Lipid metabolism during tsetse reproduction

描述（由申请人提供）：候选人（Joshua B. Benoit 博士）的长期目标是成为昆虫病媒生理学方面的权威，并提高公众对昆虫对公共卫生作用的认识。该奖学金的目标是培养候选人评估媒介繁殖的生物化学、分子生物学和生理学的能力。该培训计划提供了在分子生物学和生殖生理学之间进行广泛研究的机会。采采蝇是非洲昏睡病的唯一媒介。基于疫苗的方法尚未成功控制锥虫病。这种疾病媒介的一个独特之处是它的 K 繁殖策略。母亲通过乳腺直接向子宫内的后代提供营养，一生只生育 8-10 个后代。干扰采采蝇繁殖可以补充诱捕和其他控制方法，以减少采采蝇种群数量并减少疾病传播。目前，关于采采蝇繁殖的信息很少，特别是如何将营养物质输送给幼虫。由于这一方面在采采蝇中是独特的，因此阻断母体营养的控制技术可能是采采蝇特有的。本研究的目的是 1) 表征脂质从母亲到后代的代谢和转移，2) 破译共生细菌的存在如何影响这一过程。脂质代谢和动员的表征将重点关注脂肪运动激素（AKH；这部分已完成）和布鲁默（Bmm）脂肪酶系统，以及生殖周期中脂蛋白（Lp）的脂质转运。在幼虫宫内发育期间，将在空间和时间上评估基因表达和蛋白质合成。这些蛋白质的功能将通过 RNA 干扰来降低其表达来评估。通过分析野生型和抗生素处理的非共生果蝇（缺乏共生体的果蝇）之间的分子和生理差异，评估共生细菌在繁殖过程中脂质代谢和运动中的作用。无共生蝇是不育的，未发育的幼虫在发育周期的早期就会流产。这种表型背后的机制尚不清楚，但目前的假设是由于乳汁分泌物缺乏必需营养素而导致营养不良。可能的缺陷是牛奶中的脂质含量。将通过获取乳汁并比较乳脂的定量和定性差异来评估野生型果蝇和非共生果蝇之间的差异脂质组成。通过在血粉中添加酵母提取物可以恢复非共生果蝇的生育能力。还将对添加和不添加酵母提取物的血液的非共生果蝇的脂质组合物进行分析。为了确定膳食补充剂的遗传效应，将通过全身转录组的 Illumina 测序来比较野生型和非共生果蝇。该项目将为培养多种分子和生理学技术的熟练程度提供途径，这将为候选人成为疾病媒介分子生理学的权威奠定基础。

DESCRIPTION (provided by applicant): The long-term objectives of the candidate (Dr. Joshua B. Benoit) are to become an authority on the physiology of insect disease vectors and increase public awareness of the role of insects on public health. The goal of this fellowship is to develop the candidate's ability to assess the biochemistry, molecular biology and physiology of vector reproduction. The training plan provides opportunities to conduct extensive studies at the interface between molecular biology and reproductive physiology. Tsetse flies are the sole vector of African sleeping sickness. Vaccine-based methods have not been successful for control of trypanosomiasis. A unique aspect of this disease vector is its K-strategy of reproduction. Mothers provide nutrients, directly to their intrauterine progeny via a milk gland and only give birth to 8-10 offspring in their lifetime. Interference with tsetse reproduction can supplement trapping and other control methods used to reduce tsetse population numbers and decrease disease transmission. Currently, there is little information on tsetse reproduction, particularly how nutrients are mobilized to the larva. Since this aspect is unique in tsetse, control techniques blocking maternal nutrients could be tsetse specific. The objectives of this study are to 1) characterize the metabolism and transfer of lipids from the mother to her offspring, and 2) decipher how this process is influenced by the presence of symbiotic bacteria. Characterization of lipid metabolism and mobilization will focus on the adipokinetic hormone (AKH; this portion has been completed) and Brummer (Bmm) lipase systems, as well as lipid transport by lipophorin (Lp) during the reproductive cycle. Gene expression and protein synthesis will be assessed spatially and temporally during larval intrauterine development. Function of these proteins will be assessed utilizing RNA interference to knockdown their expression. Role of symbiotic bacteria in lipid metabolism and movement during reproduction will be assessed by analyzing molecular and physiological differences between wild type and antibiotic-treated aposymbiotic flies (flies lacking symbionts). Aposymbiotic flies are infertile and abort undeveloped larva early in their developmental cycle. The mechanism(s) underlying this phenotype are unknown, but malnourishment due to milk secretions deficient in essential nutrients is the current hypothesis. A possible deficiency is in the lipid content of the milk. Differential lipid composition between wild type and aposymbiotic flies will be assessed by obtaining milk and comparing the quantitative and qualitative differences in milk lipids. Fertility can be restored in aposymbiotic flies by supplementation of blood meals with yeast extract. Lipid compositions of aposymbiotic flies with and without yeast extract-supplemented blood will also be performed. To determine genetic effects of dietary supplementation, wild-type and aposymbiotic flies will be compared by Illumina sequencing of whole body transcriptome. This project will provide avenues to develop proficiency in multiple molecular and physiological techniques that will provide the groundwork for the candidate to become an authority on the molecular physiology of disease vectors.