Aging is generally defined as a progressive loss of physiological function associated with cellular hallmarks, such as telomere attrition, loss of proteostasis, chronic inflammation, and disabled macroautophagy (Lopez-Otin et al., 2023). One particular age-related change observed in zebrafish, mice, and Drosophila known to presage death is an increase in intestinal barrier permeability (Zane et al., 2024). This dysfunction of the intestinal barrier co-occurs with changes in the intestinal microbiota, misdifferentiation of intestinal stem cells, and increased levels of inflammation (Salazar et al., 2023). Protective measures aimed at preserving the intestinal barrier have been demonstrated to extend Drosophila life span (Salazar et al., 2018; Qin et al., 2025). The Smurf assay is an effective and simple tool used to monitor the integrity of the intestinal barrier, wherein flies are fed food containing 2.5% blue food coloring for 24 hours. Flies with a more permeable intestinal barrier and a higher-risk of mortality, the dye leaks through and turns the entire fly blue (Rera et al., 2011).

The functionality of the mitochondria is well established as important in determining longevity, and tissue-specific knockdown of electron transport chain components has been demonstrated to extend life span in Caenorhabditis elegans and Drosophila melanogaster (Copeland et al., 2009; Durieux et al., 2011). The role of the electron transport chain in neurons is noteworthy, as robust life span extension has been observed in Drosophila when the complex V component ATPsynβ L was knocked down in glutamate neurons but not other neuronal subtypes (Keppley et al., 2018; Landis et al., 2023). Glutamate neuron-specific RNAi of ATPsynβL was also associated with a pronounced increase in daytime sleep early and midway through adult life (Forrest et al., 2026).

Given the impact that glutamate neuron-specific RNAi of ATPsynβL has, we wanted to determine if it also affected the age-related degeneration of the intestinal barrier. We fed the flies blue-dyed food at 5, 30, and 50 days of age. The controls in our experiments included flies heterozygous for the glutamate neuron-specific GAL4 driver ( D42 ) or the UAS- ATPsynβL -RNAi construct. In our investigations, we noticed that loss of the intestinal barrier only occurred later, at 50 days of age, and that glutamate neuron-specific ATPsynβL RNAi protected against this barrier loss. Specifically, when we conducted the Smurf assay at 5 and 30 days of age, controls and activated RNAi flies showed a statistically equivalent range of 0 – 1.3% Smurf phenotypes ( Figure 1A, B). At 50 days of age, however, glutamate neuron-specific RNAi of ATPsynβ L protected against intestinal barrier dysfunction by 56.5% compared to the control flies ( Figure 1B ).

We validated the functionality of the RNAi construct by measuring ATPsynβL expression levels by qRT-PCR. Even though we used the D42 -GAL4 driver line to test for intestinal barrier dysfunction, we used a different GAL4 line, the ubiquitous Act5C -GAL4 driver, to simply test for ATPsynβL knockdown. Certainly, these two GAL4 lines do not have the same pattern or strength of gene expression, but the Act5C -GAL4 line offers one means of testing RNAi effectiveness on ATPsynβL specifically. Using whole flies or excised heads from a D42 -GAL4 genetic background for our qRT-PCR experiments would contain non-RNAi active cells and provide an inaccurate measurement of ATPsynβL knockdown. Immunostaining would require testing with an unproven anti- ATPsynβL antibody, a procedure beyond the limited scope of these experiments. In our qRT-PCR experiments, we noticed a significant 38 and 50% knockdown of ATPsynβL mRNA transcript from the GAL4 and RNAi control lines, respectively (P < 0.0001) ( Figure 1C ). These results demonstrate that GAL4 activation of the UAS- ATPsynβL -RNAi line does specifically knockdown ATPsynβL expression.

Changes to the intestinal microbiota are associated with age-related intestinal barrier dysfunction, and an increased number of intestinal bacteria correlates with a shorter life span (Ludington et al., 2025). To measure the changes in bacterial amounts in the ATPsynβL RNAi flies, we utilized qRT-PCR with universal primers to the bacterial 16S rRNA gene (Claesson et al., 2010). We limited our qRT-PCR experiments with dissected intestines from 50 day old flies, as the glutamate neuron-specific RNAi did not have any effects on earlier time points in adulthood. Flies with glutamate neuron-specific RNAi of ATPsynβL had a 55.1 – 61.6% (P < 0.0001) decrease in bacterial amount, when compared to the control flies ( Figure 1D ).

In this report, we explore the possible connection between the intestinal barrier and longevity in flies with glutamate neuron-specific RNAi of the complex V gene ATPsynβL . We observed that activated RNAi led to lower levels of intestinal barrier dysfunction late in the fly life, a change correlated with decreased amounts of intestinal bacteria. It is not surprising that the ATPsynβL flies have a more intact intestinal barrier, given the number of reports demonstrating the importance of the intestinal barrier on longevity. Overexpression of occluding junction proteins or induced mitochondrial activity in the intestines have been shown to protect against intestinal barrier dysfunction and dysbiosis as well as extend life span (Salazar et al., 2018; Rera et al., 2011). It is worth noting that we target glutamate neurons to perturb a complex V gene and not the intestines directly. While glutamate neurons are known to innervate the intestinal proventriculus and hindgut, it is a little surprising that they would have such a robust cell non-autonomous effect on intestinal barrier function (Kuraishi et al., 2015). It remains to be seen whether the life span extension we observe in the ATPsynβL RNAi flies is mediated solely through the protection of the intestinal barrier or through some other unknown mechanism.

General husbandry

Flies were fed standard molasses food and reared at 25°C with a 12-hour light:dark cycle. The RNAi line and GAL4 lines were backcrossed a minimum of five times to the white ¹¹¹⁸ laboratory strain to minimize hybrid vigor in our studies (Dietzl et al., 2007). The GAL4 control strains were the products of crosses between the D42 -GAL4 or Act5C -GAL4 and our white ¹¹¹⁸ strain. The RNAi control strain was the product of a cross between UAS- ATPsynβL -RNAi and white ¹¹¹⁸ . The activated RNAi line was the result of a cross between the D42 -GAL4 or Act5C -GAL4 and the UAS- ATPsynβL -RNAi line.

Smurf analysis

To test the integrity of the intestinal tract, a Smurf assay was conducted as previously described (Rera et al., 2012). Twenty females were housed with five males per vial under standard culture conditions and aged to 5, 30, or 50 days of adulthood. Aged flies were fed food containing 2.5% FD&C #1 blue food coloring (Spectrum Chemical, Gardena, CA) for 24 hours and scored for the Smurf phenotype. Measurements were conducted with at least six replicates and at least 230 females.

Quantitative real-time PCR

RNA from 5 day old flies was extracted using the RNeasy Mini protocol (Qiagen, Hilden, Germany), and isolated RNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). 700 micrograms of RNA were reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). qRT-PCR was performed on a CFX Connect Detection System (Bio-Rad, Hercules, CA) using Sso Advanced Universal SYBR Green Mix (Bio-Rad, Hercules, CA).

Each sample was analyzed with six reactions, along with controls (NTCs) without cDNA in the PCR. The specificity of each amplified reaction was verified by a dissociation curve analysis after each measurement. To determine primer efficiency, serial 2-fold dilutions of each primer set were used to generate a standard curve, and efficiencies (E) were determined based on the slope (M) of the log−linear portion of the standard curve (E = 10−1/M − 1 × 100).

Statistical analysis

Data were analyzed using GraphPad Prism (Version 10.5.0 (774), San Diego, CA) and Excel (Microsoft) software. One-way ANOVA tests (Tukey HSD) were run to determine statistical significance.

Stocks

The D42 -GAL4 (RRID: BDSC_8816) and Act5C -GAL4 (RRID: BDSC_4414) fly strains were obtained from the Bloomington Stock Center (NIH P40OD018537, Bloomington, IN). The UAS- ATPsynβL -RNAi line (VDRC ID: 22112) targeting Complex V of the electron transport chain was purchased from the Vienna Drosophila RNAi Center (Vienna, Austria).

Primers

The sequences for the 16S ribosomal, ATPsynβL , and the Act5C DNA primers have been previously reported (Forrest et al., 2026; Claesson et al., 2010; Rana et al., 2017).