Next Article in Journal
Discovery of Siderophore and Metallophore Production in the Aerobic Anoxygenic Phototrophs
Previous Article in Journal
Gut Microbiota and NAFLD: Pathogenetic Mechanisms, Microbiota Signatures, and Therapeutic Interventions
Previous Article in Special Issue
Activation of the JNK/MAPK Signaling Pathway by TGF-β1 Enhances Neonatal Fc Receptor Expression and IgG Transcytosis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 9
Issue 5
10.3390/microorganisms9050958
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

The Impact of Age on Response to Infection in Drosophila

by
Noah Sciambra
and
Stanislava Chtarbanova
*
Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(5), 958; https://doi.org/10.3390/microorganisms9050958
Submission received: 30 March 2021 / Revised: 27 April 2021 / Accepted: 28 April 2021 / Published: 29 April 2021
(This article belongs to the Special Issue Microbe–Host Interactions: From Infection to Innate Immunity Aspects)
Browse Figure
Versions Notes

Abstract

:
This review outlines the known cellular pathways and mechanisms involved in Drosophila age-dependent immunity to pathogenic microorganisms such as bacteria and fungi. We discuss the implication of host signaling pathways such as the Toll, Immune Deficiency (IMD), Janus kinase signal transducer and activator of transcription (JAK/STAT), and Insulin/Insulin Growth Factor/Target of Rapamycin (IIS/TOR) on immune function with aging. Additionally, we review the effects that factors such as sexual dimorphism, environmental stress, and cellular physiology exert on age-dependent immunity in Drosophila. We discuss potential tradeoffs between heightened immune function and longevity in the absence of infection, and we provide detailed tables outlining the various assays and pathogens used in the cited studies, as well as the age, sex, and strains of Drosophila used. We also discuss the overlapping effects these pathways and mechanisms have on one another. We highlight the great utility of Drosophila as a model organism and the importance of a greater focus on age-dependent antiviral immunity for future studies.

1. Introduction

The common fruit fly, Drosophila melanogaster, with its short lifespan, low cost of culture, and potent conserved innate immune defenses against a variety of microorganisms, serves as an excellent model for investigating the consequences of immunosenescence, a conserved process characterized by the progressive decline of the immune system’s function with age [1,2,3,4]. In humans, immunosenescence is associated with a decreased ability to defend against infections, resulting in significant morbidity and mortality among the elderly [5,6,7,8]. Despite considerable progress made towards our understanding of immunosenescence, the genetic and molecular mechanisms underlying age-dependent responses to immune challenges are areas of ongoing research. Specifically, the interplay between aging and innate immunity, which represents the first line of defense against microbial invaders, is less well understood, and often falls behind studies of the aging adaptive immune system [9]. Individuals aged 65 and older currently outnumber children under 5 globally [10] and, thus, a deeper understanding of the mechanisms underlying immunosenescence has become exceedingly important. A vast repertoire of genetic and genomic tools is available to Drosophila researchers, and the high degree of genetic homology between Drosophila and humans [11] makes the modeling of human diseases and immunosenescence in Drosophila highly translatable [12].
Drosophila lack an adaptive immune response but do have conserved pathways underlying innate immunity, which, in mammalian systems, play a key role in the age-dependent response to infections [13,14]. The Toll and Immune deficiency (IMD) pathways in Drosophila are nuclear factor kappa B (NF-κB) pathways with similarities to mammalian Toll-like receptor/interleukin (IL)-1 receptor and tumor necrosis factor receptor (TNFR) pathways, respectively. In response to fungal and bacterial infection, activation of Toll and IMD pathways leads to the transcription of downstream antimicrobial peptides (AMPs) genes (reviewed in [15]). Notably, the expression of several AMP genes increases with age [16,17,18]. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which, in mammals, is the main signaling downstream of cytokines and their receptors [19], is involved in response to Drosophila C Virus (DCV) infection in Drosophila [20]. This pathway is also activated in response to bacterial pathogens and is especially important in maintaining homeostasis in the gut, where dysbiosis exacerbates with age [21,22,23,24,25]. The Insulin/Insulin Growth Factor signaling pathway (IIS) which, together with the Target of Rapamycin (TOR) signaling pathway forms the IIS/TOR network, regulates autophagy, detoxification, and protein synthesis in Drosophila. Perhaps most importantly, the IIS/TOR pathway is key to lifespan determination in Drosophila (reviewed in [26]), and it has a linked function in Drosophila immunity due to its interaction with the Toll, IMD, and JAK/STAT pathways in Drosophila [27,28,29,30,31]. The Forkhead box, sub-group O transcription factor in Drosophila (dFOXO), a downstream target of the IIS/TOR pathway, binds to the promoter region of the AMP gene Drosomycin and induces its expression in response to starvation [27]. In Drosophila, dFOXO is required for defense against viral pathogens such as the Cricket paralysis virus (CrPV) and the Flock House virus (FHV) [32], and it plays a role in intestinal immunity to the bacterial pathogen Serratia marcescens (S. marcescens) [33].
In addition to these distinct cellular signaling pathways (Figure 1), other factors such as sexual dimorphism, environmental stress, and phagocytic efficacy affect Drosophila immunity in an age-dependent fashion. This review will focus on these key pathways and factors, as well as the relevant research assays involved in the study of age-dependent immune responses in Drosophila.

2. Pathways Affecting Age-Dependent Immunity in Drosophila

2.1. The Role of the Toll and IMD Pathways in Drosophila Age-Dependent Immunity

Drosophila have an inducible antimicrobial immune response that combats pathogenic infections with bacteria and fungi. This response is characterized by the induction of antimicrobial peptides (AMPs) [34], which is controlled by the nuclear factor-κB (NF-κB) family of transcription factors (TFs). NF-κB TFs such as Dorsal (dl), Dif, and Relish are activated through two separate signaling cascades: The Toll and IMD pathways [35,36]. The Toll pathway is primarily activated in response to Gram-positive bacterial and fungal pathogens [35,37], while the IMD pathway is mostly activated in response to Gram-negative bacteria [38,39,40,41]. These conserved innate immune pathways undergo age-dependent changes in function and gene expression. For example, Drosophila naturally display increased expression of AMP genes with age [16,17,18,42,43]. Elevated AMP expression typically allows for a more persistent induction of innate immune responses following infection with bacterial pathogens [18]. Some may hypothesize that heightened AMP expression in older individuals allows aged Drosophila to respond more efficiently to infection, but studies suggest the opposite. Older flies exposed to both live and killed bacteria (mixture of Escherichia coli (E.coli) and Micrococcus luteus (M. luteus)) induce less Diptericin, an AMP primarily induced via the IMD pathway, after a heat-killed bacterial jab than younger flies treated simultaneously. Despite the progressive upregulation of AMPs with age, the intrinsic capacity of older flies to effectively induce AMPs and defend against infection is shown to decline with age in Drosophila [18].
Age-dependent decline in immune function is conserved across species [3], but there are studies in which aged Drosophila display a lower bacterial load than younger Drosophila after infection. For example, Khan and Prasad [44] found that, following S. marcescens infection, a 13-day-old (aged) LH laboratory population of Drosophila displayed lower bacterial loads than younger flies (3 and 8 days old, respectively). However, this study did not include infection survival assays, and studies that do include infection survival assays with long-lived (~80 days median survival) Drosophila, such as chico null mutants, have found that survival to bacterial infection is not linked to AMP gene expression [45]. Additionally, there are several examples linking overactivation of Drosophila NF-κB pathways and over-expression of AMPs with reduction in lifespan [16,46,47]. Kounatidis and colleagues found that brain-specific knockdown of Relish increased Drosophila lifespan, while overexpression of the AMP genes AttacinC, Drosocin and CecropinA1 in neural tissue reduced lifespan [16]. Badinloo and colleagues showed that ubiquitous overexpression of the AMPs AttacinA, Metchnikowin, CecropinA1 and Defensin resulted in reduced lifespan [47]. In another example, Fabian and colleagues found that Drosophila longevity was increased following ubiquitous RNAi knockdown of the genes encoding for Toll (Tl) receptor and its ligand Spätzle (spz). Drosophila longevity decreased ~50% following ubiquitous RNAi knockdown of the Toll pathway inhibitor cactus (resulting in overactivation of Toll signaling). The same study also found that long-lived (median lifespan of 62.5–72 days) Drosophila caught in a peach orchard in Michigan displayed increased expression of the AMP genes Drosomycin, AttacinA, and Diptericin at a young age (5–6 days old), and decreased expression of these AMP genes at an older age (25–26 days old) following Erwinia carotovora carotovora (Ecc15) infection, in comparison to a control line [46].
Thus, it is apparent that NF-κB signaling, the ability to defend against infection, and longevity are interconnected in Drosophila. The importance of NF-κB signaling at older age from the Fabian et al., [46] study is in support of earlier findings showing that RelE20 and RelE38 Relish null mutations completely eliminate the improved ability of flies over-expressing the intracellular receptor PGRP-LE to defend against Pseudomonas aeruginosa (P. aeruginosa) infection [48]. Additionally, although fat body-specific over-expression of PGRP-LE offered enhanced pathogen resistance in both young (7 day-old) and older (40 day-old) animals, this chronic activation of NF-κB signaling resulted in a shorter lifespan in the absence of infection in comparison to Drosophila carrying a Relish null mutation [48].
All of these findings suggest a tradeoff between pathways involved in immune signaling and longevity in Drosophila. Findings from Sinam and colleagues [49] build upon this trend. The authors compared the lifespan and response to bacterial and fungal pathogens of two Cytorace lines derived from hybridization of Drosophila nasuta nasuta and Drosophila nasuta albomicans, and which were at different stages of evolutionary divergence. They showed that Cytorace-9 flies failing to upregulate expression of the Cecropin gene were more susceptible to infection with the fungal pathogens Beauveria bassiana (B. bassiana) and Metarhizium anisopliae (Table 1) in comparison to Cytorace-3 flies, which upregulate Cecropin expression after infection. Cytorace-9 flies also showed increased lifespan in the absence of infection when compared to Cytorace-3 flies. While increased AMP gene expression may offer increased pathogen resistance, especially at a younger age, this heightened immune signaling negatively impacts lifespan in the absence of infection. However, given that survival to bacterial infection was shown to not be linked to AMP gene expression in long-lived chico Drosophila mutants [45], this tradeoff between immune signaling and longevity in Drosophila is likely more complex than current research suggests [49]. Further research is necessary to fully elucidate the complex nature of the age-dependent increase of AMP expression in Drosophila and how it affects lifespan and response to infection.

2.2. The Role of the JAK/STAT Pathway in Drosophila Age-Dependent Immunity

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway is induced by the cytokines of the Unpaired family, such as Unpaired 1 (Upd1), Unpaired 2 (Upd2), and Unpaired 3 (Upd3), in response to cellular stress and damage [19,50,51]. The JAK/STAT signaling pathway plays an important role in Drosophila adult midgut homeostasis, ensuring continuous renewal of this organ throughout the animal’s lifespan [51]. The Toll pathway does not function in the Drosophila gut [52], and the JAK/STAT pathway contributes to gut antimicrobial response through the induction of Drosomycin-like peptides [24]. Given that dysfunction of the intestinal barrier is a hallmark of aging in Drosophila [53], and that a functional intestinal barrier during aging is critical to maintaining lifespan [54], it is thus understandable how JAK/STAT signaling may be involved in age-dependent immunity in Drosophila.
Intestinal epithelial renewal is a key defense against oral bacterial infection in Drosophila, and the JAK/STAT pathway is required for bacterially induced stem cell proliferation in response to infection, stress, or damage [21,22,24,54]. Salazar and colleagues [54] studied the effects of altered expression of the septate junction-specific protein Snakeskin (Ssk), in combination with the JAK/STAT pathway, and observed significant effects on intestinal barrier dysfunction, dysbiosis, lifespan, and gut morphology (Table 2). Decreased expression of intestinal Ssk resulted in increased barrier dysfunction, reduced lifespan, elevated expression levels of the AMP genes Diptericin, Drosocin, Drosomycin, and Metchnikowin, and increased upd3 mRNA levels. Restoration of Ssk expression completely reversed age-related intestinal barrier dysfunction, protected against S. marcescens infection, and increased lifespan. Thus, there is a correlation between increased upd3 expression, an activator of the JAK/STAT pathway, and junctional protein mislocalization before detectable intestinal barrier failure. This also aligns with previous studies demonstrating elevated upd3 expression in fly mutants for the human CD36 homologue croquemort (crqKO), which is involved in microbial phagocytosis and clearance. crqKO mutants are short-lived and exhibit premature aging associated with gut hyperplasia [55]. This suggests that proper regulation of the JAK/STAT pathway is central in preventing age-dependent gut dysfunction and protecting against infection.
In addition to its role in age-dependent intestinal barrier homeostasis and defense against oral bacterial infection, the JAK/STAT pathway functions in antiviral immunity, particularly in response to infection with the RNA viruses Drosophila C Virus (DCV) and Cricket paralysis virus (CrPV) [20,56], as well as the DNA virus Invertebrate iridescent Virus 6 (IIV-6) [57]. In Drosophila, the histone H3 lysine 9 methyltransferase G9a was shown to negatively regulate JAK/STAT pathway activation in response to RNA virus infection in order to limit immunopathology caused by hyperactivation of this pathway [58]. Although G9a mutants appear to have increased lifespan in comparison to respective wild-type controls, these flies succumb faster to infection with the RNA viruses FHV, CrPV, DCV and Drosophila X virus (DXV) [58]. Fabian and colleagues [46] identified upd3 as a candidate gene for longevity in Drosophila and infected long-lived (median lifespan of 62.5–72 days) wild-caught Drosophila from a Michigan peach orchard at 5–6 days of age with DCV. Flies of this long-lived strain injected with DCV survived significantly longer than random-bred control lines of the same population, suggesting that prolonged lifespan may be significantly linked to improved realized antiviral immune response. These findings further complicate the relationship between aging and immunity and the fitness tradeoffs that may arise from favoring expression of genes important for extending lifespan or for defense against infection. Maintaining epithelial junctions may be critical to prolonging lifespan and defending against infection with age, but further research is needed to fully understand the complexity of these systems and what fitness tradeoffs come as a result of age-dependent changes in JAK/STAT signaling.

2.3. The Role of the IIS/TOR Network in Drosophila Age-Dependent Immunity

The Insulin/Insulin Growth Factor (IIS) signaling pathway and its linked Target of Rapamycin (TOR) signaling pathway are vital nutritional systems that regulate growth in Drosophila [59]. Drosophila have one Insulin receptor (dInR), a singular insulin receptor substrate (chico), one downstream dFOXO transcription factor, and eight dInR ligands: insulin-like peptides (dILPs) 1–8 [60,61]. Activation of dInR induces two possible downstream signaling cascades, one via the kinase Akt1 and the other via the mitogen activated protein (MAP) kinase Rolled, also known as extracellular signal-regulated kinase (ERK). Akt1 negatively regulates dFOXO and positively regulates TOR [62,63]. Rolled (ERK) activation is implicated in cellular growth and insulin sensitivity [64,65].
IIS signaling pathway functionality is directly linked to longevity and immunity in Drosophila. The Toll, IMD, and JAK/STAT pathways interact with the IIS signaling pathway in the Drosophila fat body to regulate metabolism, growth, and immunity. dFOXO regulates AMP gene expression [27], and the Toll NF-κB transcription factor Dif can inhibit insulin signaling [28]. Additionally, IIS and subsequent Rolled/MAPK induction can activate the fly poor Imd response upon knock-in (Pirk), a gene encoding for a negative regulator of the IMD pathway [64,66,67,68,69]. Furthermore, the Drosophila fat body signals with insulin producing cells (IPCs) located in the Drosophila brain to communicate nutrient status and the release of insulin-like peptides (dILPs). This is accomplished via secretion of another cytokine from the Unpaired family, Unpaired 2 (Upd2), a ligand for the JAK/STAT pathway, when Drosophila are in a fed state [70]. This all emphasizes the possible role of the IIS signaling pathway in age-dependent immunity, given that the Toll, IMD, and JAK/STAT pathways have significant roles in Drosophila aging and immunity that have already been discussed.
Reduced IIS signaling extends lifespan [71,72], slows or delays age-dependent organ degeneration [73], and improves climbing ability in Drosophila [74]. A study from Ueda and colleagues found that increased expression of the miRNA miR-305, which usually decreases with age, accelerates aging phenotypes in Drosophila. Notably, mRNAs for insulin-like peptides also increased, along with miR-305 expression, further emphasizing the link between reduced IIS/TOR signaling and increased lifespan [75]. Growth-Blocking Peptides (GBPs) are produced as a direct result of TOR signaling. Reduced GBP expression reduces Drosophila growth rate and body size [76]. Genetic analyses from Sung and Shears [77] have linked a previously uncharacterized G Protein Coupled Receptor (GPCR), Methuselah-like receptor-10 (Mthl10), as a binding protein for GBP in Drosophila. As in other studies where reduced IIS/TOR signaling increased lifespan, Mthl10 knockdown in Drosophila also increased lifespan. This increased lifespan as a result of Mthl10 knockdown also affected pathogen defense against infection. Mthl10 knockdown increased mortality in adult Drosophila after infection with M. luteus [77]. This suggests a possible fitness tradeoff between increased longevity and age-dependent resistance to infection via IIS/TOR signaling similar to the tradeoffs seen in the Toll and IMD pathways.
In addition to its known role in regulating lifespan across numerous species, the IIS/TOR pathway also regulates homeostasis of the Drosophila lymph gland, a larval hematopoietic organ important for the generation of blood cell progenitors [78]. Despite the disappearance of the Drosophila lymph gland after the larval stage of development [79], genetic analysis of Drosophila infected with E. coli at 4 weeks of age still found genes involved with the IIS/TOR pathway to be significantly associated with bacterial clearance at older age [80]. Additionally, mutations in chico, a Drosophila insulin receptor substrate, extended lifespan and increased survival of mutant flies following E. coli and Photorhabdus luminescens (P. luminescens) infection [81]. Following E. coli and P. luminescens infection, chico mutants survived bacterial infection better than control flies, and displayed significantly lower amounts of bacterial cells at 3 and 16 h post-infection. However, chico mutants displayed higher bacterial loads at 30 h post-infection in response to E. coli infection, but not P. luminescens infection. These mutants also showed reduced transcription of the AMP genes Diptericin, CecropinA1, and Drosomycin, although pathogen- and time point post infection-based variation was observed [81]. These results suggest a link to age-dependent immunity for the IIS/TOR pathway.
Furthermore, the downstream target of the IIS/TOR signaling pathways, dFOXO, is essential to both intestinal immunity against S. marcescens and defense against viral pathogens [32,33]. In comparison to wild-type controls, dFOXO mutants die faster after oral infection with S. marcescens, and male dFOXO mutants accumulate higher bacterial loads [33]. dFOXO null mutants are also deficient in fighting off infection from both CrPV and FHV [32]. Additionally, dFOXO activity increases in response to virus infection and activated dFOXO can decrease viral load following infection [32], displaying a role for the linked IIS/TOR pathway in Drosophila innate immunity (Table 3). Interestingly, the number of FOXO-bound genes significantly decreases with age in female Drosophila (comparing 2-week-old and 5-week-old w1118 control flies), and many FOXO-targeted genes have altered transcription levels with age [82]. However, it is also notable that the FOXO transcription factor is not only influenced by the IIS/TOR pathway, and many of the observed age-dependent changes in FOXO DNA binding were independent of the Insulin signaling pathway [82]. With its role both within and independent of the IIS/TOR signaling pathway, FOXO will be a transcription factor of very high interest for future studies investigating age-dependent immunity in Drosophila.
The IIS/TOR pathway appears to be significantly involved in age-dependent immunity and the control of lifespan in Drosophila, but further research is needed to understand the complexity of these systems and the fitness tradeoffs that may come with reduced IIS/TOR signaling. Additionally, the FOXO transcription factor, its diverse mechanisms in transcriptional regulation, and its altered targeting and function related to the IIS/TOR pathway with age requires further research into the dynamics of FOXO’s roles in both aging and immunity.

3. Other Factors Affecting Drosophila Age-Dependent Immunity

There are various environmental, genetic, and physiological factors that can affect age-dependent immunity in Drosophila. One of these factors is sex [83,84]. The causes of immunosenescence can vary between sexes. Both males and females become more susceptible to infection with age, but the cause of immunosenescence can vary between sexes. Following infection with the fungus B. bassiana, age-dependent decline in immune function for males results from barrier defense deterioration, while, in females, it results from systemic senescence of immune defenses [85]. However, Khan and Prasad found that sex did not affect bacterial load levels following S. marcescens infection [44]. The true significance of sex in respect to age-dependent immunity in Drosophila specifically is yet to be determined but, given the established profound differences in immune response between males and females [84], it can be expected that future immunosenescence research may display significant sexually dimorphic results.
Environmental factors, which have sexually dimorphic effects, also have a significant impact on age-dependent immunity in Drosophila. Cold stress, subjecting Drosophila to periods of extreme low temperatures, has beneficial effects on aging, lifespan, and resistance to stresses such as severe temperature exposure and B. bassiana infection [86,87,88]. Additionally, these environmental stresses have different effects on males and females. Cold stress in male Drosophila increases longevity when applied before 4 weeks of age, while providing no positive effect in female Drosophila at any age. Meanwhile, cold stress can increase heat resistance ability at 6 weeks of age in both sexes, no matter when the cold stress was applied. This displays that exposure to environmental stress early in life can improve resistance to environmental stress later in life, regardless of sex. Prior exposure to cold stress increased survival to fungal infection (B. bassiana) at 6 weeks of age in both sexes, although males were more significantly affected. These differences in pathogen defense between sexes at old age highlights the significance of both environmental stress and sexual dimorphism on age-dependent immunity [87].
Other environmental factors, such as desiccation, also affect age-dependent immunity in Drosophila. Desiccated flies that were kept in empty vials without food for 2 h displayed an increased susceptibility to Ecc15 infection [89]. In the same study, the authors showed that desiccated flies allowed to recover prior to Ecc15 infection display higher survival rates than flies not desiccated prior to infection. This suggests that being under significant environmental stress during infection decreases resistance, but that exposure to an environmental stress with allowed recovery prior to infection increases resistance. This short desiccation period elevates levels of peptidoglycan recognition protein-LC (PGRP-LC) expression in Malpighian tubules (the equivalent of the kidney) and increases induction of the AMP genes CecropinA2, CecropinC, AttacinD, Diptericin, Defensin, and Metchnikowin [89]. This further emphasizes the impact of environmental factors on age-dependent immunity in Drosophila, and these factors should be accounted for when designing experiments relevant to aging and immunity.
In addition to environmental and physiological factors, the functionality of basic cellular mechanisms can have significant impacts on age-dependent immunity in Drosophila. Findings from Horn and colleagues highlight the effects of Drosophila phagocytic efficiency, which significantly declines with age [90]. Notably, the rate of Escherichia coli engulfment after infection does not decline, but the functional clearance of phagocytic vesicles after engulfment decreases considerably with age. Four lines from the Drosophila Genetic Reference Panel (DGRP) collection [91,92] (lines 359, 389, 437, and 589), with increased or decreased bacterial clearance with age, display the same number of phagocytic events per hemocyte, suggesting that the functional clearance of engulfed bacteria, and not engulfment itself, is an important factor in age-dependent immunity [90] (Table 4).

4. Conclusions and Future Directions

There are a variety of cellular pathways and mechanisms, as well as environmental and physiological factors, that affect the ability of aged organisms to respond to infection. Additionally, many of these pathways and factors appear to have tradeoff effects on immune function and lifespan. Toll and IMD pathways activate the NF-κB TFs in Drosophila and subsequent AMP expression [15,34], and Drosophila naturally display higher AMP expression at an older age [16,17,18,42,43,46,47]. The Toll and IMD pathways are critical to survival against bacterial, fungal, and viral infection [35,37,38,93,94,95,96], but overactivation of these pathways has been shown to decrease lifespan in the absence of infection [48]. The JAK/STAT signaling pathway contributes to antimicrobial response and homeostasis in the Drosophila gut [51]. Maintaining a functional intestinal barrier is critical to maintaining lifespan, and dysfunction of the intestinal barrier is a hallmark of aging in Drosophila [53,54]. Intestinal epithelial renewal is vital for protection against oral bacterial infection, and the JAK/STAT pathway is required for this process [21,22,24,54]. In addition to maintaining gut homeostasis and defending against bacterial infection, the JAK/STAT pathway is also important for defense against viruses such as DCV, CrPV and IIV-6 infection [20,56,57]. Upd3 has been identified as a candidate for longevity in Drosophila, and wild-caught long-lived fly strains survive longer than controls following DCV infection [46]. The IIS/TOR signaling pathways regulate growth, longevity, and defense against infection in Drosophila [59]. Reduced IIS/TOR signaling extends lifespan [71,72] and has shown contrasting effects on the ability to respond to bacterial infection in adult Drosophila [80,81]. The IIS/TOR network also interacts with the Toll (Dif), IMD (Pirk), and JAK/STAT (Upd2) pathways [28,69,70], further emphasizing its role in Drosophila age-dependent immunity. dFOXO, a transcription factor downstream of the IIS/TOR signaling pathways, has age-dependent changes in its DNA binding activity [82], and is important for defense against viral pathogens such as FHV and CrPV [32]. There are significant differences in response to infection by male and female Drosophila [84]. Different causes have been identified for the age-dependent decline in anti-fungal immune function between male and female Drosophila [85], but this does not appear universal, as another study found no significant differences in bacterial loads between male and female flies [44]. Environmental stressors such as cold stress and desiccation have positive effects on lifespan and resistance to fungal [86,87] and bacterial infection [89]. Additionally, cellular mechanisms such as phagocytic efficiency significantly decline with age following E. coli infection [90].
The studies discussed in this review illustrate at least some of the aspects the impact of aging exerts on immune function in Drosophila. However, further studies are needed to create a more complete understanding of the age-dependent factors and mechanisms that impact Drosophila immune defenses and the fitness tradeoffs between longevity and the ability to defend against infection. Additionally, given the profound impact of viral pathogens on elderly individuals, as illustrated by the ongoing Coronavirus (COVID-19) global pandemic [97,98,99,100] and the relative lack of focus on age-dependent immunity in response to viral infection, there is a great need for further research investigating the age-dependent mechanisms that defend against viral infection specifically. Drosophila can serve as a great genetic model to investigate the impact of age on innate immune responses to viral infection.
Despite well-characterized pathways involved in Drosophila antiviral immunity, such as antiviral RNAi (reviewed in [101,102]) and cellular processes such as autophagy, apoptosis, and apoptotic body clearance (reviewed in [103]), there is very limited understanding of how these antiviral immune responses are impacted by aging. Interestingly, some of the pathways already outlined in this article, which have been shown to have age-dependent effects on Drosophila immunity in other contexts, such as the JAK/STAT, Toll and IMD pathways, are also important for Drosophila antiviral immunity. The JAK/STAT pathway has been shown to mediate survival to DCV, CrPV, FHV, and DXV infection [20,56,58]. Toll pathway mutants die more rapidly following DXV infection [93], and Toll pathway genes have been shown to be important for resistance to several RNA viruses (DXV, DCV, FHV, CrPV and Nora Virus) [93,94]. IMD pathway mutants are more susceptible to CrPV and Sindbis Virus (SINV) infection [95,96]. Thus, these pathways should be of high interest in future Drosophila immunosenescence research. Additionally, antiviral cellular mechanisms, such as phagocytosis via macrophages, display an age-dependent decline in efficiency [90,104].
Another particular area of future research interest is in the importance of disease tolerance mechanisms in response to viral infection at older age. There are two strategies for organisms to defend against infection: resistance and tolerance. Disease resistance encompasses an organism’s ability to combat and prevent infection by pathogens. Disease tolerance encompasses an organism’s ability to tolerate a given level of infection (pathogen load) and limit negative effects after resistance mechanisms have been compromised (reviewed in [105]). Disease tolerance at older age may be very important for antiviral defense in Drosophila. In a recent study, Sheffield and colleagues found that, despite displaying an increased susceptibility to FHV infection, older (30 day-old) wild-type (OregonR) flies do not accumulate higher virus titers than their younger (5 day-old) counterparts [106]. Intriguingly, a similar pattern has been observed in humans following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for COVID-19. In comparison to younger individuals, older individuals display increased mortality without accumulating a higher virus titer when infected with SARS-CoV-2 [107]. Clearly, there is still much to be discovered regarding immune defenses in both humans and Drosophila, especially regarding the impact of age on these immune defenses. Drosophila can be a powerful tool in our pursuit to elucidate the intricacies of immune defense pathways and their change in efficiency over time. With the availability of advanced genetic and genomic tools in Drosophila, future findings made in this organism could lead to important translational impacts and advancements in healthcare, as well as treatment strategies that lead to improved immune response and increased longevity in aged organisms.

Author Contributions

Writing—original draft preparation, N.S.; writing—review and editing, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Aw, D.; Silva, A.B.; Palmer, D.B. Immunosenescence: Emerging challenges for an ageing population. Immunology 2007, 120, 435–446. [Google Scholar] [CrossRef]
  2. Eleftherianos, I.; Castillo, J.C. Molecular mechanisms of aging and immune system regulation in Drosophila. Int. J. Mol. Sci. 2012, 13, 9826–9844. [Google Scholar] [CrossRef] [Green Version]
  3. Min, K.J.; Tatar, M. Unraveling the Molecular Mechanism of Immunosenescence in Drosophila. Int. J. Mol. Sci. 2018, 19, 2472. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Panda, A.; Arjona, A.; Sapey, E.; Bai, F.; Fikrig, E.; Montgomery, R.R.; Lord, J.M.; Shaw, A.C. Human innate immunosenescence: Causes and consequences for immunity in old age. Trends Immunol. 2009, 30, 325–333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Solana, R.; Pawelec, G.; Tarazona, R. Aging and innate immunity. Immunity 2006, 24, 491–494. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Yoshikawa, T.T. Epidemiology and unique aspects of aging and infectious diseases. Clin. Infect. Dis. 2000, 30, 931–933. [Google Scholar] [CrossRef] [Green Version]
  7. Crossley, K.B.; Peterson, P.K. Infections in the elderly. Clin. Infect. Dis. 1996, 22, 209–215. [Google Scholar] [CrossRef] [Green Version]
  8. Kline, K.A.; Bowdish, D.M. Infection in an aging population. Curr. Opin. Microbiol. 2016, 29, 63–67. [Google Scholar] [CrossRef]
  9. Nikolich-Zugich, J. The twilight of immunity: Emerging concepts in aging of the immune system. Nat. Immunol. 2018, 19, 10–19. [Google Scholar] [CrossRef] [PubMed]
  10. United Nations, Department of Economic and Social Affairs. Population Division World Population Prospects 2019: Highlights (ST/ESA/SER.A/423). Available online: https://population.un.org/wpp/Publications/Files/WPP2019_Highlights.pdf (accessed on 21 May 2020).
  11. Reiter, L.T.; Potocki, L.; Chien, S.; Gribskov, M.; Bier, E. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res. 2001, 11, 1114–1125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Cheng, L.; Baonza, A.; Grifoni, D. Drosophila Models of Human Disease. Biomed Res. Int. 2018, 2018, 7214974. [Google Scholar] [CrossRef]
  13. Govind, S. Innate immunity in Drosophila: Pathogens and pathways. Insect. Sci. 2008, 15, 29–43. [Google Scholar] [CrossRef]
  14. Simell, B.; Vuorela, A.; Ekstrom, N.; Palmu, A.; Reunanen, A.; Meri, S.; Kayhty, H.; Vakevainen, M. Aging reduces the functionality of anti-pneumococcal antibodies and the killing of Streptococcus pneumoniae by neutrophil phagocytosis. Vaccine 2011, 29, 1929–1934. [Google Scholar] [CrossRef] [PubMed]
  15. Lemaitre, B.; Hoffmann, J. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 2007, 25, 697–743. [Google Scholar] [CrossRef] [Green Version]
  16. Kounatidis, I.; Chtarbanova, S.; Cao, Y.; Hayne, M.; Jayanth, D.; Ganetzky, B.; Ligoxygakis, P. NF-kappaB Immunity in the Brain Determines Fly Lifespan in Healthy Aging and Age-Related Neurodegeneration. Cell Rep. 2017, 19, 836–848. [Google Scholar] [CrossRef] [PubMed]
  17. Pletcher, S.D.; Macdonald, S.J.; Marguerie, R.; Certa, U.; Stearns, S.C.; Goldstein, D.B.; Partridge, L. Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster. Curr. Biol. 2002, 12, 712–723. [Google Scholar] [CrossRef] [Green Version]
  18. Zerofsky, M.; Harel, E.; Silverman, N.; Tatar, M. Aging of the innate immune response in Drosophila melanogaster. Aging Cell 2005, 4, 103–108. [Google Scholar] [CrossRef] [PubMed]
  19. Rawlings, J.S.; Rosler, K.M.; Harrison, D.A. The JAK/STAT signaling pathway. J. Cell Sci. 2004, 117, 1281–1283. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Dostert, C.; Jouanguy, E.; Irving, P.; Troxler, L.; Galiana-Arnoux, D.; Hetru, C.; Hoffmann, J.A.; Imler, J.L. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of drosophila. Nat. Immunol. 2005, 6, 946–953. [Google Scholar] [CrossRef] [PubMed]
  21. Cronin, S.J.; Nehme, N.T.; Limmer, S.; Liegeois, S.; Pospisilik, J.A.; Schramek, D.; Leibbrandt, A.; Simoes Rde, M.; Gruber, S.; Puc, U.; et al. Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection. Science 2009, 325, 340–343. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Buchon, N.; Broderick, N.A.; Poidevin, M.; Pradervand, S.; Lemaitre, B. Drosophila intestinal response to bacterial infection: Activation of host defense and stem cell proliferation. Cell Host Microbe 2009, 5, 200–211. [Google Scholar] [CrossRef] [Green Version]
  23. Jiang, H.; Patel, P.H.; Kohlmaier, A.; Grenley, M.O.; McEwen, D.G.; Edgar, B.A. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell 2009, 137, 1343–1355. [Google Scholar] [CrossRef] [Green Version]
  24. Buchon, N.; Broderick, N.A.; Chakrabarti, S.; Lemaitre, B. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 2009, 23, 2333–2344. [Google Scholar] [CrossRef] [Green Version]
  25. Clark, R.I.; Walker, D.W. Role of gut microbiota in aging-related health decline: Insights from invertebrate models. Cell Mol. Life Sci. 2018, 75, 93–101. [Google Scholar] [CrossRef]
  26. Partridge, L.; Alic, N.; Bjedov, I.; Piper, M.D. Ageing in Drosophila: The role of the insulin/Igf and TOR signalling network. Exp. Gerontol. 2011, 46, 376–381. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Becker, T.; Loch, G.; Beyer, M.; Zinke, I.; Aschenbrenner, A.C.; Carrera, P.; Inhester, T.; Schultze, J.L.; Hoch, M. FOXO-dependent regulation of innate immune homeostasis. Nature 2010, 463, 369–373. [Google Scholar] [CrossRef] [PubMed]
  28. DiAngelo, J.R.; Bland, M.L.; Bambina, S.; Cherry, S.; Birnbaum, M.J. The immune response attenuates growth and nutrient storage in Drosophila by reducing insulin signaling. Proc. Natl. Acad. Sci. USA 2009, 106, 20853–20858. [Google Scholar] [CrossRef] [Green Version]
  29. Davoodi, S.; Galenza, A.; Panteluk, A.; Deshpande, R.; Ferguson, M.; Grewal, S.; Foley, E. The Immune Deficiency Pathway Regulates Metabolic Homeostasis in Drosophila. J. Immunol. 2019, 202, 2747–2759. [Google Scholar] [CrossRef] [PubMed]
  30. Kamareddine, L.; Robins, W.P.; Berkey, C.D.; Mekalanos, J.J.; Watnick, P.I. The Drosophila Immune Deficiency Pathway Modulates Enteroendocrine Function and Host Metabolism. Cell Metab. 2018, 28, 449–462.e445. [Google Scholar] [CrossRef] [Green Version]
  31. Ahlers, L.R.H.; Trammell, C.E.; Carrell, G.F.; Mackinnon, S.; Torrevillas, B.K.; Chow, C.Y.; Luckhart, S.; Goodman, A.G. Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors. Cell Rep. 2019, 29, 1946–1960.e1945. [Google Scholar] [CrossRef] [Green Version]
  32. Spellberg, M.J.; Marr, M.T., 2nd. FOXO regulates RNA interference in Drosophila and protects from RNA virus infection. Proc. Natl. Acad. Sci. USA 2015, 112, 14587–14592. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Fink, C.; Hoffmann, J.; Knop, M.; Li, Y.; Isermann, K.; Roeder, T. Intestinal FoxO signaling is required to survive oral infection in Drosophila. Mucosal. Immunol. 2016, 9, 927–936. [Google Scholar] [CrossRef]
  34. Strominger, J.L. Animal antimicrobial peptides: Ancient players in innate immunity. J. Immunol. 2009, 182, 6633–6634. [Google Scholar] [CrossRef]
  35. Lemaitre, B.; Nicolas, E.; Michaut, L.; Reichhart, J.M.; Hoffmann, J.A. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996, 86, 973–983. [Google Scholar] [CrossRef] [Green Version]
  36. Georgel, P.; Naitza, S.; Kappler, C.; Ferrandon, D.; Zachary, D.; Swimmer, C.; Kopczynski, C.; Duyk, G.; Reichart, J.M.; Hoffmann, J.A. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 2001, 1, 503–514. [Google Scholar] [CrossRef] [Green Version]
  37. Rutschmann, S.; Kilinc, A.; Ferrandon, D. Cutting edge: The Toll pathway is required for resistance to Gram-positive bacterial infections in Drosophila. J. Immunol. 2002, 168, 1542–1546. [Google Scholar] [CrossRef] [Green Version]
  38. Leulier, F.; Rodriguez, A.; Khush, R.S.; Abrams, J.M.; Lemaitre, B. The Drosophila caspase Dredd is required to resist Gram-negative bacterial infection. EMBO Rep. 2000, 1, 353–358. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Rutschmann, S.; Jung, A.C.; Zhou, R.; Silverman, N.; Hoffmann, J.A.; Ferrandon, D. Role of Drosophila IKK gamma in a toll-independent antibacterial immune response. Nat. Immunol. 2000, 1, 342–347. [Google Scholar] [CrossRef] [PubMed]
  40. Rutschmann, S.; Jung, A.C.; Hetru, C.; Reichhart, J.M.; Hoffmann, J.A.; Ferrandon, D. The Rel protein DIF mediates the antifungal but not the antibacterial host defense in Drosophila. Immunity 2000, 12, 569–580. [Google Scholar] [CrossRef] [Green Version]
  41. Lemaitre, B.; Kromer-Metzger, E.; Michaut, L.; Nicolas, E.; Meister, M.; Georgel, P.; Reichhart, J.M.; Hoffmann, J.A. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc. Natl. Acad. Sci. USA 1995, 92, 9465–9469. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Seroude, L.; Brummel, T.; Kapahi, P.; Benzer, S. Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster. Aging Cell 2002, 1, 47–56. [Google Scholar] [CrossRef] [PubMed]
  43. Landis, G.N.; Abdueva, D.; Skvortsov, D.; Yang, J.; Rabin, B.E.; Carrick, J.; Tavare, S.; Tower, J. Similar gene expression patterns characterize aging and oxidative stress in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 2004, 101, 7663–7668. [Google Scholar] [CrossRef] [Green Version]
  44. Khan, I.; Prasad, N.G. The aging of the immune response in Drosophila melanogaster. J. Gerontol. A Biol. Sci. Med. Sci. 2013, 68, 129–135. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Libert, S.; Chao, Y.; Zwiener, J.; Pletcher, S.D. Realized immune response is enhanced in long-lived puc and chico mutants but is unaffected by dietary restriction. Mol. Immunol. 2008, 45, 810–817. [Google Scholar] [CrossRef]
  46. Fabian, D.K.; Garschall, K.; Klepsatel, P.; Santos-Matos, G.; Sucena, E.; Kapun, M.; Lemaitre, B.; Schlotterer, C.; Arking, R.; Flatt, T. Evolution of longevity improves immunity in Drosophila. Evol. Lett. 2018, 2, 567–579. [Google Scholar] [CrossRef] [Green Version]
  47. Badinloo, M.; Nguyen, E.; Suh, W.; Alzahrani, F.; Castellanos, J.; Klichko, V.I.; Orr, W.C.; Radyuk, S.N. Overexpression of antimicrobial peptides contributes to aging through cytotoxic effects in Drosophila tissues. Arch. Insect. Biochem. Physiol. 2018, 98, e21464. [Google Scholar] [CrossRef] [PubMed]
  48. Libert, S.; Chao, Y.; Chu, X.; Pletcher, S.D. Trade-offs between longevity and pathogen resistance in Drosophila melanogaster are mediated by NFkappaB signaling. Aging Cell 2006, 5, 533–543. [Google Scholar] [CrossRef]
  49. Sinam, Y.M.; Chatterjee, A.; Ranjini, M.S.; Poojari, A.; Nagarajan, A.; Ramachandra, N.B.; Nongthomba, U. A newly evolved Drosophila Cytorace-9 shows trade-off between longevity and immune response. Infect. Genet. Evol. 2016, 44, 1–7. [Google Scholar] [CrossRef]
  50. Hombria, J.C.; Brown, S.; Hader, S.; Zeidler, M.P. Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 2005, 288, 420–433. [Google Scholar] [CrossRef] [Green Version]
  51. Osman, D.; Buchon, N.; Chakrabarti, S.; Huang, Y.T.; Su, W.C.; Poidevin, M.; Tsai, Y.C.; Lemaitre, B. Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division. J. Cell Sci. 2012, 125, 5944–5949. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Ferrandon, D.; Jung, A.C.; Criqui, M.; Lemaitre, B.; Uttenweiler-Joseph, S.; Michaut, L.; Reichhart, J.; Hoffmann, J.A. A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway. EMBO J. 1998, 17, 1217–1227. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Rera, M.; Clark, R.I.; Walker, D.W. Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc. Natl. Acad. Sci. USA 2012, 109, 21528–21533. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Salazar, A.M.; Resnik-Docampo, M.; Ulgherait, M.; Clark, R.I.; Shirasu-Hiza, M.; Jones, D.L.; Walker, D.W. Intestinal Snakeskin Limits Microbial Dysbiosis during Aging and Promotes Longevity. iScience 2018, 9, 229–243. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Guillou, A.; Troha, K.; Wang, H.; Franc, N.C.; Buchon, N. The Drosophila CD36 Homologue croquemort Is Required to Maintain Immune and Gut Homeostasis during Development and Aging. PLoS Pathog. 2016, 12, e1005961. [Google Scholar] [CrossRef] [PubMed]
  56. Kemp, C.; Mueller, S.; Goto, A.; Barbier, V.; Paro, S.; Bonnay, F.; Dostert, C.; Troxler, L.; Hetru, C.; Meignin, C.; et al. Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila. J. Immunol. 2013, 190, 650–658. [Google Scholar] [CrossRef] [Green Version]
  57. West, C.; Silverman, N. p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila. PLoS Pathog. 2018, 14, e1007020. [Google Scholar] [CrossRef] [PubMed]
  58. Merkling, S.H.; Bronkhorst, A.W.; Kramer, J.M.; Overheul, G.J.; Schenck, A.; Van Rij, R.P. The epigenetic regulator G9a mediates tolerance to RNA virus infection in Drosophila. PLoS Pathog. 2015, 11, e1004692. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Kannan, K.; Fridell, Y.W. Functional implications of Drosophila insulin-like peptides in metabolism, aging, and dietary restriction. Front. Physiol. 2013, 4, 288. [Google Scholar] [CrossRef] [Green Version]
  60. Gronke, S.; Clarke, D.F.; Broughton, S.; Andrews, T.D.; Partridge, L. Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet 2010, 6, e1000857. [Google Scholar] [CrossRef] [Green Version]
  61. Nassel, D.R.; Liu, Y.; Luo, J. Insulin/IGF signaling and its regulation in Drosophila. Gen. Comp. Endocrinol. 2015, 221, 255–266. [Google Scholar] [CrossRef]
  62. Sengupta, S.; Peterson, T.R.; Sabatini, D.M. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol. Cell 2010, 40, 310–322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Dionne, M.S.; Pham, L.N.; Shirasu-Hiza, M.; Schneider, D.S. Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila. Curr. Biol. 2006, 16, 1977–1985. [Google Scholar] [CrossRef] [Green Version]
  64. Zhang, W.; Thompson, B.J.; Hietakangas, V.; Cohen, S.M. MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila. PLoS Genet 2011, 7, e1002429. [Google Scholar] [CrossRef]
  65. Texada, M.J.; Koyama, T.; Rewitz, K. Regulation of Body Size and Growth Control. Genetics 2020, 216, 269–313. [Google Scholar] [CrossRef]
  66. Kleino, A.; Myllymaki, H.; Kallio, J.; Vanha-aho, L.M.; Oksanen, K.; Ulvila, J.; Hultmark, D.; Valanne, S.; Ramet, M. Pirk is a negative regulator of the Drosophila Imd pathway. J. Immunol. 2008, 180, 5413–5422. [Google Scholar] [CrossRef] [Green Version]
  67. Lhocine, N.; Ribeiro, P.S.; Buchon, N.; Wepf, A.; Wilson, R.; Tenev, T.; Lemaitre, B.; Gstaiger, M.; Meier, P.; Leulier, F. PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 2008, 4, 147–158. [Google Scholar] [CrossRef] [Green Version]
  68. Aggarwal, K.; Rus, F.; Vriesema-Magnuson, C.; Erturk-Hasdemir, D.; Paquette, N.; Silverman, N. Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. PLoS Pathog. 2008, 4, e1000120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Ragab, A.; Buechling, T.; Gesellchen, V.; Spirohn, K.; Boettcher, A.L.; Boutros, M. Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells. EMBO J. 2011, 30, 1123–1136. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Rajan, A.; Perrimon, N. Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. Cell 2012, 151, 123–137. [Google Scholar] [CrossRef] [Green Version]
  71. Tatar, M.; Kopelman, A.; Epstein, D.; Tu, M.P.; Yin, C.M.; Garofalo, R.S. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 2001, 292, 107–110. [Google Scholar] [CrossRef] [Green Version]
  72. Clancy, D.J.; Gems, D.; Hafen, E.; Leevers, S.J.; Partridge, L. Dietary restriction in long-lived dwarf flies. Science 2002, 296, 319. [Google Scholar] [CrossRef] [Green Version]
  73. Wessells, R.J.; Fitzgerald, E.; Cypser, J.R.; Tatar, M.; Bodmer, R. Insulin regulation of heart function in aging fruit flies. Nat. Genet. 2004, 36, 1275–1281. [Google Scholar] [CrossRef]
  74. Bai, H.; Post, S.; Kang, P.; Tatar, M. Drosophila Longevity Assurance Conferred by Reduced Insulin Receptor Substrate Chico Partially Requires d4eBP. PLoS ONE 2015, 10, e0134415. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Ueda, M.; Sato, T.; Ohkawa, Y.; Inoue, Y.H. Identification of miR-305, a microRNA that promotes aging, and its target mRNAs in Drosophila. Genes Cells 2018, 23, 80–93. [Google Scholar] [CrossRef] [Green Version]
  76. Koyama, T.; Mirth, C.K. Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation. PLoS Biol. 2016, 14, e1002392. [Google Scholar] [CrossRef] [Green Version]
  77. Sung, E.J.; Shears, S.B. A genome-wide dsRNA library screen for Drosophila genes that regulate the GBP/phospholipase C signaling axis that links inflammation to aging. BMC Res. Notes 2018, 11, 884. [Google Scholar] [CrossRef] [PubMed]
  78. Benmimoun, B.; Polesello, C.; Waltzer, L.; Haenlin, M. Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila. Development 2012, 139, 1713–1717. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Grigorian, M.; Mandal, L.; Hartenstein, V. Hematopoiesis at the onset of metamorphosis: Terminal differentiation and dissociation of the Drosophila lymph gland. Dev. Genes Evol. 2011, 221, 121–131. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Felix, T.M.; Hughes, K.A.; Stone, E.A.; Drnevich, J.M.; Leips, J. Age-specific variation in immune response in Drosophila melanogaster has a genetic basis. Genetics 2012, 191, 989–1002. [Google Scholar] [CrossRef] [Green Version]
  81. McCormack, S.; Yadav, S.; Shokal, U.; Kenney, E.; Cooper, D.; Eleftherianos, I. The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila. Immun. Ageing 2016, 13, 15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Birnbaum, A.; Wu, X.; Tatar, M.; Liu, N.; Bai, H. Age-Dependent Changes in Transcription Factor FOXO Targeting in Female Drosophila. Front. Genet. 2019, 10, 312. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Troha, K.; Buchon, N. Methods for the study of innate immunity in Drosophila melanogaster. Wiley Interdiscip Rev. Dev. Biol. 2019, 8, e344. [Google Scholar] [CrossRef]
  84. Belmonte, R.L.; Corbally, M.K.; Duneau, D.F.; Regan, J.C. Sexual Dimorphisms in Innate Immunity and Responses to Infection in Drosophila melanogaster. Front. Immunol. 2019, 10, 3075. [Google Scholar] [CrossRef] [Green Version]
  85. Kubiak, M.; Tinsley, M.C. Sex-Specific Routes To Immune Senescence In Drosophila melanogaster. Sci. Rep. 2017, 7, 10417. [Google Scholar] [CrossRef] [Green Version]
  86. Le Bourg, E.; Massou, I.; Gobert, V. Cold stress increases resistance to fungal infection throughout life in Drosophila melanogaster. Biogerontology 2009, 10, 613–625. [Google Scholar] [CrossRef] [PubMed]
  87. Le Bourg, E. A cold stress applied at various ages can increase resistance to heat and fungal infection in aged Drosophila melanogaster flies. Biogerontology 2011, 12, 185–193. [Google Scholar] [CrossRef]
  88. Le Bourg, E. The NF-kB like factor DIF has weaker effects on Drosophila melanogaster immune defenses than previously thought. J. Comp. Physiol. B 2011, 181, 741–750. [Google Scholar] [CrossRef]
  89. Zheng, W.; Rus, F.; Hernandez, A.; Kang, P.; Goldman, W.; Silverman, N.; Tatar, M. Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells. BMC Biol. 2018, 16, 60. [Google Scholar] [CrossRef] [Green Version]
  90. Horn, L.; Leips, J.; Starz-Gaiano, M. Phagocytic ability declines with age in adult Drosophila hemocytes. Aging Cell 2014, 13, 719–728. [Google Scholar] [CrossRef]
  91. Huang, W.; Massouras, A.; Inoue, Y.; Peiffer, J.; Ramia, M.; Tarone, A.M.; Turlapati, L.; Zichner, T.; Zhu, D.; Lyman, R.F.; et al. Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome Res. 2014, 24, 1193–1208. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Mackay, T.F.; Richards, S.; Stone, E.A.; Barbadilla, A.; Ayroles, J.F.; Zhu, D.; Casillas, S.; Han, Y.; Magwire, M.M.; Cridland, J.M.; et al. The Drosophila melanogaster Genetic Reference Panel. Nature 2012, 482, 173–178. [Google Scholar] [CrossRef] [Green Version]
  93. Zambon, R.A.; Nandakumar, M.; Vakharia, V.N.; Wu, L.P. The Toll pathway is important for an antiviral response in Drosophila. Proc. Natl. Acad. Sci. USA 2005, 102, 7257–7262. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Ferreira, A.G.; Naylor, H.; Esteves, S.S.; Pais, I.S.; Martins, N.E.; Teixeira, L. The Toll-Dorsal Pathway Is Required for Resistance to Viral Oral Infection in Drosophila. PLoS Pathog. 2014, 10, e1004507. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  95. Avadhanula, V.; Weasner, B.P.; Hardy, G.G.; Kumar, J.P.; Hardy, R.W. A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response. PLoS Pathog. 2009, 5, e1000582. [Google Scholar] [CrossRef] [Green Version]
  96. Costa, A.; Jan, E.; Sarnow, P.; Schneider, D. The Imd pathway is involved in antiviral immune responses in Drosophila. PLoS ONE 2009, 4, e7436. [Google Scholar] [CrossRef] [Green Version]
  97. Promislow, D.E.L. A Geroscience Perspective on COVID-19 Mortality. J. Gerontol. A Biol. Sci. Med. Sci. 2020, 75, e30–e33. [Google Scholar] [CrossRef] [Green Version]
  98. Davies, N.G.; Klepac, P.; Liu, Y.; Prem, K.; Jit, M.; CMMID COVID-19 Working Group; Eggo, R.M. Age-dependent effects in the transmission and control of COVID-19 epidemics. Nat. Med. 2020, 26, 1205–1211. [Google Scholar] [CrossRef] [PubMed]
  99. Aprahamian, I.; Cesari, M. Geriatric Syndromes and SARS-Cov-2: More than Just Being Old. J. Frailty Aging 2020, 9, 127–129. [Google Scholar] [CrossRef]
  100. Mueller, A.L.; McNamara, M.S.; Sinclair, D.A. Why does COVID-19 disproportionately affect older people? Aging 2020, 12, 9959–9981. [Google Scholar] [CrossRef]
  101. Schneider, J.; Imler, J.L. Sensing and signalling viral infection in drosophila. Dev. Comp. Immunol. 2021, 117, 103985. [Google Scholar] [CrossRef]
  102. Mussabekova, A.; Daeffler, L.; Imler, J.L. Innate and intrinsic antiviral immunity in Drosophila. Cell Mol. Life Sci. 2017, 74, 2039–2054. [Google Scholar] [CrossRef] [PubMed]
  103. Lamiable, O.; Imler, J.L. Induced antiviral innate immunity in Drosophila. Curr. Opin. Microbiol. 2014, 20, 62–68. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  104. Mackenzie, D.K.; Bussiere, L.F.; Tinsley, M.C. Senescence of the cellular immune response in Drosophila melanogaster. Exp. Gerontol. 2011, 46, 853–859. [Google Scholar] [CrossRef]
  105. McCarville, J.L.; Ayres, J.S. Disease tolerance: Concept and mechanisms. Curr. Opin. Immunol. 2018, 50, 88–93. [Google Scholar] [CrossRef]
  106. Sheffield, L.; Sciambra, N.; Evans, A.; Hagedorn, E.; Goltz, C.; Delfeld, M.; Kuhns, H.; Fierst, J.L.; Chtarbanova, S. Age-dependent impairment of disease tolerance is associated with a robust transcriptional response following RNA virus infection in Drosophila. bioRxiv 2021. [Google Scholar] [CrossRef]
  107. Mahallawi, W.H.; Alsamiri, A.D.; Dabbour, A.F.; Alsaeedi, H.; Al-Zalabani, A.H. Association of Viral Load in SARS-CoV-2 Patients With Age and Gender. Front. Med. 2021, 8, 608215. [Google Scholar] [CrossRef]
Microorganisms 09 00958 g001 550
Figure 1. Pathways in Drosophila involved in aging innate immunity. The Toll pathway is traditionally activated by Gram-positive bacteria and fungi, and it is also implicated in the response to viral infection, heat shock, and cold shock stresses. Toll activation, via its ligand Spätzle (Spz), leads the degradation of the Cactus inhibitor and subsequent nuclear localization of the NF-κB transcription factors Dif and Dorsal. This leads to the transcription of antimicrobial genes such as Drosomycin and Metchnikowin. The Immune deficiency (IMD) pathway is primarily activated by Gram-negative bacteria, and is also implicated in the response to viral infection and desiccation stress. Activation of the IMD pathway leads to the nuclear localization of the NF-κB transcription factor Relish and subsequent transcription of genes, including Diptericin, Drosocin, Cecropin, Attacin, and Pirk. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is activated via Unpaired (Upd) cytokine detection in response to viral and bacterial infection, aging, and stress. This leads to the nuclear localization of the Drosophila Stat92E factor and subsequent activation of transcription. The Drosophila Insulin/Insulin Growth Factor/Target of Rapamycin (IIS/TOR) signaling network regulates growth and nutrition via binding of insulin-like peptides (dILPs) to the insulin receptor (dInR). There is a singular insulin substrate, Chico, that induces two downstream signaling cascades, one via the Akt1 kinase and the other via the extracellular signal-regulated kinase (ERK). The IIS/TOR network is impacted by viral (dFOXO specifically), Gram-positive, and Gram-negative bacterial infections, as well as starvation stress. Activation of dInR subsequently affects the activity of the Target of Rapamycin (dTOR) factor, as well as the dFOXO transcription factor.
Figure 1. Pathways in Drosophila involved in aging innate immunity. The Toll pathway is traditionally activated by Gram-positive bacteria and fungi, and it is also implicated in the response to viral infection, heat shock, and cold shock stresses. Toll activation, via its ligand Spätzle (Spz), leads the degradation of the Cactus inhibitor and subsequent nuclear localization of the NF-κB transcription factors Dif and Dorsal. This leads to the transcription of antimicrobial genes such as Drosomycin and Metchnikowin. The Immune deficiency (IMD) pathway is primarily activated by Gram-negative bacteria, and is also implicated in the response to viral infection and desiccation stress. Activation of the IMD pathway leads to the nuclear localization of the NF-κB transcription factor Relish and subsequent transcription of genes, including Diptericin, Drosocin, Cecropin, Attacin, and Pirk. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is activated via Unpaired (Upd) cytokine detection in response to viral and bacterial infection, aging, and stress. This leads to the nuclear localization of the Drosophila Stat92E factor and subsequent activation of transcription. The Drosophila Insulin/Insulin Growth Factor/Target of Rapamycin (IIS/TOR) signaling network regulates growth and nutrition via binding of insulin-like peptides (dILPs) to the insulin receptor (dInR). There is a singular insulin substrate, Chico, that induces two downstream signaling cascades, one via the Akt1 kinase and the other via the extracellular signal-regulated kinase (ERK). The IIS/TOR network is impacted by viral (dFOXO specifically), Gram-positive, and Gram-negative bacterial infections, as well as starvation stress. Activation of dInR subsequently affects the activity of the Target of Rapamycin (dTOR) factor, as well as the dFOXO transcription factor.
Microorganisms 09 00958 g001
Table
Table 1. Studies implicating the Toll and IMD pathways in age-dependent immunity.
Table 1. Studies implicating the Toll and IMD pathways in age-dependent immunity.
ReferencePathogen(s) UsedAssay(s) UsedAge/Sex of Flies Used in the StudyAltered Genes (Pathways)
Sinam et al., 2016
[49]
  • Bacilus subtilis (Gram+)
  • Beauveria bassiana (Fungus)
  • Enterobacter cloacae (Gram-)
  • Metarhizium anisopliae (Fungus)
  • Serratia marcescens (Gram-)
  • Staphylococcus aureus (Gram+)
  • Lifespan assay in the absence of infection
  • Heat stress survival assay (37 °C)
  • Oxidative stress resistance assay
  • Infection survival assay (~1012 colony forming units (CFU)/mL concentration for bacteria used in survival assays)
  • Bacterial survival in hemolymph assay (hemolymph extracted 6 h post-infection. ~100 CFU of S. marcescens and S. aureus incubated with hemolymph for 2 h at 25 °C. 100 µL then plated for bacteria growth at room temperature).
  • Male and female flies raised at 25 °C separately used for lifespan assay
  • Males and females of unspecified age separately used in heat stress assay, oxidative stress resistance assay, infection survival assay, and microbial load assay
  • Suggested Toll pathway (Cecropin expression measured via RT-qPCR)
Zerofsky et al., 2005
[18]
  • Escherichia coli (Gram-) (Concentration not specified)
  • Micrococcus luteus (Gram+)
  • (Concentration not specified)
  • Infection pricking assay (mixture of E. coli and M. luteus)
  • Immune response assay after injection (measuring Diptericin AMP expression)
  • Fecundity assay after injection (counting daily egg production)
  • Flies raised at 25 °C
  • Females aged 1, 2, 3, and 4 weeks old used in the infection assay and the immune response assay
  • Virgin females mated to males for three days used in the fecundity assay (injected at 4 d of age and newly laid eggs counted daily)
  • IMD pathway
  • (Diptericin expression after infection as a function of age; fecundity after infection in wild-type flies, hypomorph imd1-1 mutants, and null RelE38 mutants)
Fabian et al., 2018
[46]
  • Beauveria bassiana (Fungus) (Concentration not specified)
  • Drosophila C Virus (+ssRNA virus) (2 × 107 particles/mL = TCID50)
  • Enterococcus faecalis (Gram+) (ODλ = 600 nm = 8)
  • Erwinia carotovora carotovora (Gram-) (Ecc15) (ODλ = 600 nm = 200)
  • Bacterial clearance assay after infection
  • Lifespan assay following infection
  • Flies raised at 25 °C
  • Females aged to 5–6 days old and 23–25 days old used in the bacterial clearance assay (only 5–6-day-old females used for Drosophila C Virus infection)
  • Males and females raised together from eclosion to death used in the lifespan assay
  • Toll pathway (ubiquitous RNAi knockdown of Spätzle, Toll, cactus, and Dif)

  • JAK/STAT pathway (Upd3 identified as a candidate gene for longevity)
Libert et al., 2006
[48]
  • Enterobacter cloacae (Gram-) (ODλ = 600 nm = 1–70)
  • Enterococcus faecalis (Gram+) (ODλ = 600 nm = 70)
  • Pseudomonas aeruginosa (Gram-) (ODλ = 600 nm = 0.004)
  • Staphylococcus aureus (Gram+) (ODλ = 600 nm = 0.5)
  • Infection survival assay
  • Climbing assay in the absence of infection (measured using vertical vial and mechanical stimulation after 20 s)
  • Fecundity assay (eggs counted every 24 h for 7 days after mating)
  • Heat-shock tolerance assay (37 °C)
  • Lifespan assay in the absence of infection
  • Fly rearing temperature not specified
  • 10-day-old separated males and females used in the infection survival assay
  • 10-day-old flies of unspecified sex were used in the climbing assay and the heat-shock tolerance assay
  • Female flies of unspecified age used in the fecundity assay
  • Separated males and females used in the lifespan assay
  • IMD pathway (RelE20 and RelE38 null mutants, fat body-specific knockdown (FADD, TAK1 dominant-negative), and overexpression (PGRP-LE) using S106-GS-Gal4)
Libert et al., 2008
[45]
  • Pseudomonas aeruginosa (Gram-) (ODλ = 600 nm = 0.004)
  • Enterococcus faecalis (Gram+) (ODλ = 600 nm = 70)
  • Infection survival assay
  • Lifespan assay in the absence of infection
  • Immune response assay after injection (Diptericin, Drosomycin, and Attacin expression using RT-PCR)
  • Flies raised at 25 °C
  • Male and female flies of unspecified age used in the infection survival assay, lifespan assay, and immune response assay
  • NF-kB pathway (Diptericin, Drosomycin, and Attacin expression via RT-PCR)
  • Insulin signaling pathway (infection survival assay and lifespan assay using chico null mutants)
Table
Table 2. Studies implicating the JAK/STAT pathway in age-dependent immunity.
Table 2. Studies implicating the JAK/STAT pathway in age-dependent immunity.
ReferencePathogen(s) UsedAssay(s) UsedAge/Sex of Flies Used in the StudyAltered Genes (Pathways)Other Factors Investigated
Salazar et al., 2018
[54]
  • Serratia marcescens (Gram-) (ODλ = 600 nm = 1)
  • Smurf and smurf reversal assays (flies kept on blue food for 24 h before being scored for blue dye leakage)
  • Bacterial load assay via genomic DNA isolation
  • Starvation resistance assay (25 ug/mL RU486 in water only medium)
  • Triacylglyceride assay (lipids extracted from whole flies)
  • Lifespan assays with and without RU486-mediated transgene induction
  • Flies raised at 25 °C
  • 10- and 17-day-old female flies used in the bacterial load assay
  • 3-day-old females used in the starvation resistance assay
  • 9-, 13-, and 30-day-old female flies used in fluorescence microscopy and ratios for Ssk, Dlg, Mesh, and Coracle localization
  • Snakeskin junction protein (knockdown of Ssk using RNAi)
  • JAK/STAT (elevated upd3 expression following depletion of Ssk)
Intestinal junction proteins (Ssk, Dlg, Mesh, and Coracle localization) in Smurf and non-Smurf flies
Fabian et al., 2018
[46]
  • Beauveria bassiana (Fungus) (Concentration not specified)
  • Drosophila C Virus (+ssRNA virus) (2 × 107 particles/mL = TCID50)
  • Enterococcus faecalis (Gram+) (ODλ = 600 nm = 8)
  • Erwinia carotovora carotovora (Ecc15) (Gram-) (ODλ = 600 nm = 200)
  • Bacterial clearance assay after infection
  • Lifespan assay following infection
  • Flies raised at 25 °C
  • Females aged 5–6 and 23–25 days old used in the bacterial clearance assay
  • 1–4-day-old females used for DCV infection
  • Males and females raised together from eclosion to death used in the lifespan assay
  • JAK/STAT pathway (upd3 identified as a candidate gene for longevity)
  • Toll pathway (ubiquitous knockdown of Spätzle, Toll, cactus, and Dif using RNAi)
Table
Table 3. Studies implicating the IIS/TOR network and dFOXO Transcription factor in age-dependent immunity.
Table 3. Studies implicating the IIS/TOR network and dFOXO Transcription factor in age-dependent immunity.
ReferencePathogen(s) UsedAssay(s) UsedAge/Sex of Flies Used in the StudyAltered Genes (Pathways)Other Factors Investigated
Felix et al., 2012
[80]
  • Escherichia coli (Gram-) (ODλ = 600 nm = 1, ~5.5 × 108 CFUs/mL)
  • Bacterial clearance assay after infection
  • Genome Wide Association Study (GWAS) utilizing DGRP lines
  • Flies raised at 25 °C
  • 1- and 4-week-old flies of unspecified sex were used in the bacterial clearance assay
  • Insulin signaling/TOR pathway (alterations in Akt and mos are associated with variation in bacterial clearance ability at older age)
  • IMD pathway (Relish, AttacinA, AttacinB, AttacinD, CecropinB and C, DiptericinB, PGRP-SD, PGRP-LF, PGRP-SB1 found to be upregulated with age)
  • Metabolism (several candidate genes involved in metabolic functions associated with bacterial clearance ability at older age)
McCormack et al., 2016
[81]
  • Escherichia coli (Gram-) (non-pathogenic K12 strain) (100–300 CFU)
  • Photorhabdus luminescens (Gram-) (Strain TT01) (100–300 CFU)
  • Infection survival assay
  • Bacterial load assay after infection (measured at 3, 16, and 30 h post-infection. CFU estimated from the standard curves generated from E. coli and P. luminescens)
  • Phenoloxidase activity assay after infection (measured at 3 h post-infection. OD measured at 492 nm against a blank control)
  • Phagocytosis estimation assay after infection (measured 1-h post-infection via fluorescent imaging of the fly dorsal surface)
  • Metabolic activity assay after infection (measured at 3- and 18 h post-infection. Absorbance measured at 562 nm and protein concentration of the samples calculated from a standard curve)
  • Flies raised at 25 °C
  • 7–10-day-old males and females raised together were infected in the infection survival assay, bacterial load assay, phenoloxidase activity assay, phagocytosis estimation assay, and the metabolic activity assay
  • Insulin-like growth factor signaling (chicoKG00032 mutants)
  • IMD pathway (measured Diptericin and Cecropin-A1 gene expression via RT-qPCR in chicoKG00032 mutants)
  • Toll pathway (measured Drosomycin gene expression via RT-qPCR in chicoKG00032 mutants)
Sung et al., 2018
[77]
  • Micrococcus luteus (Gram+) (Concentrated culture, CFU not listed)
  • Infection survival assay
  • Lifespan assay in the absence of infection
  • Flies raised at 25 °C
  • Adult males of unspecified age used in the infection survival assay
  • Females used in the lifespan assay
  • Methuselah-like receptor-10 (Mthl10) (ubiquitous knockdown of Mthl10 using RNAi)
  • Insulin-like Peptide (ILP) secretion (ubiquitous knockdown of Growth-Blocking Peptide cytokine with Actin-Gal4>UAS-GBPRNAi line)
  • NF-kB pathway (measurement of Metchnikowin gene expression in Mthl10 RNAi lines)
Spellberg et al., 2015
[32]
  • Cricket Paralysis Virus (CrPV) (+ssRNA) (100 and 300 TCID50)
  • Flock House Virus (FHV) (+ssRNA) (5 * 105 PFU)
  • Infection survival assay
  • Heat Shock assay in the absence of infection (flies placed at 37 °C for 1h and then incubated at 25 °C for 6h)
  • Cell culture (Drosophila S2 cells with inducible constitutively active FOXO plated at 1 × 106 cells per mL)
  • Fly rearing temperature not specified
  • 5–7-day-old flies of unspecified sex were used in the infection survival assay
  • Male flies of unspecified age were used in the heat shock assay
  • dFOXO null mutants (FOXOΔ94) used in the infection survival assay
  • Heat shock-mediated knockdown of dFOXO using RNAi
Ueda et al., 2018
[75]
  • No pathogen used
  • Lifespan assay in the absence of infection
  • Climbing assay (measured via vertical vial and mechanical stimulation after 6 s)
  • RNA-seq (performed in miR-305 over-expressing flies), analysis for possible miR-305 target mRNAs)
  • Immunostaining (targeting poly-ubiquitinated proteins and abnormal protein aggregates)
  • Flies raised at 28 °C
  • Male adult flies (24 h after eclosion-death) were used in the lifespan assay
  • Male adult flies ranging from 0 to 45 days old were used in the climbing assay.
  • Insulin-like growth factor signaling (IIS) pathway (RNAseq analysis showed that tobi, dilp2, and dilp5 mRNA increased upon miR-305 over-expression and that mRNA for dilp6 and dilp8 decreased upon miR-305 over-expression).
  • Toll pathway (reported that miR-305 targets ModSP mRNA, which is part of the Toll signaling cascade)
Table
Table 4. Studies implicating other factors in age-dependent immunity.
Table 4. Studies implicating other factors in age-dependent immunity.
ReferencePathogen(s) UsedAssay(s) UsedAge/Sex of Flies Used in the StudyAltered Genes (Pathways)Other Factors Investigated
Khan et al., 2013
[44]
  • Serratia marcescens (ATCC 13880) (Gram-) (ODλ = 600 nm = 1)
  • Bacterial load assay
  • Flies raised at 25 °C
  • 3-, 10-, and 13-day-old males and females raised separately used in the bacterial load assay
  • Sexual dimorphism (No significant difference in bacterial load by sex)
  • Age-dependent immunity (lower bacterial load in older flies (13 day-old) than younger flies (3 day-old))
Horn et al., 2014
[90]
  • Escherichia coli (Gram-) (heat-killed, 6 × 109 particles/mL diluted 1:6)
  • Whole-fly fluorescence assay after infection
  • Hemocyte fluorescence assay after infection
  • In-vivo quantitative phagocytosis assay (imaged 90 min after infection)
  • Phagocytic ability assay after infection (measured as the amount of fluorescent E. coli per active hemocyte 90 min after infection)
  • Bead engulfment assay (flies infected with fluorescent plastic beads which cannot be broken down) (Alexa Fluor 568-labeled 1-µm beads)
  • Fly rearing temperature not indicated
  • 1- and 5-week-old females used in the whole-fly fluorescence assay, hemocyte fluorescence assay, and bead engulfment assay
  • Adult virgin female Canton-S flies of unspecified age were used in the in-vivo quantitative phagocytosis assay
  • 5–7-day, 35–39-day, 1-week, and 5-week-old female flies used in the phagocytic ability assay
  • Phagocytosis
  • RNAi knockdown (eater and nimrod C1) and overexpression (Rab5) in hemocytes using Hemese (He)-Gal4, UAS-GFP.
Le Bourg et al., 2009
[86]
  • Beauveria bassiana (Fungus) (Concentration not specified)
  • Hypergravity assay pretreatment before infection (flies kept at 3 or 5 g hypergravity for 2 weeks starting at 2 days of age with 20-min pauses twice a week for vial changes)
  • Heat shock assay pretreatment before infection (flies placed at 37 °C 5 min daily for 5 successive days starting at 5 days of age)
  • Cold Shock assay pretreatment before infection (placed at 0 °C 60 min daily for two periods of 5 days starting at 5 days of age)
  • Climbing assay after infection (measured via vertical vial and mechanical stimulation after 20 s)
  • Lifespan assay following infection
  • Flies raised at 25 °C
  • 18-day-old separated males and females infected in the hypergravity assay pre-treatment
  • 12-day-old separated males and females infected in the heat shock assay pre-treatment
  • 19-, 26-, 33-, and 40-day-old separated males and females infected in in the cold shock assay pre-treatment and in the lifespan assay
  • 3–4-week-old separated males and females infected in the climbing assay (scores measured 3 or 7 days after infection)
  • Suggested role for the Toll pathway
  • Environmental stress (hypergravity, heat shock, and cold shock effects on response to infection)
Le Bourg et al., 2011
[87]
  • Beauveria bassiana (Fungus) (Concentration not specified)
  • Infection survival assay with and without cold shock pre-treatment
  • Cold shock survival assay in the absence of infection (flies kept at 0 °C for 60 min per day for two 5-day periods separated by a 2-day period without cold shock)
  • Heat shock survival assay with and without cold shock pre-treatment (flies placed at 37 °C and observed every 5 min)
  • Flies raised at 25 °C
  • 41-day-old separated males and females used in the infection survival assay with cold shock pre-treatment
  • 1-,2-, 3-, 4-, 5-, and 6-week-old separated males and females used in the cold shock survival assay in the absence of infection
  • 40–44-day-old separated males and females were used in the heat shock survival assay
  • Sexual dimorphism (cold stress increased longevity of males but not females. Cold stress increased resistance to infection in males but not in females)
  • Age-dependent response to environmental stress (negative effect of cold stress on longevity of 5–6-week-old flies)
Le Bourg et al., 2011b
[88]
  • Beauveria bassiana (Fungus) (Concentration not specified)
  • Cold shock assay pre-treatment (flies kept at 0 °C for 16 or 18 h overnight)
  • Infection survival assay with and without cold shock pre-treatment
  • Lifespan assay with and without cold shock pre-treatment
  • Heat shock survival assay with and without cold shock pre-treatment (kept at 37 °C and observed every 5 min until dead)
  • Climbing assay with and without cold shock pretreatment (measured via vertical vial and mechanical stimulation after 20 s)
  • Flies raised at 25 °C
  • 12-day-old separated males and females used in the infection assay following cold shock pre-treatment
  • Separated males and females used in the lifespan assay following cold shock pre-treatment (from 13 days of age until death)
  • 13-day-old separated males and females used in the heat shock survival assay
  • 2-, 3-, and 4-week-old separated males and females used in the climbing assay
  • Toll pathway (Dif1 mutants used in the climbing assay, heat shock assay, lifespan assay, and infection survival assay)
  • Environmental Stress
Zheng et al., 2018
[89]
  • Erwinia carotovora carotovora 15 (Gram-) (~6 × 105 CFUs of Ecc15)
  • Infection survival assay by pricking after desiccation
  • Desiccation assay (flies kept in empty vials without food for 2 h)
  • Flies raised at 25 °C
  • 7- and 40-day-old females
  • Toll/IMD/NF-kB pathway (PGRP-LC, PGRP-LE, 20E)
  • Environmental Stress
Kubiak et al., 2017
[85]
  • Beauveria bassiana (Fungus)
  • 1- and 4-week-old males and females
  • Flies raised at 25 °C
  • Barrier Defenses
  • Sexual Dimorphism
  • Injection assay (cuticle inoculation)
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Sciambra, N.; Chtarbanova, S. The Impact of Age on Response to Infection in Drosophila. Microorganisms 2021, 9, 958. https://doi.org/10.3390/microorganisms9050958

AMA Style

Sciambra N, Chtarbanova S. The Impact of Age on Response to Infection in Drosophila. Microorganisms. 2021; 9(5):958. https://doi.org/10.3390/microorganisms9050958

Chicago/Turabian Style

Sciambra, Noah, and Stanislava Chtarbanova. 2021. "The Impact of Age on Response to Infection in Drosophila" Microorganisms 9, no. 5: 958. https://doi.org/10.3390/microorganisms9050958

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop