Nanoliter sampling and analysis of Drosophila melanogaster hemolymph

Developing tools to understand the accurate blood chemical composition of organisms is vital to the study of human health. Animal models are also often used due to cost and ethical considerations. Drosophila melanogaster, commonly known as the fruit fly, is an extensively used insect animal model. For decades, the extremely small size of the organism (about 3 mm) has complicated the collection of its blood (hemolymph) for chemical analysis. This dissertation presents techniques for sampling and analysis of nano-volumes of hemolymph from indivdual larval and adult fruit flies.;The developed sampling techniques enable collection of 50-300 nL hemolymph volumes from individual Drosophila larvae and 8-45 nL hemolymph volumes from individual fruit flies within 30 s. Capillary electrophoresis with laserinduced fluorescence detection and MALDITOF-MS techniques were utilized for the chemical analysis. This is the first study to report qualitative and quantitative chemical analysis of hemolymph from individual fruit-flies and larvae.;Amino acids content was determined from the hemolymph of stressed and non-stressed wild type and seven mutant fly alleles to better understand the physiological consequences of genetic mutations. There is a significant influence of the sampling conditions on observed hemolymph composition. In particular, stressed flies showed glutamate elevations and flies with strenuous motions showed elevated arginine levels in hemolymph. While these glutamate and arginine responses were common among the genotypes, genotype-specific behavior-related hemolymph chemical changes were also observed. As a step toward monitoring hemolymph composition, the sampling technique was modified to allow collection of two hemolymph samples from the same adult fly.;The analytical tools for hemolymph analysis presented in this dissertation are low cost, efficient, and robust. The method enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, alterations, and effects of evaporation compared to the traditional fruit fly sampling techniques. The results demonstrate the tools for analysis of nano-volume samples and contribute means to obtain fruit fly chemical information that may play significant roles in solving human health problems.