title: Introducing Commensal Bacteria from Caenorhabditis elegans to Drosophila melanogaster
title: Effects on Health, Ageing and Insulin-like Signalling
titleType: Subtitle
Creators: name: Ng, Tze Chang (Justin)
Publisher: name: Lancaster University
Year: 2023
Subjects: Ageing
Caenorhabditis elegans
Drosophila melanogaster
Insulin/insulin-like signaling pathway
Gut Microbiome
CeMbio
Fluorescence microscopy
Behaviour
Survival analysis
Exploratory walking
Negative geotaxis
Sleep analysis
Gut permeability
Fecundity
Oviposition

description: Background: With the rise of age-related diseases and challenges associated with ageing, there is a growing demand for innovative approaches to promote healthspan. The gut microbiome, essential for health and homeostasis in vertebrates and invertebrates, emerges as a promising avenue for enhancing overall health and addressing age-related disorders. However, little is known about how gut commensals affect host biological processes, let alone what constitutes a healthy gut microbiome. Purpose: Given the shared ecological environments and co-evolutionary history between nematode roundworms and fruit flies, this study introduces Caenorhabditis elegans commensal bacteria (derived from the CeMbio database) to Drosophila melanogaster to probe for bacterial impacts on ageing and interactions with evolutionarily conserved nutrient-sensing pathway—insulin/insulin-like signalling pathway (IIS). Methods: To ascertain successful bacteria colonisation of the fly gut, fly media was adapted for bacteria growth and candidate bacteria were fluorescently transformed to enable direct observation under fluorescence microscopy. Using the optimal experimental conditions and bacterial combination, the impacts of the introduced bacteria on fly health and ageing were evaluated by assaying fly lifespan, exploratory walking behaviour, gut integrity, sleep, neuromuscular function, and fecundity. To gain insights into potential crosstalk between bacteria and IIS, bacteria were also introduced to flies with impaired IIS achieved through either ablating cells that produce Drosophila insulin‐like peptides 2-3 (d2-3GAL4/UAS-rpr) or downregulating insulin receptors expression in serotonergic neurons (trhGAL4/UAS-InRDN). Results: From 16 CeMbio bacteria, 11 bacteria grow on fly media in contingent that antifungals were omitted. Thus, all subsequent experiments utilised additive-free fly media. Out of 24 transformations, 6 new fluorescent bacteria were generated and validated for their correct identity. Three fluorescent bacteria—Ochrobactrum vermis (MYb71-sfGFP), Enterobacter ludwigii (MYb174-dTomato), and Enterobacter cloacae (CEent1-mPlum)— successfully colonised the fly gut. When introduced together, these three bacteria reduced fly median lifespan but increased early-age egg laying, resulting in earlier egg exhaustion. Across life, these bacteria attenuated changes to exploratory walking and sleep behaviour induced by IIS reduction. However, fly gut permeability and neuromuscular function remained unaffected. Conclusions: MYb71-sfGFP, MYb174-dTomato, and CEent1-mPlum may benefit adult flies during early age but be detrimental later as bacterial load increases. Regardless, these bacterial-host interactions crosstalk with IIS to affect complex behaviours like exploratory walking and sleep. These findings provide evidence that the study of ecologically relevant commensal bacteria from C. elegans can be translated onto D. melanogaster to further probe for interactions affecting evolutionarily conserved biological pathways and complex behaviours.