Jan L. Bilbao-Del Valle¹, Patricia V. Pujols¹, Kerianalee Rivera-Cruz¹, Imilce A. Rodriguez-Fernandez¹

¹College of Natural Sciences, Department of Biology, University of Puerto Rico Rio Piedras

INTRODUCTION: The microbiome is an unseen “organ” consisting of all the microorganisms that naturally inhabit an organism, including bacteria, fungi, viruses, archaea, and eukaryotes. This complex microbiome serves essential functions such as maintaining homeostasis, supporting the immune system, and producing vital substances like vitamin B12 and vitamin K, as well as protecting against pathogens. However, factors like age, diet, infections, antibiotics, and alcohol consumption can disrupt the gut-microbiota brain communication system.

METHODS: The objective of this research is to evaluate three methods of different complexities to identify the most informative when measuring changes in the gut microbiome. These are Colony Forming Units (CFUs), Nanopore sequencing technology and Biolog® Ecoplates. As a read-out, we used a paradigm showing that exposing flies to 50% ethanol vapor is enough to increase microbiota (preliminary data from the lab). To test these three methods, we took wild-type (Canton-S) young flies (7d old) and exposed them to water (control) or one time to 50% ethanol vapor.

RESULTS: We found that a single exposure to ethanol significantly increases intestinal Lactobacilli, Acetobacter, and Enterobacteria CFUs. For Nanopore sequencing we took young (7d old) and old flies (50d old) exposed to water (control), 50% ethanol once or twice. Intestines were flash frozen, DNA extracted and the entire 16s rRNA gene (~1500 bp) was amplified by PCR. Then, barcodes will be ligated, and sequences will be analyzed using Nanopore technology. We find that the Biolog® EcoPlate which measures bacteria utilization of 31 carbon sources remained unchanged when tested with Drosophila gut homogenates compared to a positive control.

CONCLUSION: Out of the three methods CFUs and Nanopore seem to be the most informative whereas EcoPlates failed to detect bacteria found in the fly intestine. Ongoing experiments are aiming at confirming the CFUs results with the Nanopore sequencing.

ACKNLOWLEDGMENTS: Thanks to Dra. Imilce Lab, NIH-GREAT (IDGeNe) program and its respective coordinators Dr. Jose E. Garcia Arrarás and Dr. Humberto Ortiz Zuazaga, and Miguel Urdaneta for teaching us about Nanopore sequencing and for providing his Nanopore machine.