Effect of gut microbes on olfactory behavior of Drosophila melanogaster larva

Abstract

The symbiotic relationship between an animal and its gut microbiota is known to influence host neural function and behavior. The mechanisms by which gut microbiota influence brain function are not well understood. This study measures the impact of gut microbiota on olfactory behavior of Drosophila larvae and explores possible mechanisms by which gut microbiota communicate with neural circuits. The microbiota load in Drosophila larvae was altered by treating them with antibiotics or probiotics. Control larvae and larvae with altered microbiota loads were subjected to olfactory assays to analyze the chemotaxis response of larvae to odorants. Larvae treated with antibiotics had reduced microbiota load and exhibited reduced chemotaxis response toward odorants compared to control animals. This behavioral phenotype was partially rescued in larvae treated with probiotics that resulted in partial recovery of microbiota loads. Expression levels of several olfactory genes in larvae subjected to different treatments were analyzed. The results suggest that the expression of certain components of the GABA signaling pathway is sensitive to microbiota load. The study concludes that the microbiota influences homeostatic mechanisms in the host that control GABA production and GABA-receptor expression, which are known to impact host olfactory behavior. These results have implications for understanding the bidirectional communication between a host organism and its microbiota as well as for understanding the modulation of olfactory neuron function.

Keywords: GABA signaling; olfaction; olfactory receptor neurons.

Figure 1.. Monitoring microbiota load in larvae using a bacterial growth medium.

Each quadrant represents homogenate from a single larvae grown on normal food (A), larvae grown on antibiotic food (B), larvae from antibiotic-treated parents grown on normal food (C), and probiotic food (D). The image is a representative image from 8 separate trials.

Figure 2.. Monitoring presence of microbiota in larvae using multiplex PCR of Drosophila and bacterial DNA.

Multiplex PCR reactions containing primers for Drosophila (grey arrows) and bacterial (black arrows) targets were performed on larval DNA samples. Nonspecific bacterial 16S (A), L. brevis (B), L. fructivoran (C), A. pomorum (D), L. plantarum (E), and A. tropicalis (F) were targeted separately. Lanes were loaded as follows: larvae grown on normal food (lanes 2–4), larvae grown on antibiotic food (lanes 5–7), and larvae from antibiotic-treated parents grown on normal (lanes 8–10), and probiotic food (lanes 11–13) minus-template control (lane 14), DNA ladder (lanes 1, 15).

Figure 3.. Microbiota load impacts larval olfactory behavior as assayed in a two-choice small-format paradigm.

Two-choice behavior paradigm were performed on all larval treatments for odors 4-Hexene-3-one, ethyl acetate, and pentyl acetate; mean response indices (RI) are shown. Each bar represents RI ± SEM (n = 10). ANOVA with a Bonferroni post-hoc test (* = p < 0.05, *** = p < 0.001) was performed to test for significance.

Figure 4.. Microbiota load impacts expression levels of GABA signaling components measured using qRT-PCR analysis.

Drosophila larvae grown on normal untreated food (black bars), antibiotic-treated food (white bars), and larvae grown on probiotic food from antibiotic-treated parents (grey bars) are shown. Bars represent average fold change in expression (relative to three neuronal genes) ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.