Methods for Collection, Quantification and Identification of Biomolecules from Drosophila melanogaster

The development of methods to generate quantitative chemical content information from precise tissue locations is needed to understand fundamental cellular and tissue physiology. This work describes a method to perfuse the extracellular fluid of fly brains in vivo using μLFPP for quantitative chemical content determinations, as well as a preliminary study of npc1a mutant flies. Miniaturization of push-pull perfusion probe designs allowed the development of methods for probe tip placement into and sampling from the fruit fly’s brain. Perfusate analysis identified and quantified arginine, octopamine, histidine, taurine, glycine, glutamate, and aspartate. Perfusate protein content was quantified to yield an average of 0.57 ± 0.092 µg of protein per fly and 159 unique proteins identified over the course of this study. The perfusate data did not exhibit any statistical differences based on sex. The perfusate analysis was compared to hemolymph samples to confirm probe placement in fly brain tissues. Appearance of probe placement into the brain space was confirmed with the following observations. Hemolymph and perfusate samples were found to contain analytes unique to each sample type. Quantitated levels of perfusate were not a simple dilution of hemolymph content. Further, the discovery of perfusates with composition similar to both hemolymph and brain perfusate when damage was intentionally inflicted supports the observation that perfusates are distinct from hemolymph. The probe placement into the fly brain was also confirmed by several protein IDs. The analysis of perfusate collected for greater than an hour of sampling exhibits the possibility of monitoring applications. Tissue and hemolymph collection were performed to determine lipid compositional differences between control and npc1a mutant flies. Experimental results suggests that there are compositional differences in each group’s lipidome and how the downregulation of LPC 16:0/0:0 could be evidence of how ineffectively lipids are transported around the npc1a fly body. Altogether, this work demonstrates the viability of performing μ low-flow push−pull perfusion for in vivo studies of fly brain tissues to identify and quantitate neurotransmitter content, as well as the potential for annotating lipid content and shifts that correspond with specific biological mechanisms in fruit flies.