Larval mosquito feeding and microbial interactions

描述（由申请人提供）：水生幼虫栖息地的成蚊的生长和生产受到可折射有机物质转化为可消化和可消化微生物生物量的限制。有机物转化为微生物生物量的速率受到颗粒基质质量的影响，这表现为抑制和刺激微生物和幼虫生长的双重作用。我们的长期目标是描述蚊子生产的生物学基础和限制因素，确定这些资源的利用效率，并制定一个现实的幼虫生长模型。我们确定了树洞生态系统作为研究对象，树洞生态系统中栖息着三角纹螟、日本纹螟和淡纹库蚊，它们分别是人类病原体的媒介和外来入侵的媒介。研究方法包括一个无菌（无菌）实验系统，其中可以提供包括微生物在内的单个食物来源；稳定同位素分析（13C/12C; 15N/14N）追踪幼虫食物来源；微生物群落结构的分子分析；编码大分子降解酶的微生物基因的功能基因阵列分析，包括木聚糖酶、纤维素酶和几丁质酶；在微观和自然实验中，微生物生物量和代谢活动相对于幼虫生长进行了量化。提出了一种生长模型，其中发育时间和质量，蜕变（即化蛹）被假设为灵活的生活史特征，其最小值受到营养和遗传的限制，成为定义体型和影响成虫存活率的化蛹窗口；两者都是关键适应度参数，也是向量容量的重要组成部分。实验将描述它们之间的关系，并揭示导致它们变异的环境和遗传因素。目前对幼虫生长速率的理解要求转化效率（每摄入毫克食物同化的毫克食物）远远高于观察到的，因此我们将迭代测试相对于不同品质的天然微生物食物来源的生长响应，将观察到的生长数据拟合到模型中，并相应地修改转化效率估计。将比较共享相同幼虫栖息地和资源的三种蚊子，以评估在激烈的种内和种间竞争条件下的生长速度，既可以预测竞争结果和生态位移的潜力，又可以量化幼虫食物利用的相对效率。利用分子遗传工具操纵黄杆菌菌群的主要组成部分来表达编码具有幼虫毒素和生长抑制特性的蛋白质的基因，包括来自芽孢杆菌的哭毒素基因和几丁质酶。蚊子负责传播大量人类病原体，包括引起疟疾、丝虫病和虫媒病毒感染的病原体。成蚊、雌蚊（吸血和传播病原体阶段）的丰度和寿命取决于幼虫环境中的营养等因素，这些因素的制约因素是本文研究的重点。

DESCRIPTION (provided by applicant): The growth and production of adult mosquitoes from the aquatic larval habitats is constrained by conversion of refractile organic material into ingestible and digestible microbial biomass. Rates of conversion of organic matter into microbial biomass are tempered by the quality of particulate substrates that is manifested in dual roles of inhibition and stimulation of microbial and larval growth. Our long-term goal is to delineate the biological basis for and constraints on mosquito production, to identify the efficiency of utilization of these resources, and to elaborate a realistic model of larval growth. We have identified the tree hole ecosystem for study, harboring the species Ochlerotatus triseriatus, Ochlerotatus japonicus, and Culex pipiens, two vectors of human pathogens and one an exotic invader. Research approaches include an axenic (germ-free) experimental system into which individual food sources including microorganisms can be provisioned; stable isotopic analyses (13C/12C; 15N/14N) to track larval foods to their sources; molecular analyses of microbial community structure; a functional gene array analysis of microbial genes encoding enzymes involved in large biomolecule degradation, including xylanases, cellulases, and chitinases; and microcosm and naturalistic experiments in which microbial biomass and metabolic activity relative to larval growth are quantified. A growth model is proposed in which developmental time to, and mass at, metamorphosis (i.e., pupation) are postulated to be flexible life history traits whose minima are nutritionally and genetically constrained into a pupation window defining body size and affecting adult survivorship; both are key fitness parameters and both important components of vectorial capacity. Experiments will delineate relationships among them, and reveal environmental and genetic components contributing to their variation. The current understanding of larval growth rates requires conversion efficiencies (mg food assimilated per mg food ingested) far higher than observed, thus we will iteratively test growth responses relative to natural microbial food sources of various qualities, fit observed growth data to models, and revise conversion efficiency estimates accordingly. The three mosquito species sharing the same larval habitat and resources will be compared to assess growth rates under conditions of intense intra- and interspecific competition, both to make predictions of competitive outcomes and potential for ecological displacement, and to quantify comparative efficiency of utilization of larval foods. Manipulation of the Flavobacterium component of the microbial community a dominant member with molecular genetic tools to express genes encoding proteins having larval toxin and growth inhibiting properties will be conducted, including cry toxin genes from Bacillus, and chitinases. PUBLIC HEALTH RELEVANCE Mosquitoes are responsible for transmission of large number of human pathogens, including those causing malaria, filariasis, and arboviral infections. Abundance and longevity of adult, female mosquitoes (the blood feeding and pathogen transmitting stage) are dependent upon the nutritional and other factors operating in the larval environment, the constraints of which are the focus of the research here.