Treatment with mitochondrial targeting antibiotics improves survival outcomes after Flock House virus infection in young and aged Drosophila melanogaster

Treatment with mitochondrial targeting antibiotics improves survival outcomes after Flock House virus infection in young and aged Drosophila melanogaster | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Treatment with mitochondrial targeting antibiotics improves survival outcomes after Flock House virus infection in young and aged Drosophila melanogaster Dean Bunnell, Madelyn Buhl, Justin McGee, Grace Milas, Stanislava Chtarbanova This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6816306/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aged organisms are more susceptible to infectious diseases, including infections with RNA viruses. Mitochondrial dysfunction is one of many hallmarks of aging that could affect this increased susceptibility, as the relationship between immunity and metabolism is crucial to manage infections. Using Drosophila melanogaster - Flock House virus (FHV) host-virus interactions model system, previous work has identified differences in young and aged flies’ ability to modulate oxygen consumption rates (OCR). Here, we hypothesized that interventions that reduce OCR could improve survival of FHV, as observed in young flies. Tetracycline (TTC) and rifampicin (RIF) antibiotics disrupt mitochondrial translation and transcription respectively because of mitochondria's bacterial ancestry. The mitochondrial unfolded protein response (UPR mt ) is activated by mitochondrial stressors, including reactive oxygen species, defects in oxidative phosphorylation, and mitonuclear protein imbalance. UPR mt activation initiates retrograde signaling to the nucleus, prompting transcription, translation, and import of nuclear proteins to resolve stress. We showed TTC or RIF treatment extended survival in young and aged flies after FHV infection, independently of virus load modulation. Furthermore, we demonstrate that bacterial loads are not significantly different between FHV-infected flies and controls, and that the protective effect of TTC likely occurs independently of its antimicrobial properties. We observed increased expression of genes involved in the UPR mt , glycolysis, and oxidative stress response with TTC treatment. Our results suggest perturbing mitonuclear protein balance with TTC or RIF could activate the UPR mt and improve outcomes of virus infection. Drosophila melanogaster innate immunity aging virus infection antibiotics mitochondrial unfolded protein response Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Living organisms are constantly exposed to stressors capable of altering physiological functions that may have implications on their survival. These stressors include but are not limited to exposure to variable environments, nutrient limitations, injury, oxidative stress, and disease states [ 1 ]. Cells, tissues, and organisms possess mechanisms of ‘adaptive homeostasis’ [ 2 ] capable of detecting these constant and variable stressors and generating the appropriate response to restore homeostasis and in many cases promote survival. A key component of this response is the ability to employ these adaptive mechanisms quickly and inactivate the response when the stress or damage has been resolved. An abundance of evidence in many model organisms has demonstrated that aging results in a functional decline in adaptive homeostatic mechanisms [ 3 ]. The immune system also experiences a progressive functional decline with age, or immunosenescence [ 4 ]. Ultimately, this decline renders organisms more susceptible to infections, including RNA virus infections [ 5 – 8 ]. A major gap exists in our understanding of the underlying factors implicated in older individuals’ increased susceptibility to infections. The world population of people 65 years or older is projected to more than double by 2050 [ 9 ], so identifying strategies to promote aged organisms’ ability to survive infection and improve health will be critical in the near future. Drosophila melanogaster and FHV provide a powerful aged host-RNA virus interaction model to elucidate mechanisms that improve the aged host’s survival. Although invertebrates such as Drosophila lack the adaptive immunity found in vertebrates, they provide a unique model to investigate innate immunity. In vertebrates, innate immunity is the first line of defense when faced with infection and aids adaptive immune system activation, as the two branches must act synergistically to fight infection. FHV is an insect virus with a positive sense, RNA genome composed of two single-stranded RNA molecules copackaged in a virion. Its genetic simplicity has made it a widely used model in host-virus interactions, and its replication cycle is well characterized [ 10 ]. It replicates within replication spherules formed on the outer mitochondrial membrane [ 11 – 13 ]. FHV infection is pathogenic to flies and results in mortality within days [ 12 , 14 – 16 ]. During pathogenic infections, two evolutionarily conserved defense strategies must act in conjunction to limit pathology. Resistance involves detection of the pathogen by recognition of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) followed by activation of effector mechanisms capable of reducing pathogen load. while disease tolerance limits tissue damage and consequently disease severity without affecting pathogen load. In Drosophila , several antiviral resistance mechanisms have been identified in response to FHV infection, including RNA interference (RNAi) [ 15 ], apoptosis [ 17 ], and hemocyte-mediated phagocytosis [ 18 ]. Disease tolerance mechanisms have not been characterized as extensively as resistance mechanisms. The epigenetic regulator, Histone H3 lysine 9 (H3K9) methyltransferase G9-alpha, has been shown to play a role in disease tolerance to FHV by preventing hyperactivation of the JAK-STAT pathway [ 11 ]. Our lab previously showed that FHV infection causes mortality in aged flies’ significantly faster than in young flies without observing an increase in viral titers [ 14 ], suggesting an age-related decline in disease tolerance to FHV. Transcriptomic analysis revealed many genes whose products are involved in metabolic processes, mitochondrial structure, and mitochondrial respiration were regulated to a stronger extent in older flies 48h p.i. [ 14 ]. This finding implicated host metabolism as a potential factor differentially regulated with age that may increase susceptibility to FHV. Previously, we conducted a longitudinal, single-fly respirometry study to measure whole organismal oxygen consumption rates (OCR) in single flies as a proxy for metabolic rate and mitochondrial function. In this study, we determined if metabolic rates differed with age after FHV infection and whether differences may be correlated with mortality [ 19 ]. We found young flies reduced OCR after FHV infection, but aged flies showed no significant changes in OCR, providing more evidence that metabolism could impact age-related susceptibility to FHV [ 19 ]. In fact, the reduction of OCR in young flies is indicative of an evolutionarily conserved strategy of metabolic rate depression or hypometabolism, which has been reported as a survival strategy in the presence of various stressors across organisms (reviewed in [ 20 , 21 ]), including the promotion of disease tolerance [ 22 ]. Several classes of antibiotics have demonstrated protective effects in the context of viral infections, cancer, sepsis, and inflammatory disorders that are not attributed to direct antimicrobial effects [ 23 – 27 ]. Since mitochondria retain many characteristics of their bacterial ancestors, many classes of antibiotics can act via similar mechanisms on eukaryotic mitochondria and cause dysfunction [ 28 , 29 ]. In bacteria, tetracycline inhibits bacterial protein synthesis by occupying the A-site of the bacterial 30S ribosomal subunit and blocking recruitment of the aminoacyl-tRNA [ 30 ]. Rifampicin inhibits bacterial DNA transcription by inhibiting RNA polymerase [ 31 ]. In eukaryotes like Drosophila , these antibiotics alter the balance of nuclear-encoded and mitochondrial-encoded proteins, which is crucial for mitochondrial electron transport chain function. High-resolution respirometry has shown doxycycline, a tetracycline class antibiotic, decreases OCR in flies [ 29 ], and Drosophila’s mitochondrial RNA polymerase was shown to be inhibited by RIF [ 32 ]. In Drosophila S2 cells, rifampicin was shown to have an antiviral effect against Drosophila C Virus (DCV) and Cricket Paralysis Virus (CrPV), which was associated with increased expression of detoxification enzymes of the cytochrome P450 family [ 33 ]. Drosophila ND23 is a nuclear gene that encodes a highly conserved subunit of the mitochondrial ETC Complex I. Previous studies have shown that ND23 60114 mutants display abnormal mitochondrial morphology, reduced ATP levels and have a significantly shorter lifespan in comparison to wild type controls [ 34 ]. Additionally, ND23 60114 mutants show increased expression of Hsp22 [ 35 ], a chaperone protein that has been identified to play a role in the Drosophila UPR mt [ 36 ]. Modest perturbation of the mitochondrial electron transport chain (ETC) function has been shown to increase lifespan in Drosophila , C. elegans , and mice [ 37 – 39 ]. Additionally, the mitochondrial unfolded protein response (UPR mt ) can be activated by various stressors including mitonuclear protein imbalance, misfolded protein accumulation, reactive oxygen species (ROS), mtDNA depletion, or mitochondrial toxins, and involves retrograde signaling to the nucleus to mount a transcriptional response to relieve mitochondrial stress [ 40 – 43 ]. This includes the transcription of genes that encode for mitochondrial chaperones, proteases, antioxidants, and glycolytic genes, all of which function to alleviate mitochondrial stress (reviewed in [ 44 ]). UPR mt activation has been shown to improve disease tolerance mechanisms in mammalian studies [ 24 , 25 ]. Therefore, many studies indicate that the presence of mitochondrial stress and subsequent activation of the UPR mt represents a hormetic response, where a mild amount of stress can have beneficial effects [ 24 , 25 , 45 , 46 ]. In this study, we sought to investigate whether antibiotics that impair mitochondrial metabolism improve survival of FHV infection with aging. We observed that TTC and RIF extended survival of FHV infection in both young and older flies without reducing virus. Survival extension occurred independently of antimicrobial properties after TTC treatment. In virus infected flies of both age cohorts, we found that TTC treatment affected the expression of genes implicated in the UPR mt , glycolysis, and oxidative stress response. Additionally, in comparison to wild type controls, ND23 60114 mutants displayed extended end point survival when infected with FHV. Overall, our findings indicate that mitochondrial dysfunction and resulting UPR mt activation improves survival outcomes of FHV infection likely by functioning as part of a disease tolerance mechanism. Materials and Methods Drosophila stocks, handling and aging Oregon R stock (#2376) was obtained from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Flies were maintained in vials containing Nutri-Fly Bloomington formulation food (Genesee Scientific, Cat #: 66–113) in a 25°C incubator with controlled 12 h:12 h light:dark cycle. For experimentation, Oregon R male flies were collected, aged, and flipped every 3–4 days into vials of fresh food until reaching the desired age. Young and aged cohorts were 5–9 days-old and 28–32 days-old, respectively. ND23 60114 stock was kindly provided by Dr. Barry Ganetzky and Canton S (#64349) was obtained from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Heterozygous controls were obtained by crossing Canton S males with ND23 60114 virgin females (Control: n = 53, FHV: n = 47) and the reciprocal cross (Control: n = 49, FHV = 56). Flies were aged to 5–9 days in vials containing Nutri-Fly Molasses formulation food (Genesee Scientific, Cat #: 66–116) in a 25°C incubator with controlled 12 h:12 h light:dark cycle. All fly lines were confirmed Wolbachia- negative prior to experimentation. Virus stock and injections FHV stock was propagated by infecting low passaged Schneider’s Drosophila line 1 (DL-1) cells. Virus stock solution was stored at − 80°C. All injections were conducted with a Nanoject II or III (Drummond Scientific), as described previously [ 14 ]. Flies were individually injected at the appropriate age with either 59.8 nL of cell culture media (Schneider’s media, 10% fetal bovine serum, and 1:100 penicillin-streptomycin (pen/strep U/mL); control injection) or FHV (2.57x 10 5 50% Tissue Culture Infective Dose (TCID 50 )/mL). The control-injected cohort was injected prior to the FHV-injected cohort to prevent cross-contamination. Flies were anesthetized with CO 2 on a fly pad (Genesee Scientific) and manipulated with a paintbrush during injections. Injected flies of the same experimental condition were then placed in groups of 10–15 flies per vial and allowed to rest at room temperature for 1 hour after injection before being placed in a 22°C incubator. For survival assays, survival status was recorded daily, and flies were flipped into vials of fresh food every other day. Antibiotic Food Preparation Nutri-Fly Bloomington formulation food (Genesee Scientific, Cat #: 66–113) or Corn-syrup/Soy media (Archon Scientific, W1) was prepared per manufacturer’s directions and supplemented with the appropriate dose of antibiotic or equal volume of vehicle serving as a control for each antibiotic treatment. Tetracycline (ThermoFisher Scientific, Alfa Aesar, Cat#: J61714) was supplemented to the food at the final concentrations of 0.05 mg/mL [ 47 ], 0.005 mg/mL, and 0.0005 mg/mL. The tetracycline stock solution was prepared using 80% ethanol, which served as the vehicle. Rifampicin (Tokyo Chemical Industry Co., Ltd., Product#: R0079) was supplemented to the food at a final concentration of 500 mg/L [ 48 ]. The rifampicin stock solution was prepared using DMSO, which served as the vehicle. Flies were placed on vehicle- or antibiotic-supplemented food only after they have been injected with either cell culture media (control) or FHV. Reverse transcription quantitative real time PCR (RT-qPCR) Total RNA was extracted from 5 whole flies (24, 72, or 120h p.i.) using the Quick-RNA MiniPrep kit (Zymo Research) and cDNA was prepared from 500 ng total RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Triplicate cDNA samples were amplified using the PowerTrack™ SYBR Green Master Mix (Applied Biosystems) with a StepOnePlus Real time PCR system according to the manufacturers’ protocols. Primers for all genes analyzed can be found in Table S1 . The ΔΔCt method was used to analyze the expression of UPR mt , metabolic, and oxidative stress response genes. The expression of each gene of interest was normalized to expression of ribosomal protein L32 (RpL32) and to expression of control-injected flies to observe changes resulting from TTC treatment. The ΔCt method was used to analyze the expression of FHV RNA1 for virus load and copy number of bacterial 16S rRNA for bacterial load. Each value was normalized to expression of ribosomal protein L32 (RpL32). Bacterial load assay and real time quantitative PCR (qPCR) for 16S rRNA gene Young and aged Oregon R males were injected with 59.8 nL of either cell culture media (control) or FHV 5 days post injection (d p.i.), 10 whole flies were collected and frozen per sample. To facilitate DNA extraction from gram-positive bacteria, samples were homogenized in 1,200 uL of Enzymatic Lysis Buffer with Lysozyme for 1 hr at 37 ° C and centrifuged for 2 minutes at 16,000 g and supernatant removed [ 49 ]. Then, the Wizard Genomic DNA Purification kit’s (Promega, A1120) protocol for Isolation of Genomic DNA from Gram Positive and Gram-Negative Bacteria was followed. DNA concentrations were determined using a NanoDrop One Spectrophotometer. The StepOnePlus Real time PCR system was utilized for all qPCR reactions. Each reaction in a 96-well plate contained 1 uL diluted DNA and 9 uL SYBR Green-containing Mastermix (Applied Biosystems). Three technical replicates were run for each sample and each set of primers. 8FM and Bact515R were the primers used to amplify the bacterial 16S rRNA gene. RpL32 forward and RpL32 reverse primers were used to amplify RpL32 . Primer sequences may be found in Table S1 . The thermal cycling protocol for amplification was 50°C for 2 minutes, denaturation for 10 minutes at 95°C, then 40 cycles of 30 seconds at 95°C, 1 minute at 59°C and 30 seconds at 72°C. Gene levels of 16S rRNA were normalized to RpL32 . Generation of axenic flies Male and female Oregon R flies were placed in an egg laying chamber and allowed to lay eggs overnight onto apple juice plates with a smear of yeast paste. Sterile water and paintbrush were used to collect eggs from the plate. Working within a biological safety cabinet, collected eggs were placed in 10% bleach for 5 minutes for dechorionation. Next, the eggs were washed 3 times in 70% ethanol, followed by 3 washes in sterile water. Eggs were collected and transferred to autoclaved, NutriFly Bloomington formulation food. Validation of treatment was performed by extracting DNA from individual flies [ 50 ] and subsequently used for PCR with universal bacterial 16S rRNA primers (listed in Table S1 ) [ 51 ] and by plating a single fly homogenate on LB agar plates and placed at 37°C overnight to monitor bacterial colony formation ( Fig. S1 ). Male flies were only collected from vials that confirmed successful axenic treatment 4 days post-eclosion and aged to 5-9d for injections. Statistical analysis Statistical analyses were performed with GraphPad Prism software (version 10.4.0) for MAC. The Log-rank (Mantel-Cox) test was used to compare survival curves. Significance of gene expression, virus load, and bacterial load was determined by two-way ANOVA test followed by a Tukey’s multiple comparisons test. For all comparisons, p < 0.05 was considered significant. Results Tetracycline and rifampicin extend survival of FHV infection without reducing virus load To determine how treatment with mitochondrial targeting antibiotics affected survival of FHV infection, we individually injected young and aged, wild-type Oregon R male flies with cell culture media (control) or FHV, then placed the flies on either vehicle- or antibiotic food recording the number of living flies each day. We found tetracycline (TTC) treatment significantly extends survival of young ( p < 0.0001) and aged ( p < 0.0001) flies after FHV infection (Fig. 1 A). The median survival of young flies receiving vehicle food was 10 days. TTC treatment extended the median survival of young flies to 12 days, a 20% extension. The median survival of aged flies receiving vehicle food was 8 days. TTC treatment extended the median survival of aged flies to 10 days, a 25% extension. Aged flies fed TTC food ( p = 0.1768) displayed the same median survival (10d) as young flies fed on vehicle food, demonstrating that TTC treatment of aged flies rescued the age-dependent susceptibility to FHV. Feeding flies on lower doses of TTC revealed a dose-dependent survival extension by TTC in young and aged flies ( Fig. S2 ), as flies fed lower doses did not show extended survival. Rifampicin (RIF) treatment also significantly extended survival of young ( p < 0.0001) and aged ( p < 0.0001) flies (Fig. 1 B). The median survival of young flies receiving vehicle food was 8 days. Rifampicin treatment extended the median survival of young flies to 10 days, a 25% extension. In aged flies, the median survival of flies receiving vehicle food was 6 days. Rifampicin treatment extended the median survival of aged flies to 7 days, a 16.7% extension. Next, we sought to determine if the observed protection from each antibiotic treatment was a result of reducing virus load. We measured virus loads via RT-qPCR at 5d p.i. for TTC and 4d p.i. for RIF. At these respective timepoints, we began to observe differences in survival rates between young and aged flies fed the appropriate vehicle food. Conversely, survival rates between young and aged flies receiving TTC or RIF remained similar, demonstrating a protective effect of each antibiotic compared to its vehicle (Fig. 1 A, B). For tetracycline’s vehicle (80% EtOH) at 5d p.i., the survival rate of young flies after infection was 100%, while aged flies’ survival rate was 84.5%. Young and aged flies receiving tetracycline had similar survival rates at this timepoint (93 and 97%, respectively). For rifampicin’s vehicle (DMSO) at 4d p.i., the survival rate of young flies after infection was 94.4%, while aged flies’ survival rate was 78.6%. Young and aged flies receiving rifampicin had similar survival rates at this timepoint (95.2 and 91.4%, respectively). We observed no significant difference in FHV RNA1 ( FHV1 ) expression between flies receiving vehicle and TTC or RIF treatment, and independent of age (Fig. 1 C, D). Together, these results show that TTC and RIF extend survival without reducing FHV load at timepoints where each antibiotic begins to demonstrate its protective effect. Therefore, it is likely that treatment with these antibiotics improves disease tolerance to FHV infection. Antimicrobial properties do not explain tetracycline-mediated survival extension To account for the possibility that antibiotic treatments may extend survival by mitigating secondary bacterial infections, we determined bacterial load by measuring the relative bacterial 16S rRNA copy number [ 49 ]. We found that FHV infection did not significantly alter bacterial loads in young ( p = 0.9567) or aged ( p = 0.2025) flies ( Fig. 2A ), so the presence of secondary bacterial infections with FHV infection is not likely. Consistent with previous studies, we found a significant increase in bacterial loads with age regardless of treatment (Control: p = 0.0005, FHV: p = 0.0029) [ 52 ]. To address potential confounding effects of antibiotic treatment on the host microbiome, we generated axenic Oregon R males to assess their survival after FHV infection with vehicle- or TTC treatment. We found that TTC treatment extended the survival of FHV-infected axenic flies ( p < 0.0001). The median survival of axenic flies receiving vehicle food was 11 days. TTC treatment extended the median survival of axenic flies to 12 days, a 9.1% extension. Our findings indicate that survival extension after TTC treatment occurs independently of potential alterations to the composition of the host’s microbiome. Tetracycline treatment upregulates genes involved in the UPR mt , glycolysis, and oxidative stress response after FHV infection Due to its ability to induce mitochondrial stress, we hypothesized the protection with TTC treatment may result from expression changes of genes involved in UPR mt activation, glucose metabolism, or the response to oxidative stress. Using RT-qPCR, we measured the expression of genes involved in the Drosophila UPR mt [ 36 , 53 ] or orthologs involved in the mammalian UPR mt [ 54 – 56 ], glycolysis [ 57 – 68 ], and oxidative stress response at various timepoints (24h, 72h, and 120h p.i.). We analyzed genes involved in the oxidative stress response that we previously observed age-dependent differences in expression after FHV infection [ 14 ]. We observed an increase in expression of genes involved in the UPR mt after TTC treatment ( Fig. 3A, B ). We found that TTC treatment significantly increased Hsp60 expression in both young and aged flies ( p = 0.0343 and p = 0.0095, respectively) 120 hpi ( Fig. 3A ). We also observed an increase in Hsc-70-5 expression with TTC treatment in aged flies 120 hpi ( p = 0.0052). Although we did not observe relevant changes in gene expression with the other UPR mt genes analyzed ( Fig. S2 ), Hsp60 and Hsc-70-5 are UPR mt markers encoding highly conserved mitochondrial chaperone proteins that function to restore mitochondrial homeostasis [ 56 , 69 ]. These results demonstrate that UPR mt activation occurs with TTC treatment after FHV infection, and its activation may be more pronounced in aged flies. We observed increased expression of lactate dehydrogenase ( Ldh) , responsible for converting pyruvate to lactate and promoting glycolysis, after TTC treatment ( Fig. 3C, D) . At 72 hpi, this expression increase was specific to aged flies ( p = 0.0010). At 120 hpi, we observed significantly increased Ldh expression with TTC treatment regardless of age (young: p = 0.0079, aged: p = 0.00393). This is consistent with previous studies that described increased expression of glycolysis genes with UPR mt activation [ 70 – 72 ], indicating a metabolic shift from oxidative phosphorylation to glycolysis. We did not observe significant changes in expression of the remaining genes that we tested ( Fig. S4) , but this may be explained by the fact that many glycolytic enzymes are regulated post transcriptionally [ 73 ]. We also observed an increase in expression of genes involved in the oxidative stress response after TTC treatment ( Fig. 3E-G ). At 24 hpi, we observed increased expression of MsrA ( p = 0.0.379) in aged flies ( Fig. 3E ). At 120 hpi, dj-1beta and Mrp4 expression was significantly increased after TTC treatment ( Fig. 3F, G ). A significant increase in expression of dj-1beta was observed in aged flies with TTC treatment; young flies also showed increased expression with treatment, but this was not statistically significant. Mrp4 expression significantly increased regardless of age with TTC treatment (young: p = 0.0019, aged: p = 0.0005). We did not observe significant changes in expression of the remaining genes we tested ( Fig. S5 ). These findings suggest that TTC treatment improved the oxidative stress response. ND23 60114 mutants display longer end-point survival of FHV compared to wild type controls As an additional approach, we sought to determine whether mitochondrial stress resulting from a genetic mutation could also improve survival outcomes after FHV infection. To do so, we monitored survival of FHV-infected ND23 60114 homozygous mutants relative to wild type ( WT ) Canton S and Canton S/ND23 60114 ( ND23 60114 /+ ) heterozygotes. We found no significant differences in FHV survival of ND23 60114 homozygous mutants compared to Canton S ( p = 0.2490) or ND23 60114 /+ heterozygotes ( p = 0.1117). However, we did find that the ND23 60114 /+ heterozygotes survived FHV infection significantly better than Canton S flies ( p < 0.0001, Fig. 4 ). This result suggests that one mutated copy of the ND23 gene could potentially induce a milder mitochondrial stress resulting in a hormetic effect. This survival improvement aligns with previous work showing ND23 60114 /+ heterozygotes had significantly increased lifespan compared to Canton S [ 34 ]. Although the median survival of ND23 60114 homozygotes was shorter (7d) compared to Canton S and heterozygotes (both 9d), ND23 60114 mutants had a later end-point survival. At 11d, the survival rate of ND23 60114 mutants was 18.1%, while Canton S and heterozygote survival rates were 0.8% and 8.7%, respectively. It is worth noting that male ND23 60114 mutants exhibit a significantly shorter lifespan in comparison to Canton S and heterozygous ND23 60114 /+ flies (~ 20d vs 50d and 60d, respectively [ 34 ]), suggesting that the later time point extension of FHV survival in the homozygous mutant is significant. Discussion In this study, we tested whether inducing mitochondrial dysfunction could extend survival in young and aged Drosophila after FHV infection. We found that treatment with mitochondrial targeting antibiotics extended survival in Drosophila melanogaster regardless of age. We observed greater protection of FHV infection with TTC compared to RIF (young: TTC = 12d vs RIF = 10d, aged: TTC = 10d vs RIF = 7d). This could be a result of the different mechanisms of action of these antibiotics or simply that the doses of antibiotics tested did not induce an equivalent level of stress. Therefore, it appears that the type of mitochondrial stressor or intensity of stress could influence the strength of the adaptive response and impact survival outcomes after infection. This notion was further supported by the observed dose-dependent survival improvement of TTC, as we found that lower doses reduced the survival extension or did not provide protection ( Fig. S2 ). Interestingly, TTC provided better protection of FHV to aged flies, as we observed a 25% median survival increase compared to 20% in young flies. Conversely, RIF provided better protection to young flies, resulting in a 25% survival increase compared to 16.7% in aged flies. Future experiments could compare the effects of both antibiotics on mitochondrial function using high-resolution respirometry. We showed that TTC and RIF extend survival without reducing FHV load, indicating improved disease tolerance is likely responsible for the observed protection. A recent study reported that rifampicin has antiviral effects in cultured S2 cells infected with two other RNA viruses, significantly reducing virus load [ 33 ]. However, this was observed after DCV and CrPV infection, which are both picorna-like viruses, unlike FHV which is a nodavirus. Haas et al. pretreated the cells for 24h with increasing RIF doses of 0, 25, 50, and 100 µM. Meanwhile, in this study we only tested RIF at 500 mg/L or 607.6 µM. FHV has been shown to significantly downregulate Cyp6a8 expression in aged flies at 24h and 48 p.i.[ 14 ], which is one of the genes that Haas et al. reported to be upregulated after RIF treatment [ 33 ]. While we did not observe significant changes in FHV load after RIF treatment, we cannot exclude the possibility that feeding the antibiotic to FHV-infected flies results in changes of Cyp6a8 expression and is associated with the observed improved outcomes. Further studies are needed to confirm this in vivo and in the context of FHV infection. Future studies should also investigate whether rifampicin has an antiviral effect in vivo following infection with viruses such as DCV and CrPV. UPR mt activation after TTC treatment has been shown to improve disease tolerance mechanisms in mice models of bacterial sepsis and influenza virus infection [ 24 , 25 ]. In the context of sepsis, the lungs and liver showed decreased tissue damage with TTC treatment, which was associated with distinct tissue-specific transcriptional signatures that improved survival by promoting tissue repair or reprogramming metabolism [ 24 ]. Likewise, the improved tolerance after influenza virus infection was associated with induction of genes involved in lung epithelial cell and cilia function, as well as downregulated inflammatory and immune genes in the lungs, liver, and kidneys to reduce immunopathology [ 25 ]. Based on our findings and existing literature, we could investigate tissue-specific disease tolerance mechanisms (reviewed in [ 74 ]) in future studies to elucidate potential mechanisms that improve survival. Tissue-specific transcriptomic analyses of tissues with known FHV tropism could identify genes implicated in disease tolerance to FHV induced by TTC or RIF. We concluded that the survival extension of FHV was not likely a result of TTC’s antimicrobial properties. We found bacterial loads did not significantly increase with FHV infection, which suggests antimicrobial properties of the antibiotics were not mitigating potential secondary bacterial infections. We also showed survival extension in TTC-treated axenic flies exposed to FHV, so TTC’s survival extension was not likely a result of altering the host’s microbiome. We recognize that assessing bacterial load by quantifying bacterial 16S rRNA copy number is an approach with limitations. Therefore, future studies could aim to characterize the host microbiome with 16S rRNA gene amplicon sequencing, allowing us to characterize microbial composition and relative abundance. Alternatively, we could repeat similar experiments with 9- tert -butyl doxycycline, a derivative of TTC shown to have minimal antimicrobial properties but maintained ability to induce the UPR mt [ 25 ]. These approaches could allow us to conclude more convincingly that TTC’s protection is not a result of antimicrobial properties. Since TTC treatment extended survival better in aged flies relative to young, we sought to determine if there were differences in expression of genes involved in the UPR mt , glycolytic metabolism, and oxidative stress response following TTC treatment. Hsp60 and Hsc-70-5 are evolutionarily conserved across C. elegans , mammals, and flies as mitochondrial chaperone proteins involved in the UPR mt , and are considered one of the best assessments of UPR mt activation [ 42 , 75 ]. We found increased expression of these UPR mt markers with TTC treatment. We observed increased expression of Ldh after TTC treatment. This increase in Ldh expression is also correlated with our expected reduction of OCR with TTC treatment, as it indicates a metabolic shift from oxidative phosphorylation to glycolysis. This metabolic shift has been shown to be involved in UPR mt activation. Together, these results suggest that TTC treatment activates the UPR mt , perhaps with better induction in aged flies. We observed increases in expression in oxidative stress response genes with TTC treatment. This is consistent with previous reports of increased expression of oxidative stress response genes with UPR mt activation [ 72 ]. The gene products of dj-1beta and MsrA are highly conserved antioxidants [ 76 , 77 ]. In response to paraquat-induced oxidative stress, Mrp4 was shown to be necessary and sufficient for transcription of JNK-dependent antioxidant genes. The increased expression that we observed of these genes suggests an improved oxidative stress response after TTC treatment limits oxidative damage and promotes cell survival, offering another potential mechanism to extend survival. We demonstrated that ND23 60114 mutants had a longer end-point survival to FHV compared to wildtype flies, despite having a significantly shorter lifespan. Alternatively, we could knockdown ND23 or ND75 ubiquitously using the Gal4/UAS system combined with the Gal80 ts factor to induce knockdown in adults, as ubiquitous knockdown is lethal when induced in development [ 78 ]. This approach could resolve the difficulties of making comparisons between mutants and controls with such significant lifespan differences and perhaps provide more convincing conclusions. Our results confirmed the hypothesis that interventions reducing metabolic rates, or inducing hypometabolism, serve as a disease tolerance mechanism that improves survival outcomes after FHV infection. However, we do not completely understand the mechanisms by which this survival extension occurs. Is this simply a result of UPR mt activating protective stress responses that allow cellular survival? Is this a result of a metabolic shift that serves to reduce mitochondrial stress and promote function? Since the Drosophila UPR mt remains relatively uncharacterized compared to the UPR mt of C. elegans and mammals, it may be difficult to elucidate the mechanisms that are conferring protection at this point. For example, different mitochondrial stressors have been shown to activate different transcription factors in C. elegans and mammals, implicating multiple branches of the UPR mt (reviewed in [ 79 ]). Various experimental models have described UPR mt activation to be protective in disease states or extend longevity, which has made it a popular target for therapeutics. While the evidence is considerable, there are negative consequences that come with prolonged UPR mt activation [ 80 – 82 ]. Additionally, C. elegans lifespan studies have shown that the timing of UPR mt activation can affect its protective effect. UPR mt activation during development resulted in lifespan extension; however, induction after adulthood did not extend lifespan [ 83 ]. Further research is necessary to better understand which mitochondrial stressors are activating specific branches of the UPR mt . However, many studies suggest that activating cellular and mitochondrial stress responses could be a valuable therapeutic target in many different contexts. Declarations Funding S.C. acknowledges NIH for funding (grants R01AG079962 and R15AI169146). D.B. acknowledges funding support from the University of Alabama graduate school. Author Contribution S.C. and D.B. designed the study. D.B., M.B., J.MG. and G.M. performed experiments. D.B., M.B. and J.MG. analyzed data. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6816306","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468598377,"identity":"c1c96f40-e524-485c-9ecc-3ff771fdf0f8","order_by":0,"name":"Dean Bunnell","email":"","orcid":"","institution":"University of Alabama","correspondingAuthor":false,"prefix":"","firstName":"Dean","middleName":"","lastName":"Bunnell","suffix":""},{"id":468598379,"identity":"2276d619-1108-4848-a585-05e2bd2f3b98","order_by":1,"name":"Madelyn Buhl","email":"","orcid":"","institution":"University of Alabama","correspondingAuthor":false,"prefix":"","firstName":"Madelyn","middleName":"","lastName":"Buhl","suffix":""},{"id":468598380,"identity":"bdcf589b-2b06-45ca-9d61-f11aed3330da","order_by":2,"name":"Justin McGee","email":"","orcid":"","institution":"University of Alabama","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"McGee","suffix":""},{"id":468598381,"identity":"aab34f54-245b-4fdd-866c-986761a8bd21","order_by":3,"name":"Grace Milas","email":"","orcid":"","institution":"University of Alabama","correspondingAuthor":false,"prefix":"","firstName":"Grace","middleName":"","lastName":"Milas","suffix":""},{"id":468598382,"identity":"025d3e7b-c80c-432c-82ac-8f856570b01a","order_by":4,"name":"Stanislava Chtarbanova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACHhBxAML+ACYTCGphbIBpYZxBshZmHmK0yLefPf7g55478ubsPYafbdvsGPjZcwzwajE4k5fY2PPsmeHOnjPG0rltyQySPW8IaJHgMWzgOXCYccONtASgFmYGgxsEbJGfwWPY+OfAYXugluTflm31DPaEtDDc4DFsBtqSuOFG8jFpxrbDQHsJ+iXHcLbMgWfJG84cPmbZc+44j8SZZwX4HdZ+xuDjmwN3bDccb2y+8aOsWo6/PXkDfoehAx7SlI+CUTAKRsEowAoAHVxMxN5MZ8UAAAAASUVORK5CYII=","orcid":"","institution":"University of Alabama","correspondingAuthor":true,"prefix":"","firstName":"Stanislava","middleName":"","lastName":"Chtarbanova","suffix":""}],"badges":[],"createdAt":"2025-06-04 04:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6816306/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6816306/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84307644,"identity":"2b0a5a0f-4d9a-400d-903c-f67b09e4c7aa","added_by":"auto","created_at":"2025-06-10 11:39:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":570826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTetracycline and rifampicin antibiotics extend survival after FHV infection without reducing virus load.\u003c/strong\u003e (\u003cstrong\u003eA, B\u003c/strong\u003e) Survival curves of young and aged male \u003cem\u003eOregon R \u003c/em\u003eflies infected with FHV or control-injected with the same volume of cell culture media. The graphs compare the survival curves of young and aged males from 8 independent injection experiments (\u003cstrong\u003eA\u003c/strong\u003e) and 5 independent injection experiments (\u003cstrong\u003eB\u003c/strong\u003e). After injection, flies were then placed on TTC or vehicle food (\u003cstrong\u003eA\u003c/strong\u003e) and RIF or vehicle food (\u003cstrong\u003eB\u003c/strong\u003e). Statistics of FHV survival are based on a Log-Rank (Mantel-Cox) test. ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. (\u003cstrong\u003eC, D\u003c/strong\u003e) Virus load determined by \u003cem\u003eFHV1 \u003c/em\u003eexpression reveals comparable titers between young and aged flies. Comparable titers were also observed between flies receiving the appropriate vehicle food and TTC (\u003cstrong\u003eC\u003c/strong\u003e) or RIF (\u003cstrong\u003eD\u003c/strong\u003e). Graphs represent mean +/- SEM from 3 biological replicates from groups of 5 flies. Statistics for virus load are based on two-way ANOVA followed by Tukey post-test to correct for multiple comparisons. ns= non-significant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/6e1bcadd7806fdbcf61f402a.png"},{"id":84307670,"identity":"8f69a13c-1747-448d-8ca8-5584bca2c7fd","added_by":"auto","created_at":"2025-06-10 11:39:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival extension by tetracycline is likely a result of non-antimicrobial properties.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Bacterial load 5d p.i. determined by bacterial \u003cem\u003e16S rRNA \u003c/em\u003ecopy number reveals comparable bacterial loads between control-injected and FHV-injected flies and significantly increased bacterial loads with age. Graphs represent +/- SEM of 3 biological replicates from groups of 10 flies. Statistics for bacterial load are based on two-way ANOVA followed by Tukey post-test to correct for multiple comparisons. ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ns= non-significant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05). (\u003cstrong\u003eB\u003c/strong\u003e) Survival curves of axenic \u003cem\u003eOregon R \u003c/em\u003emales infected with FHV or control-injected with the same volume of cell-culture media. The graph compares the survival curves of axenic flies fed vehicle or TTC food from 5 independent injection experiments. ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/a7f3d8c59fb77b7af5b457f0.png"},{"id":84307680,"identity":"87fa4b3b-06c0-40a2-bdb6-ce4abf64ebc3","added_by":"auto","created_at":"2025-06-10 11:39:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTTC treatment increased expression of genes involved in the UPR\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emt\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e, glycolysis, and oxidative stress response after FHV infection\u003c/strong\u003e. (\u003cstrong\u003eA-G\u003c/strong\u003e) Gene expression determined by RT-qPCR after FHV infection and comparing flies fed vehicle or TTC. Graphs depict +/- SEM of three biological replicates from groups of 5 flies. Statistics are based on a two-way ANOVA followed by Tukey post-test to correct for multiple comparisons. *\u003cem\u003ep\u0026lt;\u003c/em\u003e0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u0026lt;\u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/b5ee3f3ab19d62e6968d8bef.png"},{"id":84307678,"identity":"a50452df-fe26-45be-ae29-f1820bd81ec4","added_by":"auto","created_at":"2025-06-10 11:39:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":242152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eND23\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e60114\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutants are protected following FHV infection.\u003c/strong\u003e Survival curves of \u003cem\u003eCanton S, ND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u0026gt;Canton S \u003c/em\u003eheterozygous\u003cem\u003e \u003c/em\u003eflies infected with FHV or control-injected with the same volume of cell culture media. The graphs compare the survival curves from at least 3 independent injection experiments. ****\u003cem\u003ep\u0026lt;\u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/4b2fb628d1f1b446b09002e4.png"},{"id":84308843,"identity":"0fec172d-0b40-4eee-8ccf-f0fd83d456f1","added_by":"auto","created_at":"2025-06-10 11:55:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2055590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/3ca7bc3d-5fb9-4f40-a8d2-e6d2f203238e.pdf"},{"id":84307689,"identity":"4eab7931-8d42-43be-a03b-f4b27a9170ba","added_by":"auto","created_at":"2025-06-10 11:39:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2016906,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6816306/v1/2e4d9387e298150245f9a3bc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Treatment with mitochondrial targeting antibiotics improves survival outcomes after Flock House virus infection in young and aged Drosophila melanogaster","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiving organisms are constantly exposed to stressors capable of altering physiological functions that may have implications on their survival. These stressors include but are not limited to exposure to variable environments, nutrient limitations, injury, oxidative stress, and disease states [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cells, tissues, and organisms possess mechanisms of \u0026lsquo;adaptive homeostasis\u0026rsquo; [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] capable of detecting these constant and variable stressors and generating the appropriate response to restore homeostasis and in many cases promote survival. A key component of this response is the ability to employ these adaptive mechanisms quickly and inactivate the response when the stress or damage has been resolved. An abundance of evidence in many model organisms has demonstrated that aging results in a functional decline in adaptive homeostatic mechanisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The immune system also experiences a progressive functional decline with age, or immunosenescence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Ultimately, this decline renders organisms more susceptible to infections, including RNA virus infections [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A major gap exists in our understanding of the underlying factors implicated in older individuals\u0026rsquo; increased susceptibility to infections. The world population of people 65 years or older is projected to more than double by 2050 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], so identifying strategies to promote aged organisms\u0026rsquo; ability to survive infection and improve health will be critical in the near future.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDrosophila melanogaster\u003c/em\u003e and FHV provide a powerful aged host-RNA virus interaction model to elucidate mechanisms that improve the aged host\u0026rsquo;s survival. Although invertebrates such as \u003cem\u003eDrosophila\u003c/em\u003e lack the adaptive immunity found in vertebrates, they provide a unique model to investigate innate immunity. In vertebrates, innate immunity is the first line of defense when faced with infection and aids adaptive immune system activation, as the two branches must act synergistically to fight infection. FHV is an insect virus with a positive sense, RNA genome composed of two single-stranded RNA molecules copackaged in a virion. Its genetic simplicity has made it a widely used model in host-virus interactions, and its replication cycle is well characterized [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It replicates within replication spherules formed on the outer mitochondrial membrane [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. FHV infection is pathogenic to flies and results in mortality within days [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring pathogenic infections, two evolutionarily conserved defense strategies must act in conjunction to limit pathology. Resistance involves detection of the pathogen by recognition of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) followed by activation of effector mechanisms capable of reducing pathogen load. while disease tolerance limits tissue damage and consequently disease severity without affecting pathogen load. In \u003cem\u003eDrosophila\u003c/em\u003e, several antiviral resistance mechanisms have been identified in response to FHV infection, including RNA interference (RNAi) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and hemocyte-mediated phagocytosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Disease tolerance mechanisms have not been characterized as extensively as resistance mechanisms. The epigenetic regulator, Histone H3 lysine 9 (H3K9) methyltransferase G9-alpha, has been shown to play a role in disease tolerance to FHV by preventing hyperactivation of the JAK-STAT pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur lab previously showed that FHV infection causes mortality in aged flies\u0026rsquo; significantly faster than in young flies without observing an increase in viral titers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting an age-related decline in disease tolerance to FHV. Transcriptomic analysis revealed many genes whose products are involved in metabolic processes, mitochondrial structure, and mitochondrial respiration were regulated to a stronger extent in older flies 48h p.i. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This finding implicated host metabolism as a potential factor differentially regulated with age that may increase susceptibility to FHV. Previously, we conducted a longitudinal, single-fly respirometry study to measure whole organismal oxygen consumption rates (OCR) in single flies as a proxy for metabolic rate and mitochondrial function. In this study, we determined if metabolic rates differed with age after FHV infection and whether differences may be correlated with mortality [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We found young flies reduced OCR after FHV infection, but aged flies showed no significant changes in OCR, providing more evidence that metabolism could impact age-related susceptibility to FHV [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In fact, the reduction of OCR in young flies is indicative of an evolutionarily conserved strategy of metabolic rate depression or hypometabolism, which has been reported as a survival strategy in the presence of various stressors across organisms (reviewed in [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]), including the promotion of disease tolerance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral classes of antibiotics have demonstrated protective effects in the context of viral infections, cancer, sepsis, and inflammatory disorders that are not attributed to direct antimicrobial effects [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Since mitochondria retain many characteristics of their bacterial ancestors, many classes of antibiotics can act via similar mechanisms on eukaryotic mitochondria and cause dysfunction [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In bacteria, tetracycline inhibits bacterial protein synthesis by occupying the A-site of the bacterial 30S ribosomal subunit and blocking recruitment of the aminoacyl-tRNA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Rifampicin inhibits bacterial DNA transcription by inhibiting RNA polymerase [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In eukaryotes like \u003cem\u003eDrosophila\u003c/em\u003e, these antibiotics alter the balance of nuclear-encoded and mitochondrial-encoded proteins, which is crucial for mitochondrial electron transport chain function. High-resolution respirometry has shown doxycycline, a tetracycline class antibiotic, decreases OCR in flies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and \u003cem\u003eDrosophila\u0026rsquo;s\u003c/em\u003e mitochondrial RNA polymerase was shown to be inhibited by RIF [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In \u003cem\u003eDrosophila\u003c/em\u003e S2 cells, rifampicin was shown to have an antiviral effect against \u003cem\u003eDrosophila\u003c/em\u003e C Virus (DCV) and Cricket Paralysis Virus (CrPV), which was associated with increased expression of detoxification enzymes of the cytochrome P450 family [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eDrosophila ND23\u003c/em\u003e is a nuclear gene that encodes a highly conserved subunit of the mitochondrial ETC Complex I. Previous studies have shown that \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants display abnormal mitochondrial morphology, reduced ATP levels and have a significantly shorter lifespan in comparison to wild type controls [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants show increased expression of \u003cem\u003eHsp22\u003c/em\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], a chaperone protein that has been identified to play a role in the \u003cem\u003eDrosophila\u003c/em\u003e UPR\u003csup\u003emt\u003c/sup\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eModest perturbation of the mitochondrial electron transport chain (ETC) function has been shown to increase lifespan in \u003cem\u003eDrosophila\u003c/em\u003e, \u003cem\u003eC. elegans\u003c/em\u003e, and mice [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, the mitochondrial unfolded protein response (UPR\u003csup\u003emt\u003c/sup\u003e) can be activated by various stressors including mitonuclear protein imbalance, misfolded protein accumulation, reactive oxygen species (ROS), mtDNA depletion, or mitochondrial toxins, and involves retrograde signaling to the nucleus to mount a transcriptional response to relieve mitochondrial stress [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This includes the transcription of genes that encode for mitochondrial chaperones, proteases, antioxidants, and glycolytic genes, all of which function to alleviate mitochondrial stress (reviewed in [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]). UPR\u003csup\u003emt\u003c/sup\u003e activation has been shown to improve disease tolerance mechanisms in mammalian studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, many studies indicate that the presence of mitochondrial stress and subsequent activation of the UPR\u003csup\u003emt\u003c/sup\u003e represents a hormetic response, where a mild amount of stress can have beneficial effects [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we sought to investigate whether antibiotics that impair mitochondrial metabolism improve survival of FHV infection with aging. We observed that TTC and RIF extended survival of FHV infection in both young and older flies without reducing virus. Survival extension occurred independently of antimicrobial properties after TTC treatment. In virus infected flies of both age cohorts, we found that TTC treatment affected the expression of genes implicated in the UPR\u003csup\u003emt\u003c/sup\u003e, glycolysis, and oxidative stress response. Additionally, in comparison to \u003cem\u003ewild type\u003c/em\u003e controls, \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants displayed extended end point survival when infected with FHV. Overall, our findings indicate that mitochondrial dysfunction and resulting UPR\u003csup\u003emt\u003c/sup\u003e activation improves survival outcomes of FHV infection likely by functioning as part of a disease tolerance mechanism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eDrosophila\u003c/span\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003estocks, handling and aging\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eOregon R\u003c/em\u003e stock (#2376) was obtained from Bloomington \u003cem\u003eDrosophila\u003c/em\u003e Stock Center (Bloomington, IN, USA). Flies were maintained in vials containing Nutri-Fly Bloomington formulation food (Genesee Scientific, Cat #: 66\u0026ndash;113) in a 25\u0026deg;C incubator with controlled 12 h:12 h light:dark cycle. For experimentation, \u003cem\u003eOregon R\u003c/em\u003e male flies were collected, aged, and flipped every 3\u0026ndash;4 days into vials of fresh food until reaching the desired age. Young and aged cohorts were 5\u0026ndash;9 days-old and 28\u0026ndash;32 days-old, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eND23\u003c/em\u003e \u003csup\u003e \u003cem\u003e60114\u003c/em\u003e \u003c/sup\u003e stock was kindly provided by Dr. Barry Ganetzky and \u003cem\u003eCanton S\u003c/em\u003e (#64349) was obtained from Bloomington \u003cem\u003eDrosophila\u003c/em\u003e Stock Center (Bloomington, IN, USA). Heterozygous controls were obtained by crossing \u003cem\u003eCanton S\u003c/em\u003e males with \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e virgin females (Control: n\u0026thinsp;=\u0026thinsp;53, FHV: n\u0026thinsp;=\u0026thinsp;47) and the reciprocal cross (Control: n\u0026thinsp;=\u0026thinsp;49, FHV\u0026thinsp;=\u0026thinsp;56). Flies were aged to 5\u0026ndash;9 days in vials containing Nutri-Fly Molasses formulation food (Genesee Scientific, Cat #: 66\u0026ndash;116) in a 25\u0026deg;C incubator with controlled 12 h:12 h light:dark cycle. All fly lines were confirmed \u003cem\u003eWolbachia-\u003c/em\u003enegative prior to experimentation.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVirus stock and injections\u003c/h2\u003e \u003cp\u003eFHV stock was propagated by infecting low passaged Schneider\u0026rsquo;s \u003cem\u003eDrosophila\u003c/em\u003e line 1 (DL-1) cells. Virus stock solution was stored at \u0026minus;\u0026thinsp;80\u0026deg;C. All injections were conducted with a Nanoject II or III (Drummond Scientific), as described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Flies were individually injected at the appropriate age with either 59.8 nL of cell culture media (Schneider\u0026rsquo;s media, 10% fetal bovine serum, and 1:100 penicillin-streptomycin (pen/strep U/mL); control injection) or FHV (2.57x 10\u003csup\u003e5\u003c/sup\u003e 50% Tissue Culture Infective Dose (TCID\u003csub\u003e50\u003c/sub\u003e)/mL). The control-injected cohort was injected prior to the FHV-injected cohort to prevent cross-contamination. Flies were anesthetized with CO\u003csub\u003e2\u003c/sub\u003e on a fly pad (Genesee Scientific) and manipulated with a paintbrush during injections. Injected flies of the same experimental condition were then placed in groups of 10\u0026ndash;15 flies per vial and allowed to rest at room temperature for 1 hour after injection before being placed in a 22\u0026deg;C incubator. For survival assays, survival status was recorded daily, and flies were flipped into vials of fresh food every other day.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibiotic Food Preparation\u003c/h3\u003e\n\u003cp\u003eNutri-Fly Bloomington formulation food (Genesee Scientific, Cat #: 66\u0026ndash;113) or Corn-syrup/Soy media (Archon Scientific, W1) was prepared per manufacturer\u0026rsquo;s directions and supplemented with the appropriate dose of antibiotic or equal volume of vehicle serving as a control for each antibiotic treatment. Tetracycline (ThermoFisher Scientific, Alfa Aesar, Cat#: J61714) was supplemented to the food at the final concentrations of 0.05 mg/mL [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], 0.005 mg/mL, and 0.0005 mg/mL. The tetracycline stock solution was prepared using 80% ethanol, which served as the vehicle. Rifampicin (Tokyo Chemical Industry Co., Ltd., Product#: R0079) was supplemented to the food at a final concentration of 500 mg/L [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The rifampicin stock solution was prepared using DMSO, which served as the vehicle. Flies were placed on vehicle- or antibiotic-supplemented food only after they have been injected with either cell culture media (control) or FHV.\u003c/p\u003e\n\u003ch3\u003eReverse transcription quantitative real time PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from 5 whole flies (24, 72, or 120h p.i.) using the Quick-RNA MiniPrep kit (Zymo Research) and cDNA was prepared from 500 ng total RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Triplicate cDNA samples were amplified using the PowerTrack\u0026trade; SYBR Green Master Mix (Applied Biosystems) with a StepOnePlus Real time PCR system according to the manufacturers\u0026rsquo; protocols. Primers for all genes analyzed can be found in \u003cb\u003eTable S1\u003c/b\u003e. The ΔΔCt method was used to analyze the expression of UPR\u003csup\u003emt\u003c/sup\u003e, metabolic, and oxidative stress response genes. The expression of each gene of interest was normalized to expression of \u003cem\u003eribosomal protein L32 (RpL32)\u003c/em\u003e and to expression of control-injected flies to observe changes resulting from TTC treatment. The ΔCt method was used to analyze the expression of \u003cem\u003eFHV RNA1\u003c/em\u003e for virus load and copy number of bacterial \u003cem\u003e16S rRNA\u003c/em\u003e for bacterial load. Each value was normalized to expression of \u003cem\u003eribosomal protein L32 (RpL32).\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eBacterial load assay and real time quantitative PCR (qPCR) for\u003c/span\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e16S rRNA\u003c/span\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003egene\u003c/span\u003e\u003c/p\u003e \u003cp\u003eYoung and aged \u003cem\u003eOregon R\u003c/em\u003e males were injected with 59.8 nL of either cell culture media (control) or FHV 5 days post injection (d p.i.), 10 whole flies were collected and frozen per sample. To facilitate DNA extraction from gram-positive bacteria, samples were homogenized in 1,200 uL of Enzymatic Lysis Buffer with Lysozyme for 1 hr at 37 \u0026deg; C and centrifuged for 2 minutes at 16,000 g and supernatant removed [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Then, the Wizard Genomic DNA Purification kit\u0026rsquo;s (Promega, A1120) protocol for Isolation of Genomic DNA from Gram Positive and Gram-Negative Bacteria was followed. DNA concentrations were determined using a NanoDrop One Spectrophotometer. The StepOnePlus Real time PCR system was utilized for all qPCR reactions. Each reaction in a 96-well plate contained 1 uL diluted DNA and 9 uL SYBR Green-containing Mastermix (Applied Biosystems). Three technical replicates were run for each sample and each set of primers. 8FM and Bact515R were the primers used to amplify the bacterial \u003cem\u003e16S rRNA\u003c/em\u003e gene. \u003cem\u003eRpL32\u003c/em\u003e forward and \u003cem\u003eRpL32\u003c/em\u003e reverse primers were used to amplify \u003cem\u003eRpL32\u003c/em\u003e. Primer sequences may be found in \u003cb\u003eTable S1\u003c/b\u003e. The thermal cycling protocol for amplification was 50\u0026deg;C for 2 minutes, denaturation for 10 minutes at 95\u0026deg;C, then 40 cycles of 30 seconds at 95\u0026deg;C, 1 minute at 59\u0026deg;C and 30 seconds at 72\u0026deg;C. Gene levels of \u003cem\u003e16S rRNA\u003c/em\u003e were normalized to \u003cem\u003eRpL32\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eGeneration of axenic flies\u003c/h3\u003e\n\u003cp\u003eMale and female \u003cem\u003eOregon R\u003c/em\u003e flies were placed in an egg laying chamber and allowed to lay eggs overnight onto apple juice plates with a smear of yeast paste. Sterile water and paintbrush were used to collect eggs from the plate. Working within a biological safety cabinet, collected eggs were placed in 10% bleach for 5 minutes for dechorionation. Next, the eggs were washed 3 times in 70% ethanol, followed by 3 washes in sterile water. Eggs were collected and transferred to autoclaved, NutriFly Bloomington formulation food. Validation of treatment was performed by extracting DNA from individual flies [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and subsequently used for PCR with universal bacterial \u003cem\u003e16S rRNA\u003c/em\u003e primers (listed in \u003cb\u003eTable S1\u003c/b\u003e) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and by plating a single fly homogenate on LB agar plates and placed at 37\u0026deg;C overnight to monitor bacterial colony formation (\u003cb\u003eFig. S1\u003c/b\u003e). Male flies were only collected from vials that confirmed successful axenic treatment 4 days post-eclosion and aged to 5-9d for injections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with GraphPad Prism software (version 10.4.0) for MAC. The Log-rank (Mantel-Cox) test was used to compare survival curves. Significance of gene expression, virus load, and bacterial load was determined by two-way ANOVA test followed by a Tukey\u0026rsquo;s multiple comparisons test. For all comparisons, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eTetracycline and rifampicin extend survival of FHV infection without reducing virus load\u003c/h2\u003e\n \u003cp\u003eTo determine how treatment with mitochondrial targeting antibiotics affected survival of FHV infection, we individually injected young and aged, wild-type \u003cem\u003eOregon R\u003c/em\u003e male flies with cell culture media (control) or FHV, then placed the flies on either vehicle- or antibiotic food recording the number of living flies each day. We found tetracycline (TTC) treatment significantly extends survival of young (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and aged (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) flies after FHV infection (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The median survival of young flies receiving vehicle food was 10 days. TTC treatment extended the median survival of young flies to 12 days, a 20% extension. The median survival of aged flies receiving vehicle food was 8 days. TTC treatment extended the median survival of aged flies to 10 days, a 25% extension. Aged flies fed TTC food (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1768) displayed the same median survival (10d) as young flies fed on vehicle food, demonstrating that TTC treatment of aged flies rescued the age-dependent susceptibility to FHV. Feeding flies on lower doses of TTC revealed a dose-dependent survival extension by TTC in young and aged flies (\u003cstrong\u003eFig. S2\u003c/strong\u003e), as flies fed lower doses did not show extended survival. Rifampicin (RIF) treatment also significantly extended survival of young (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and aged (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) flies (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The median survival of young flies receiving vehicle food was 8 days. Rifampicin treatment extended the median survival of young flies to 10 days, a 25% extension. In aged flies, the median survival of flies receiving vehicle food was 6 days. Rifampicin treatment extended the median survival of aged flies to 7 days, a 16.7% extension.\u003c/p\u003e\n \u003cp\u003eNext, we sought to determine if the observed protection from each antibiotic treatment was a result of reducing virus load. We measured virus loads via RT-qPCR at 5d p.i. for TTC and 4d p.i. for RIF. At these respective timepoints, we began to observe differences in survival rates between young and aged flies fed the appropriate vehicle food. Conversely, survival rates between young and aged flies receiving TTC or RIF remained similar, demonstrating a protective effect of each antibiotic compared to its vehicle (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). For tetracycline\u0026rsquo;s vehicle (80% EtOH) at 5d p.i., the survival rate of young flies after infection was 100%, while aged flies\u0026rsquo; survival rate was 84.5%. Young and aged flies receiving tetracycline had similar survival rates at this timepoint (93 and 97%, respectively). For rifampicin\u0026rsquo;s vehicle (DMSO) at 4d p.i., the survival rate of young flies after infection was 94.4%, while aged flies\u0026rsquo; survival rate was 78.6%. Young and aged flies receiving rifampicin had similar survival rates at this timepoint (95.2 and 91.4%, respectively). We observed no significant difference in \u003cem\u003eFHV RNA1\u003c/em\u003e (\u003cem\u003eFHV1\u003c/em\u003e) expression between flies receiving vehicle and TTC or RIF treatment, and independent of age (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Together, these results show that TTC and RIF extend survival without reducing FHV load at timepoints where each antibiotic begins to demonstrate its protective effect. Therefore, it is likely that treatment with these antibiotics improves disease tolerance to FHV infection.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAntimicrobial properties do not explain tetracycline-mediated survival extension\u003c/h3\u003e\n\u003cp\u003eTo account for the possibility that antibiotic treatments may extend survival by mitigating secondary bacterial infections, we determined bacterial load by measuring the relative bacterial \u003cem\u003e16S rRNA\u003c/em\u003e copy number [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. We found that FHV infection did not significantly alter bacterial loads in young (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9567) or aged (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2025) flies (\u003cstrong\u003eFig.\u0026nbsp;2A\u003c/strong\u003e), so the presence of secondary bacterial infections with FHV infection is not likely. Consistent with previous studies, we found a significant increase in bacterial loads with age regardless of treatment (Control: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005, FHV: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0029) [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eTo address potential confounding effects of antibiotic treatment on the host microbiome, we generated axenic \u003cem\u003eOregon R\u003c/em\u003e males to assess their survival after FHV infection with vehicle- or TTC treatment. We found that TTC treatment extended the survival of FHV-infected axenic flies (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The median survival of axenic flies receiving vehicle food was 11 days. TTC treatment extended the median survival of axenic flies to 12 days, a 9.1% extension. Our findings indicate that survival extension after TTC treatment occurs independently of potential alterations to the composition of the host\u0026rsquo;s microbiome.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTetracycline treatment upregulates genes involved in the UPR\u003c/span\u003e \u003csup\u003e\u0026nbsp;\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003emt\u003c/span\u003e\u0026nbsp;\u003c/sup\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eglycolysis, and oxidative stress response after FHV infection\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eDue to its ability to induce mitochondrial stress, we hypothesized the protection with TTC treatment may result from expression changes of genes involved in UPR\u003csup\u003emt\u003c/sup\u003e activation, glucose metabolism, or the response to oxidative stress. Using RT-qPCR, we measured the expression of genes involved in the \u003cem\u003eDrosophila\u003c/em\u003e UPR\u003csup\u003emt\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e] or orthologs involved in the mammalian UPR\u003csup\u003emt\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e], glycolysis [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e], and oxidative stress response at various timepoints (24h, 72h, and 120h p.i.). We analyzed genes involved in the oxidative stress response that we previously observed age-dependent differences in expression after FHV infection [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eWe observed an increase in expression of genes involved in the UPR\u003csup\u003emt\u003c/sup\u003e after TTC treatment (\u003cstrong\u003eFig.\u0026nbsp;3A, B\u003c/strong\u003e). We found that TTC treatment significantly increased \u003cem\u003eHsp60\u003c/em\u003e expression in both young and aged flies (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0343 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0095, respectively) 120 hpi (\u003cstrong\u003eFig.\u0026nbsp;3A\u003c/strong\u003e). We also observed an increase in \u003cem\u003eHsc-70-5\u003c/em\u003e expression with TTC treatment in aged flies 120 hpi (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0052). Although we did not observe relevant changes in gene expression with the other UPR\u003csup\u003emt\u003c/sup\u003e genes analyzed (\u003cstrong\u003eFig. S2\u003c/strong\u003e), \u003cem\u003eHsp60\u003c/em\u003e and \u003cem\u003eHsc-70-5\u003c/em\u003e are UPR\u003csup\u003emt\u003c/sup\u003e markers encoding highly conserved mitochondrial chaperone proteins that function to restore mitochondrial homeostasis [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]. These results demonstrate that UPR\u003csup\u003emt\u003c/sup\u003e activation occurs with TTC treatment after FHV infection, and its activation may be more pronounced in aged flies.\u003c/p\u003e\n\u003cp\u003eWe observed increased expression of lactate dehydrogenase (\u003cem\u003eLdh)\u003c/em\u003e, responsible for converting pyruvate to lactate and promoting glycolysis, after TTC treatment (\u003cstrong\u003eFig.\u0026nbsp;3C, D)\u003c/strong\u003e. At 72 hpi, this expression increase was specific to aged flies (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0010). At 120 hpi, we observed significantly increased \u003cem\u003eLdh\u003c/em\u003e expression with TTC treatment regardless of age (young: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0079, aged: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.00393). This is consistent with previous studies that described increased expression of glycolysis genes with UPR\u003csup\u003emt\u003c/sup\u003e activation [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e], indicating a metabolic shift from oxidative phosphorylation to glycolysis. We did not observe significant changes in expression of the remaining genes that we tested (\u003cstrong\u003eFig. S4)\u003c/strong\u003e, but this may be explained by the fact that many glycolytic enzymes are regulated post transcriptionally [\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eWe also observed an increase in expression of genes involved in the oxidative stress response after TTC treatment (\u003cstrong\u003eFig.\u0026nbsp;3E-G\u003c/strong\u003e). At 24 hpi, we observed increased expression of \u003cem\u003eMsrA\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0.379) in aged flies (\u003cstrong\u003eFig.\u0026nbsp;3E\u003c/strong\u003e). At 120 hpi, \u003cem\u003edj-1beta\u003c/em\u003e and \u003cem\u003eMrp4\u003c/em\u003e expression was significantly increased after TTC treatment (\u003cstrong\u003eFig.\u0026nbsp;3F, G\u003c/strong\u003e). A significant increase in expression of \u003cem\u003edj-1beta\u003c/em\u003e was observed in aged flies with TTC treatment; young flies also showed increased expression with treatment, but this was not statistically significant. \u003cem\u003eMrp4\u003c/em\u003e expression significantly increased regardless of age with TTC treatment (young: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0019, aged: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0005). We did not observe significant changes in expression of the remaining genes we tested (\u003cstrong\u003eFig. S5\u003c/strong\u003e). These findings suggest that TTC treatment improved the oxidative stress response.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e\u003cstrong\u003eND23\u003c/strong\u003e\u003c/span\u003e\u003cstrong\u003e\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e60114\u003c/span\u003e\u0026nbsp;\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cstrong\u003emutants display longer end-point survival of FHV compared to\u003c/strong\u003e\u003c/span\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e\u003cstrong\u003ewild type\u003c/strong\u003e\u003c/span\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e\u003cstrong\u003econtrols\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAs an additional approach, we sought to determine whether mitochondrial stress resulting from a genetic mutation could also improve survival outcomes after FHV infection. To do so, we monitored survival of FHV-infected \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e homozygous mutants relative to \u003cem\u003ewild type\u003c/em\u003e (\u003cem\u003eWT\u003c/em\u003e) \u003cem\u003eCanton S\u003c/em\u003e and \u003cem\u003eCanton S/ND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/+\u003c/em\u003e) heterozygotes. We found no significant differences in FHV survival of \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e homozygous mutants compared to \u003cem\u003eCanton S\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2490) or \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/+\u003c/em\u003e heterozygotes (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.1117). However, we did find that the \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/+\u003c/em\u003e heterozygotes survived FHV infection significantly better than \u003cem\u003eCanton S\u003c/em\u003e flies (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e). This result suggests that one mutated copy of the \u003cem\u003eND23\u003c/em\u003e gene could potentially induce a milder mitochondrial stress resulting in a hormetic effect. This survival improvement aligns with previous work showing \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/+\u003c/em\u003e heterozygotes had significantly increased lifespan compared to \u003cem\u003eCanton S\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although the median survival of \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e homozygotes was shorter (7d)\u003c/p\u003e\n\u003cp\u003ecompared to \u003cem\u003eCanton S\u003c/em\u003e and heterozygotes (both 9d), \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants had a later end-point survival. At 11d, the survival rate of \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants was 18.1%, while \u003cem\u003eCanton S\u003c/em\u003e and heterozygote survival rates were 0.8% and 8.7%, respectively. It is worth noting that male \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants exhibit a significantly shorter lifespan in comparison to \u003cem\u003eCanton S\u003c/em\u003e and heterozygous \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/+\u003c/em\u003e flies (~\u0026thinsp;20d vs 50d and 60d, respectively [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]), suggesting that the later time point extension of FHV survival in the homozygous mutant is significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we tested whether inducing mitochondrial dysfunction could extend survival in young and aged \u003cem\u003eDrosophila\u003c/em\u003e after FHV infection. We found that treatment with mitochondrial targeting antibiotics extended survival in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e regardless of age. We observed greater protection of FHV infection with TTC compared to RIF (young: TTC\u0026thinsp;=\u0026thinsp;12d vs RIF\u0026thinsp;=\u0026thinsp;10d, aged: TTC\u0026thinsp;=\u0026thinsp;10d vs RIF\u0026thinsp;=\u0026thinsp;7d). This could be a result of the different mechanisms of action of these antibiotics or simply that the doses of antibiotics tested did not induce an equivalent level of stress. Therefore, it appears that the type of mitochondrial stressor or intensity of stress could influence the strength of the adaptive response and impact survival outcomes after infection. This notion was further supported by the observed dose-dependent survival improvement of TTC, as we found that lower doses reduced the survival extension or did not provide protection (\u003cb\u003eFig. S2\u003c/b\u003e). Interestingly, TTC provided better protection of FHV to aged flies, as we observed a 25% median survival increase compared to 20% in young flies. Conversely, RIF provided better protection to young flies, resulting in a 25% survival increase compared to 16.7% in aged flies. Future experiments could compare the effects of both antibiotics on mitochondrial function using high-resolution respirometry.\u003c/p\u003e \u003cp\u003eWe showed that TTC and RIF extend survival without reducing FHV load, indicating improved disease tolerance is likely responsible for the observed protection. A recent study reported that rifampicin has antiviral effects in cultured S2 cells infected with two other RNA viruses, significantly reducing virus load [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, this was observed after DCV and CrPV infection, which are both picorna-like viruses, unlike FHV which is a nodavirus. Haas et al. pretreated the cells for 24h with increasing RIF doses of 0, 25, 50, and 100 \u0026micro;M. Meanwhile, in this study we only tested RIF at 500 mg/L or 607.6 \u0026micro;M. FHV has been shown to significantly downregulate \u003cem\u003eCyp6a8\u003c/em\u003e expression in aged flies at 24h and 48 p.i.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which is one of the genes that Haas et al. reported to be upregulated after RIF treatment [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. While we did not observe significant changes in FHV load after RIF treatment, we cannot exclude the possibility that feeding the antibiotic to FHV-infected flies results in changes of \u003cem\u003eCyp6a8\u003c/em\u003e expression and is associated with the observed improved outcomes. Further studies are needed to confirm this \u003cem\u003ein vivo\u003c/em\u003e and in the context of FHV infection. Future studies should also investigate whether rifampicin has an antiviral effect \u003cem\u003ein vivo\u003c/em\u003e following infection with viruses such as DCV and CrPV. UPR\u003csup\u003emt\u003c/sup\u003e activation after TTC treatment has been shown to improve disease tolerance mechanisms in mice models of bacterial sepsis and influenza virus infection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the context of sepsis, the lungs and liver showed decreased tissue damage with TTC treatment, which was associated with distinct tissue-specific transcriptional signatures that improved survival by promoting tissue repair or reprogramming metabolism [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Likewise, the improved tolerance after influenza virus infection was associated with induction of genes involved in lung epithelial cell and cilia function, as well as downregulated inflammatory and immune genes in the lungs, liver, and kidneys to reduce immunopathology [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Based on our findings and existing literature, we could investigate tissue-specific disease tolerance mechanisms (reviewed in [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]) in future studies to elucidate potential mechanisms that improve survival. Tissue-specific transcriptomic analyses of tissues with known FHV tropism could identify genes implicated in disease tolerance to FHV induced by TTC or RIF.\u003c/p\u003e \u003cp\u003eWe concluded that the survival extension of FHV was not likely a result of TTC\u0026rsquo;s antimicrobial properties. We found bacterial loads did not significantly increase with FHV infection, which suggests antimicrobial properties of the antibiotics were not mitigating potential secondary bacterial infections. We also showed survival extension in TTC-treated axenic flies exposed to FHV, so TTC\u0026rsquo;s survival extension was not likely a result of altering the host\u0026rsquo;s microbiome. We recognize that assessing bacterial load by quantifying bacterial \u003cem\u003e16S rRNA\u003c/em\u003e copy number is an approach with limitations. Therefore, future studies could aim to characterize the host microbiome with \u003cem\u003e16S rRNA\u003c/em\u003e gene amplicon sequencing, allowing us to characterize microbial composition and relative abundance. Alternatively, we could repeat similar experiments with 9-\u003cem\u003etert\u003c/em\u003e-butyl doxycycline, a derivative of TTC shown to have minimal antimicrobial properties but maintained ability to induce the UPR\u003csup\u003emt\u003c/sup\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These approaches could allow us to conclude more convincingly that TTC\u0026rsquo;s protection is not a result of antimicrobial properties.\u003c/p\u003e \u003cp\u003eSince TTC treatment extended survival better in aged flies relative to young, we sought to determine if there were differences in expression of genes involved in the UPR\u003csup\u003emt\u003c/sup\u003e, glycolytic metabolism, and oxidative stress response following TTC treatment. \u003cem\u003eHsp60\u003c/em\u003e and \u003cem\u003eHsc-70-5\u003c/em\u003e are evolutionarily conserved across \u003cem\u003eC. elegans\u003c/em\u003e, mammals, and flies as mitochondrial chaperone proteins involved in the UPR\u003csup\u003emt\u003c/sup\u003e, and are considered one of the best assessments of UPR\u003csup\u003emt\u003c/sup\u003e activation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. We found increased expression of these UPR\u003csup\u003emt\u003c/sup\u003e markers with TTC treatment. We observed increased expression of \u003cem\u003eLdh\u003c/em\u003e after TTC treatment. This increase in \u003cem\u003eLdh\u003c/em\u003e expression is also correlated with our expected reduction of OCR with TTC treatment, as it indicates a metabolic shift from oxidative phosphorylation to glycolysis. This metabolic shift has been shown to be involved in UPR\u003csup\u003emt\u003c/sup\u003e activation. Together, these results suggest that TTC treatment activates the UPR\u003csup\u003emt\u003c/sup\u003e, perhaps with better induction in aged flies.\u003c/p\u003e \u003cp\u003eWe observed increases in expression in oxidative stress response genes with TTC treatment. This is consistent with previous reports of increased expression of oxidative stress response genes with UPR\u003csup\u003emt\u003c/sup\u003e activation [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The gene products of \u003cem\u003edj-1beta\u003c/em\u003e and \u003cem\u003eMsrA\u003c/em\u003e are highly conserved antioxidants [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In response to paraquat-induced oxidative stress, \u003cem\u003eMrp4\u003c/em\u003e was shown to be necessary and sufficient for transcription of JNK-dependent antioxidant genes. The increased expression that we observed of these genes suggests an improved oxidative stress response after TTC treatment limits oxidative damage and promotes cell survival, offering another potential mechanism to extend survival.\u003c/p\u003e \u003cp\u003eWe demonstrated that \u003cem\u003eND23\u003c/em\u003e\u003csup\u003e\u003cem\u003e60114\u003c/em\u003e\u003c/sup\u003e mutants had a longer end-point survival to FHV compared to \u003cem\u003ewildtype\u003c/em\u003e flies, despite having a significantly shorter lifespan. Alternatively, we could knockdown \u003cem\u003eND23\u003c/em\u003e or \u003cem\u003eND75\u003c/em\u003e ubiquitously using the Gal4/UAS system combined with the Gal80\u003csup\u003ets\u003c/sup\u003e factor to induce knockdown in adults, as ubiquitous knockdown is lethal when induced in development [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. This approach could resolve the difficulties of making comparisons between mutants and controls with such significant lifespan differences and perhaps provide more convincing conclusions.\u003c/p\u003e \u003cp\u003eOur results confirmed the hypothesis that interventions reducing metabolic rates, or inducing hypometabolism, serve as a disease tolerance mechanism that improves survival outcomes after FHV infection. However, we do not completely understand the mechanisms by which this survival extension occurs. Is this simply a result of UPR\u003csup\u003emt\u003c/sup\u003e activating protective stress responses that allow cellular survival? Is this a result of a metabolic shift that serves to reduce mitochondrial stress and promote function? Since the \u003cem\u003eDrosophila\u003c/em\u003e UPR\u003csup\u003emt\u003c/sup\u003e remains relatively uncharacterized compared to the UPR\u003csup\u003emt\u003c/sup\u003e of \u003cem\u003eC. elegans\u003c/em\u003e and mammals, it may be difficult to elucidate the mechanisms that are conferring protection at this point. For example, different mitochondrial stressors have been shown to activate different transcription factors in \u003cem\u003eC. elegans\u003c/em\u003e and mammals, implicating multiple branches of the UPR\u003csup\u003emt\u003c/sup\u003e (reviewed in [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]). Various experimental models have described UPR\u003csup\u003emt\u003c/sup\u003e activation to be protective in disease states or extend longevity, which has made it a popular target for therapeutics. While the evidence is considerable, there are negative consequences that come with prolonged UPR\u003csup\u003emt\u003c/sup\u003e activation [\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Additionally, \u003cem\u003eC. elegans\u003c/em\u003e lifespan studies have shown that the timing of UPR\u003csup\u003emt\u003c/sup\u003e activation can affect its protective effect. UPR\u003csup\u003emt\u003c/sup\u003e activation during development resulted in lifespan extension; however, induction after adulthood did not extend lifespan [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Further research is necessary to better understand which mitochondrial stressors are activating specific branches of the UPR\u003csup\u003emt\u003c/sup\u003e. However, many studies suggest that activating cellular and mitochondrial stress responses could be a valuable therapeutic target in many different contexts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eS.C. acknowledges NIH for funding (grants R01AG079962 and R15AI169146). D.B. acknowledges funding support from the University of Alabama graduate school.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.C. and D.B. designed the study. D.B., M.B., J.MG. and G.M. performed experiments. D.B., M.B. and J.MG. analyzed data. D.B wrote the initial manuscript draft with input from S.C. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRajan A and Perrimon N. Drosophila as a Model for Interorgan Communication: Lessons from Studies on Energy Homeostasis. 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PLOS Biology. 2007; 5(10):e259.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Drosophila melanogaster, innate immunity, aging, virus infection, antibiotics, mitochondrial unfolded protein response","lastPublishedDoi":"10.21203/rs.3.rs-6816306/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6816306/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAged organisms are more susceptible to infectious diseases, including infections with RNA viruses. Mitochondrial dysfunction is one of many hallmarks of aging that could affect this increased susceptibility, as the relationship between immunity and metabolism is crucial to manage infections. Using \u003cem\u003eDrosophila melanogaster\u003c/em\u003e- Flock House virus (FHV) host-virus interactions model system, previous work has identified differences in young and aged flies\u0026rsquo; ability to modulate oxygen consumption rates (OCR). Here, we hypothesized that interventions that reduce OCR could improve survival of FHV, as observed in young flies. Tetracycline (TTC) and rifampicin (RIF) antibiotics disrupt mitochondrial translation and transcription respectively because of mitochondria's bacterial ancestry. The mitochondrial unfolded protein response (UPR\u003csup\u003emt\u003c/sup\u003e) is activated by mitochondrial stressors, including reactive oxygen species, defects in oxidative phosphorylation, and mitonuclear protein imbalance. UPR\u003csup\u003emt\u003c/sup\u003e activation initiates retrograde signaling to the nucleus, prompting transcription, translation, and import of nuclear proteins to resolve stress. We showed TTC or RIF treatment extended survival in young and aged flies after FHV infection, independently of virus load modulation. Furthermore, we demonstrate that bacterial loads are not significantly different between FHV-infected flies and controls, and that the protective effect of TTC likely occurs independently of its antimicrobial properties. We observed increased expression of genes involved in the UPR\u003csup\u003emt\u003c/sup\u003e, glycolysis, and oxidative stress response with TTC treatment. Our results suggest perturbing mitonuclear protein balance with TTC or RIF could activate the UPR\u003csup\u003emt\u003c/sup\u003e and improve outcomes of virus infection.\u003c/p\u003e","manuscriptTitle":"Treatment with mitochondrial targeting antibiotics improves survival outcomes after Flock House virus infection in young and aged Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 11:38:35","doi":"10.21203/rs.3.rs-6816306/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"061e21b6-f428-4962-a53e-90293a4fb22e","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-10T11:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 11:38:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6816306","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6816306","identity":"rs-6816306","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}