizados em outros países e nas pesquisas conduzidas pelos autores do texto.

Deciphering proteome dynamics using cell-type selective metabolic protein labeling in the fruit fly Drosophila melanogaster

Otto-von-Guericke-Universität Magdeburg, Fakultät für Naturwissenschaften, Dissertation, 2016

Genetic architecture and sexual conflict in the life history of Drosophila

Because males and females of a species express many homologous traits, sex-specific selection on these traits can shift the opposite sex away from its phenotypic optimum. This mode of sexually antagonistic selection, known as intralocus sexual conflict (IaSC), arises when the evolution of sexual dimorphism is constrained by the two sexes sharing a common gene pool. As IaSC has been historically overlooked, many outstanding questions remain. For example, what is its contribution in maintaining genetic variation for fitness in populations? What characters underlie this variation in fitness? How does the selection history of the population influence the standing genetic variation? I used the model organism Drosophila melanogaster to attempt to resolve some of these questions. The first part of my Master’s project involved assessing the detectability of sexually antagonistic alleles in populations at different stages of adaptation to the laboratory. For the second part of my Master’s project, I looked for evidence of conflict during the development of body size, a well-known sexually dimorphic trait. While the first part of my thesis proved inconclusive, the second part revealed a surprising source of sexual conflict in pre-adult stages of D. melanogaster.

Suscetibilidade de bagas de genótipos de videira pela infestação por Drosophila suzukii (Diptera: Drosophilidae).

O objetivo deste trabalho foi avaliar a suscetibilidade de genótipos de videira à drosófila‑da‑asa‑ manchada, Drosophila suzukii (Diptera: Drosophilidae), em bagas de uva intactas e em bagas infestadas após a ocorrência de puncturas, causadas pela oviposição da mosca‑das‑frutas sul‑americana [Anastrepha fraterculus(Diptera: Tephritidae)] ou por danos mecânicos (alfinetes). Os experimentos foram realizados em laboratório, a 22±1°C, umidade relativa de 65±10% e fotófase de 12 horas. A suscetibilidade foi avaliada para 18 genótipos de videira, em bagas intactas submetidas às fêmeas de D. suzukii. O potencial de interação foi verificado em bagas de uva 'Italia', cuja epiderme foi danificada por puncturas de A. fraterculusou por alfinete, em comparação a frutos de morango 'Albion'. As cultivares de Vitis labrusca 'Niagara Rosada' e 'Concord' não foram infestadas por D. suzukii, e cinco dos oito genótipos que foram infestados são cultivares melhoradas. A infestação de D. suzukii em bagas de uva 'Italia' com danos mecânicos, feitos com um alfinete ou pela oviposição de A. fraterculus, foi semelhante à de bagas íntegras. Há baixa adequação hospedeira de videiras a D. suzukii, mesmo com a presença de danos. As cultivares 'Benitaka', 'BRS Vitória' e 'BRS Morena' são as mais suscetíveis a D. suzukii.

Susceptibility and Interactions of Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae) in Damaging Strawberry.

Resumo: Drosophila suzukii (Matsumura) foi recentemente detectado causando danos idade para morangos no Brasil. Infestação na cultura de morango tem frequentemente foi observado conjuntamente com a presença de Zaprionus indianus Gupta. este estudo investigou a suscetibilidade de morangos em três amadurecimento estágios para infestação de D. suzukii e Z. indianus e sua interação. Abstracts: Drosophila suzukii (Matsumura) has been recently detected causing damage to strawberries in Brazil. Infestation in strawberry culture has often been observed jointly with the presence of Zaprionus indianus Gupta. This study investigated the susceptibility of strawberries at three ripening stages to infestation of D. suzukii and Z. indianus and their interaction. In the laboratory, strawberries cv. Albion at different ripening stages (green, semi-ripe and ripe) were exposed to D. suzukii and Z. indianus for 24 h in choice and no-choice bioassays. Additionally, we evaluated the effects of mechanical damage incurred artificially or by D. suzukii ovi-position on Z. indianus infestation. In no-choice bioassay, there were no significant differences in fruit susceptibility to D. suzukii infestation at different ripening stages. However, in choice bioassay, D. suzukii adults preferred to oviposit on R fruit. The presence of mechanical damage did not increase susceptibility of fruit to D. suzukii oviposition. For Z. indianus , there was greater susceptibility of R fruit in relation to SR and G fruit in both the choice and no-choice bioassays. There was a significant and positive interaction of mechanical damage and damage caused by D. suzukii to R fruit and infestation by Z. indianus , which was not observed in SR and G fruit. Although infestation of Z. indianus is related to attack damaged or decaying fruit, this work shows that this species has the ability to oviposit and develop in healthy strawberry fruit with and increased infestation level when the fruit has damage to its epidermis.

Eficiência do atrativo a base de fermento biológico com diferentes tempos de fermentação na captura de Drosophila suzukii.

Drosophila suzukii é uma espécie nativa da Ásia com elevada capacidade de ocasionar danos em frutos sadios de uma diversidade de espécies vegetais, especialmente nos pequenos frutos. A falta de ferramentas adequadas para o monitoramento é fator limitante no desenvolvimento de métodos de manejo e controle da praga. A utilização de um atrativo a base de fermento biológico, açúcar e água tem se mostrado eficiente e seletivo na captura de D. suzukii em áreas de pequenos frutos. Porém, há necessidade de informações mais precisas sobre o tempo necessário para fermentação do atrativo antes do seu uso no campo. Assim, este estudo teve como objetivo avaliar o tempo de fermentação necessário para uso do atrativo F1 (fermento, açúcar e água) no monitoramento de D. suzukii em pomar de pequenos frutos.

Movimentos populacionais de Drosophila suzukii (Matsumura, 1931) (Diptera, Drosophilidae) entre agroecossistemas de mata de pinus e pomar de pequenos frutos.

Nos últimos cinco anos, a mosca D. suzukii tem se alastrado rapidamente por varias regiões do mundo. No Brasil, foi constatada causando danos em frutos de morangueiro no município de Vacaria-RS e, posteriormente, atacando amora e framboesa. Por se tratar de uma praga recente no Brasil, há necessidade de investigações sobre a bioecologia da praga, com vista à elaboração de planos de manejo para a espécie. Assim, o objetivo do presente estudo foi determinar os movimentos populacionais de D. suzukii entre agroecossistemas de produção de comercial de pequenas frutas (Amora e Framboesa) e área adjacente com mata de Pinus (Lat.:28°47?S; Lon.:50°97?W e 932 m de altitude.).