Larval Mosquito Feeding and Microbial Interactions

描述(由申请人提供)：自该项目开始以来，我们的全球假设是，蚊子幼虫栖息地的微生物群落动态控制着幼虫的生长和随后的成虫羽化，并通过幼虫营养的直接和间接影响影响蚊子种群动态和媒介能力。通过了解微生物群落在支持或抑制幼虫生长方面施加的控制因素，将阐明对蚊子生态和媒介控制方法的新见解，包括通过基因转化方法操纵微生物群落的方法。在这个相互竞争的延续方案中，我们将直接以我们最近的研究进展为基础，提出新的框架假设和新的方向，涉及三个相互关联的具体目标，涵盖以下目标：(1.1)开发一个无菌(无菌)实验系统，用于直接分析单个微生物成分对幼虫生长反应的影响，在野外实验中使用稳定的同位素示踪剂，在实验室实验中使用放射性同位素示踪剂；(1.2)分析与微生物生物膜有关的胞外多糖以及溶解和胶体有机化合物对幼虫营养和生长的贡献；(1.3)估计天然微生物食物的同化效率；(2.1)利用16S rDNA序列文库和单一碳源利用表型分析生物膜相关微生物群落的分子特征；(2.2)真菌和原生动物群落以及与底物生物膜相关的生物量的特征；(2.3)磷限制对微生物生物量生产和蚊子生长的作用的分析；(3.1)在选择培养基上分离优势黄杆菌、丛枝杆菌和棘假单胞菌；(3.2)利用微型Tn5转座子通过染色体整合将Cryl 1A杀虫毒素基因转化这些细菌菌株；(3.3)研究转化子对蚊子幼虫的杀灭效果，以及在模拟蚊子栖息地中的持久性。

DESCRIPTION (provided by the applicant): Since the inception of this project, our global hypothesis has been that microbial community dynamics in larval mosquito habitats govern larval growth and consequent adult emergence, and through direct and indirect influences of larval nutrition affect mosquito population dynamics and vectorial capacity. By understanding the controlling factors imposed by the microbial community in support or suppression of larval growth, new insights into mosquito ecology and vector control methods will be elucidated, including means of manipulating the microbial community with genetic transformation methods. In this competing continuation proposal, we will build directly upon our recent research advances with newly-framed hypotheses and new directions that involve 3 inter-related Specific Aims covering the following objectives: (1.1) Development of an axenic (germ-free) experimental system for direct analysis of the effects of individual microbial components to larval growth responses, using stable isotope tracers in field experiments and radio-isotope tracers in laboratory experiments; (1.2) Analysis of the contribution of extracellular polysaccharides associated with microbial biofilms, as well as dissolved and colloidal organic compounds, to larval nutrition and growth; (1 .3) Estimation of assimilation efficiencies of natural microbial foods; (2.1) Molecular-based characterizations of the biofilm-associated microbial community using 16S rDNA sequence libraries, and sole-carbon source utilization phenotype analysis; (2.2) Characterization of the fungat and protozoan community and biomass associated with substrate biofllms; (2.3) Analysis of the role of phosphorous limitation to microbial biomass production and mosquito growth; (3.1) Isolation on selective media of dominant Flavobacterium, Comamonas, and Pseudomonas spinosa strains; (3.2) Transformation of these bacterial strains with Cryl 1A larvicidal toxin genes by chromosomal integration using mini-TN5 transposons; and (3.3) Studies of transformant efficacy against mosquito larvae, and persistence in simulated mosquito habitats.