Introduction

The immune system is made up of a network of tissues and circulating cells whose primary function is to prevent and limit pathogenic infections (Danilova, 2006; Pradeu et al., 2024; Schmid-Hempel, 2021). Although immunity has been studied in many animal species, it has been extensively characterized in only a few, such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, zebrafish, rodents, and humans. In recent years, this traditional understanding of the immune system as a specialized group of cells and tissues has gradually changed to a more complex picture of highly redundant multilevel defense modules. The last few decades have revealed a great deal about the molecular mechanisms regulating each of these immune modules, from recognition and signaling to effector production (e.g. phagocytosis, antimicrobial peptides) (Pradeu et al., 2024). However, an immune response encompasses the concomitant activation of several modules and there has been little investigation of how modules contribute collectively to host defense. Immune research has traditionally focused on modules in isolation due to technical limitations. This article aims to fill this gap by analyzing the specific contributions of the main immune modules to the collective Drosophila systemic response.

The Drosophila systemic immune response consists of a set of humoral and cellular reactions in the hemolymph (insect blood) that occur when flies are systemically infected by bacteria, viruses, or fungi (Liegeois and Ferrandon, 2022; Westlake et al., 2024). A first facet of the systemic immune response is the production of host-defense effectors by the fat body and hemocytes that are secreted into the hemolymph. Among the best-characterized effectors are the antimicrobial peptides (AMPs), whose expression is rapidly induced to incredibly high levels upon infection in a race to control invading microbes (Hanson et al., 2019; Imler and Bulet, 2005). AMPs are small cationic peptides that exhibit antimicrobial activity and directly participate in microbial killing. Beyond AMPs, many other proteins and host defense peptides are secreted into the hemolymph, creating a hostile environment for invading pathogens through a variety of mechanisms. The systemic antimicrobial response is predominantly regulated at the transcriptional level by the Toll and Imd pathways (De Gregorio et al., 2002; Lemaitre et al., 1996). In Drosophila, the Toll pathway is activated by cell wall components (fungal glucans and Lysine-type peptidoglycan), as well as microbial proteases (Gottar et al., 2006; Issa et al., 2018; Leulier et al., 2003; Vaz et al., 2019). Toll signaling culminates in the activation of two NF-κB transcription factors, Dif and Dorsal, which regulate a large set of immune genes (Ip, 1993; Lemaitre et al., 1996). These encode small peptides such as the antifungal peptide Drosomycin, the Toll-regulated Bomanin peptides, and many other proteins (e.g. serine proteases, serpins, lipases) (Clemmons et al., 2015; De Gregorio et al., 2002; Fehlbaum et al., 1994; Ligoxygakis et al., 2003). Flies lacking the Toll pathway are viable but highly susceptible to infection by Gram-positive bacteria and fungi (Buchon et al., 2009; Lemaitre et al., 1996; Rutschmann et al., 2002). The Imd pathway is activated by DAP-type peptidoglycan produced by Gram-negative bacteria and a subset of Gram-positive bacteria (e.g. Bacillus) (Kaneko et al., 2004; Lemaitre et al., 1995; Leulier et al., 2003). The binding of peptidoglycan to receptors of the Peptidoglycan Recognition Protein family (PGRP-SD, PGRP-LC, PGRP-LE) initiates an intracellular signaling cascade, which ultimately activates the NF-κB factor Relish (Choe et al., 2005; Iatsenko et al., 2016; Kaneko et al., 2006; Kaneko et al., 2006; Westlake et al., 2024). The Imd pathway regulates the expression of many genes, including those encoding antibacterial peptides and serine proteases (De Gregorio et al., 2002; Lemaitre et al., 1997). Imd deficient flies are viable but susceptible to Gram-negative bacterial infection.

In addition to humoral pathways, melanization and phagocytosis are two complementary mechanisms that contribute to host survival upon systemic infection. Melanization is an arthropod-specific immune response resulting in rapid deposition of the black pigment melanin at wound or infection sites and the concomitant production of microbicidal reactive oxygen species (Cerenius et al., 2008; Nappi et al., 2009; Westlake et al., 2024). The melanization and Toll pathways are co-activated at the level of the serine proteases Hayan and Persephone, which in turn activate Spatzle-processing enzyme (SPE) and serine proteases critical to cleavage of prophenoloxidases (PPOs) (Dudzic et al., 2019; Nakano et al., 2023; Shan et al., 2023). This is collectively referred to as the Toll-PO SP cascade (Westlake et al., 2024). The melanization reaction itself relies on the oxidation of phenols, resulting in the polymerization of melanin. Two PPOs (PPO1 and PPO2) are the primary catalysts for melanin synthesis during the melanization response to systemic infection. Flies deficient for both PPO1 and PPO2 are viable, but lack hemolymph phenoloxidase (PO) activity and exhibit susceptibility to certain Gram-positive bacteria and fungi (Binggeli et al., 2014; Dudzic et al., 2015).

Phagocytosis by both sessile and circulating plasmatocytes (Drosophila macrophage-equivalent cells) is thought to provide a complementary and important host defense (Melcarne et al., 2019; Ulvila et al., 2011). The use of hemocyte-deficient flies (via targeted expression of a pro-apoptotic gene in plasmatocytes) has shown that hemocytes contribute to survival upon systemic infection to certain bacterial species (Charroux and Royet, 2009; Defaye et al., 2009; Stephenson et al., 2022). Phagocytosis of bacteria greatly relies on two transmembrane receptors, NimC1 and Eater (Bretscher et al., 2015; Kocks et al., 2005; Kurucz et al., 2007; Melcarne et al., 2019). NimC1, Eater double mutant larvae are viable and their hemocytes can effectively encapsulate and melanize macroparasites; however, their hemocytes are non-sessile and nearly totally phagocytosis deficient. While NimC1, Eater larvae have elevated hemocyte numbers, adult flies have few hemocytes and fail to perform phagocytosis, providing a good tool to assess the role of cellular response (Melcarne et al., 2019; Melcarne, 2020).

We have scattered circumstantial evidence that each of these four immune modules are critical for defense against certain pathogens. However, we lack a global view of how, when, and to what extent each of these four immune modules contribute to host defense against specific pathogens, as they have never been simultaneously assessed. Moreover, the extent to which individual findings can be generalized to broad classes of pathogens remains unclear.

In this article, we have used a specific set of mutations principally affecting phagocytosis (NimC1, Eater), melanization (PPO1, PPO2), the Toll pathway (spz), and the Imd pathway (Rel) in a controlled genetic background to address their individual roles in host defense. We then recombined these and other mutations to generate double and even quadruple mutants of the four immune modules. This set of tools allowed us to systematically compare the survival of flies deficient in these responses against a broad panel of viruses, fungi, Gram-positive bacteria, and Gram-negative bacteria, to reveal the relative contributions of each immune mechanism to resistance against these pathogens.

Materials and methods

Insect stocks

Precise details of Drosophila stocks used in this study are provided in Table 1. To minimize the influence of genetic background as a variable in this study, mutations were isogenized into the DrosDel iso w¹¹¹⁸ genetic background (BDSC #5905) as described by Ferreira et al. (Ferreira et al., 2014; Ryder et al., 2004). Isogenized lines were generated and used for every mutant except the double mutant Toll and Imd (Rel^E20, spz^rm7) line, for which homozygous isogenized flies were not viable in our hands. Double mutants for NimC1¹; Eater¹ were poorly viable when isogenized in the DrosDel iso w¹¹¹⁸ background, and so we used both isogenic and non-isogenic lines over the course of our study to facilitate investigation. Furthermore, NimC1¹; spz^rm7, Eater¹ triple mutants were homozygous lethal regardless of isogenization, and so could not be included. In addition to the immune module mutants, the ΔAMP14 line bears a knockout of 14 different AMP genes, which includes the AMP families Drosomycin, Metchnikowin, Cecropin, Defensin, Drosocin, Attacin, and Diptericin, as described previously (Carboni et al., 2022; Hanson et al., 2019). The Bom^1′55C mutation was characterized in Clemmons et al. (Clemmons et al., 2015) and isogenic flies were generated in Hanson et al. (Hanson et al., 2019). For larval experiments, GFP-labelled CyO or TM3 balancers were used to enable selection of homozygous mutant larvae.

Table 1:

Microorganism cultures

Microbe strain information, microorganism classifications, and culture conditions are listed in Table S1. Bacterial cultures and Candida albicans were grown overnight shaking in appropriate media at 200rpm. Aspergillus fumigatus fungus was grown on Malt Agar at room temperature until sporulation. The entomopathogenic fungus Beauveria bassiana strain R444 was provided by Andermatt as spore preparations (BB-PROTEC) which were directly used in natural infections or dissolved in PBS for septic injury. Viruses DCV and FHV were kindly provided by Carla Saleh (Pasteur Institute, Paris), produced in S2 cells. The supernatant of S2 cells was titrated before being used for fly infections. Viruses SINV, DXV and IIV-6 were kindly provided by Ronald van Rij (Radboud University Medical Center, Nijmegen).

Heat-killed microbes were prepared by two repeats of boiling at 95°C for 30 minutes then freezing at −20°C for 30 minutes, before storage long term at −20°C. Microbe preparations were streaked onto agar plates to verify full efficiency of heat-killing before use in experiments.

Gene expression

Flies were inoculated by pricking at the junction of thoracic pleura with a needle dipped in a mixed pellet containing a 1:1 mixture of OD₆₀₀ = 200 heat-killed Escherichia coli and Micrococcus luteus (final OD₆₀₀ = 100 for each), and frozen at −20°C 6h, 12h and 24h post-infection. Total RNA was then extracted from pooled samples of five flies using TRIzol reagent per manufacturer’s protocol and resuspended in MilliQ dH2O. Reverse transcription was performed using PrimeScript RT (Takara) with random hexamer and oligo dT primers. Quantitative PCR was performed on a LightCycler 480 (Roche) in 96-well plates using Applied Biosystems PowerUP SYBR green Master Mix (Applied Biosystems). Data points represent pooled samples from three replicate experiments. Error bars represent one standard deviation from the mean. Statistical analyses were performed using one-way ANOVA with Holms-Sidak multiple test correction. Primers used in this study were:

• Drs-F 5’- CGTGAGAACCTTTTCCAATATGAT-3’

• Drs-R 5’- TCCCAGGACCACCAGCAT-3’

• DptA-F: 5’-GCTGCGCAATCGCTTCTACT-3’

• DptA-R: 5’-TGGTGGAGTGGGCTTCATG-3’

• RPL32-F: 5’-GACGCTTCAAGGGACAGTATCTG-3’

• RPL32-R: 5’-AAACGCGGTTCTGCATGAG-3’

Ex vivo larval hemocyte phagocytosis assays

Ex vivo phagocytosis assays were performed using a mix of equal volumes of E. coli and Staphylococcus aureus AlexaFluorTM488 BioParticlesTM (Invitrogen), following manufacturer’s instructions; and see (Melcarne et al., 2019). Five L3 wandering larvae from our newly generated mutant lines, or carrying the Hml-Gal4, UAS-GFP hemocyte marker as a control, were bled into 150 uL of Schneider’s insect medium (Sigma-Aldrich) containing 1 uM phenylthiourea (PTU; Sigma-Aldrich). The hemocyte suspension was then transferred to 1.5 mL low binding tubes (Eppendorf, SigmaAldrich), and AlexaFluorTM488 bacteria BioParticlesTM were added. The samples were vortexed, incubated at room temperature for 2 hours to allow phagocytosis and then placed on ice to stop the reaction. The fluorescence of extracellular particles was quenched by adding 0.4% trypan blue (Sigma-Aldrich) diluted to 1/3 concentration. Phagocytosis was quantified using a flow cytometer (BD Accuri C6) to measure the fraction of cells phagocytosing, and their fluorescence intensity. Isogenic wild-type iso w¹¹¹⁸larvae and Hml-Gal4, UAS-GFP larvae with or without bacterial particles were used to define the gates for hemocyte counting and the thresholds for phagocytosed particle emission. The phagocytic index was calculated as follows:

Finally, due to experimenter differences in total hemocytes collected and knock-on effects to calculating phagocytic index, phagocytic index was normalized with the wild-type as a reference set to 100% within experiment batch.

Wounding experiment

Clean injury was performed with a needle sterilized with an EtOH and PBS wash. For imaging of the melanization reaction upon pricking, the thorax of 3-8 days old flies was pricked using a sterile needle (diameter ∼0.1mm). Pictures were taken 24h post-pricking and categorized into normal, weak, or no melanization blackening seen at the injury site per Dudzic et al. (Dudzic et al., 2019).

Systemic infections and survival

Systemic infections were performed by pricking 3- to 5-day-old adult males in the thorax with a 0.1mm-thick needle dipped into a concentrated pellet of bacteria, yeast, or fungal spores. For natural infections with A. fumigatus, flies were anesthetized and then shaken on a sporulating plate of fungi for 30 seconds. For B. bassiana, flies were shaken for 30s in a tube containing an excess of commercial spores sufficient to coat all flies uniformly, of which the flies later remove the vast majority through grooming. At least two replicate survival experiments were performed for each infection, though in specific cases some genotypes may have been included in only one or no experiments. 20-35 flies were included per vial when possible, and maintained on cornmeal fly media containing (per 1L): 6.2g agar, 58.8g cornmeal, 58.8g yeast, 60mL grape fruit juice, 4.83mL propionic acid, and 26.5mL 100g/L moldex in pure ethanol. Survivals were scored daily, and flies were flipped to new food vials 3 times per week. Due to challenges working with genotypes of different viability, we could not include all genotypes in all experimental replicates. Moreover, comparisons across genotypes required multiple levels of consideration. Given the complexity of genotype-vs-reference-by-pathogen comparisons in our study, needing to compare infections both to clean injury within genotype, and to wild-type (WT, DrosDel iso w¹¹¹⁸) or ΔITPM (flies lacking all four immune modules) across treatments, we chose to highlight survival differences only when they were: i) consistent across experimental replicates; ii) of a consistent logic across comparable genotypes; for instance, compound mutants containing ΔMel (e.g. ΔIMD, ΔMel) should be as or more susceptible to infections as ΔMel alone if melanization is truly critical for defense; and iii) of a mean lifespan difference ≥1.0 days after accounting for comparisons with unchallenged or clean-injury data. Total experiments (N exp) and total flies per genotype are reported to provide summary statistics per pathogen-by-genotype interaction. The mean lifespan reported in survival summary data has a maximum of 7 or 10 days depending on experimental conditions. Kaplan-Meier survival curves are provided in the supplementary information.

Quantification of microbial load for growth kinetics

3-8 days old flies were infected with the indicated microbe and concentration per OD₆₀₀ as described in Table S1, and allowed to recover. At the indicated time post-infection, flies were anesthetized using CO₂ and surface sterilized by washing them in 70% ethanol. Ethanol was removed, and then flies were homogenized using a Precellys bead beater at 6500 rpm for 30 s in LB broth (Providencia rettgeri), BHI (S. aureus), or YPG (C. albicans) with 500 μL as pools of 5 flies. These homogenates were serially diluted and 100uL was plated on LB, BHI or YPG agar. Plates were incubated overnight, and colony-forming units (CFUs) were counted using a Interscience Scan 500 plate scanning colony counter, and validated independently by manual counts for a subset of plates to ensure accuracy. Statistical differences were tested using Brown-Forsythe and Welch ANOVA tests with Dunnett’s multiple test correction, which consider unequal variance.

Results

A set of immune-deficient lines in the iso Drosdel background

Mutations affecting only one of the four main modules of the Drosophila systemic immune response have been described. These include Relish^E20(Rel^E20, deficient for the Imd pathway, referred to here as ΔIMD), spatzle^rm7 (spz^rm7, deficient for the Toll pathway, referred to here as ΔTOLL), PPO1^Δ, PPO2^Δ (no melanization, referred to here as ΔMel), and NimC1¹; eater¹ (reduced phagocytosis, referred to here as ΔPhag). In addition to blocking phagocytosis, ΔPhag flies have a defect in hemocyte sessility and a decreased number of hemocytes at the adult stage (Bretscher et al., 2015; Melcarne et al., 2019; Melcarne, 2020). We have preferred the use of NimC1¹; eater¹ to study the contribution of hemocytes to defense rather than other approaches with important indirect effects such as the pre-injection of latex beads to saturate phagocytes (Elrod-Erickson et al., 2000), or expression of pro-apoptotic genes in plasmatocytes (Charroux and Royet, 2009; Defaye et al., 2009).

All the mutations in this study were isogenized for at least seven generations into the DrosDel iso w¹¹¹⁸ genetic background (Ferreira et al., 2014; Ryder et al., 2004). We then generated by recombination six of the seven possible combinations of flies simultaneously lacking two immune modules, with the exception of ΔTOLL, ΔPhag as NimC1¹; eater¹, spz^rm7 flies were not viable (see fly strains in Table 1). Of note, in our hands recombinant Rel^E20, spz^rm7 flies were not viable in the DrosDel isogenic background, necessitating use of non-iso flies to assess host defense in ΔIMD, ΔTOLL flies. Over the course of our study, isogenic NimC1¹;eater¹ flies also displayed viability issues, and specific experiments used non-isogenic flies to improve confidence in phenotypes; these experiments are noted in figure captions. We used two approaches to simultaneously delete the Toll and melanization modules: i) PPO1^Δ, PPO2^Δ; spz^rm7 flies (referred to as ΔTOLL, ΔMel^PPS) and ii) flies carrying a small viable deletion Hayan-psh^Def(referred to as ΔTOLL, ΔMel^HP) which removes two clustered serine protease genes, Hayan, and persephone, blocking the TOLL-PO SP cascade that regulates both humoral-Toll and melanization (Dudzic et al., 2019; Westlake et al., 2024). This effectively deletes the Toll and melanization pathways using only one deletion event on the X chromosome. Excitingly, we succeeded in generating a fly line deficient for all four immune modules with the genotype: Hayan-psh^Def; NimC1¹; eater¹, Rel^E20 hereafter named ΔITPM (ΔIMD, ΔTOLL, ΔPhag, ΔMel). These flies were homozygous viable (very poor viability), and fertile, despite major deficiencies in these four important systemic immune mechanisms. These flies allow us to monitor survival kinetics and pathogen growth in the near-total absence of an immune system. Finally, we added two iso fly lines in our analysis that delete key host defense peptides downstream of the Toll and Imd pathways: i) Bom^Δ55C, a short deletion removing ten Bomanins at position 55C, which causes a major susceptibility to infection by Gram-positive bacteria and fungi (Clemmons et al., 2015), and ii) ΔAMP14, a compound fly line of eight mutations removing 14 AMP genes (4 Cecropins, 4 Attacins, 2 Diptericins, Drosocin, Defensin, Drosomycin, and Metchnikowin), which causes a major susceptibility to infection by Gram-negative bacteria (Carboni et al., 2022; Hanson et al., 2019). This set of immune-deficient fly lines in a defined genetic background provides a unique tool kit to systematically address the individual and collective contributions of immune modules to defense against a variety of pathogens.

Imd, Toll, Phagocytosis, and Melanization modules function largely independently

The Imd, Toll, Phagocytosis, and Melanization modules are interconnected (Westlake et al., 2024). For instance, the Toll and Imd pathways both regulate a subset of genes involved in melanization (De Gregorio et al., 2002; Ligoxygakis et al., 2002). Moreover, one module can indirectly affect another. For instance, a lack of melanization could increase the bacterial load after infection, resulting in higher activation of the Toll and Imd pathways (Binggeli et al., 2014). In the first step, we analyzed the activation of each of the four modules in single- and multiple-module deficient flies using classical readouts of these modules. This also allowed monitoring of the possible effects of deleting one module on the activity of another. We analyzed the immune response in single module deficient flies by monitoring i) the expression of the antibacterial peptide gene Diptericin (DptA - a target of the Imd pathway), and ii) the antifungal peptide gene Drosomycin (Drs - a target of the Toll pathway) upon systemic infection with a mixture of heat-killed Escherichia coli and Micrococcus luteus, iii) ex vivo phagocytosis of E. coli and Staphylococcus aureus coated bioparticles incubated with larval hemocytes, and iv) melanization at the injury site of adult flies. This analysis first confirmed that ΔIMD, ΔTOLL, ΔPhag, and ΔMel flies are deficient for Imd, Toll, phagocytosis, and melanization respectively (Figure 1A-D). We note that the expression level of DptA was roughly similar to the wild-type in ΔTOLL and ΔMel flies, however, ΔPhag flies show reduced DptA induction at 6h compared to WT (∼50%) (Figure 1A). This represents an immense induction of DptA after infection, but may suggest either a lag or lesser overall Imd response in ΔPhag flies. DptA expression in ΔPhag was, however, comparable to WT at 12h and 24h. On the other hand, Drs induction at 6h in ΔMel flies was significantly higher than WT (∼1.6x), as found previously using live M. luteus infection (Binggeli et al., 2014) (Figure 1B). The phagocytic assay confirmed that the ΔPhag mutant was significantly impaired in its ability to phagocytose bacterial bioparticles. Furthermore, ΔIMD, and ΔMel mutant larvae had a phagocytic index similar to WT (Figure 1C), while ΔTOLL had a higher phagocytic index, but with high variance. As expected, there was no apparent melanization at the site of the injury in ΔMel flies while blackening was readily observed in the wild-type and in other single-module mutants (Figure 1D). Collectively, this analysis indicates that each of the four immune modules can be selectively blocked in the isogenic DrosDel background without interfering with the competence of the others. We did note in some cases a modest change in module activity in the absence of another (higher Toll activity in ΔMel, lower IMD activity in ΔPhag, and higher phagocytic activity in ΔTOLL), findings that require further exploration to establish consistency.

Figure 1:

We then repeated our analysis using our set of module compound mutants. Unsurprisingly, all compound flies displayed the expected immune deficiencies, validating the lines we constructed. This analysis confirms that ΔITPM flies, although viable, are indeed fully immune deficient for the four host defense modules. We did not detect any striking synergistic or antagonistic effects where deleting two modules had a vastly different impact compared to the additive effect of deleting the modules individually. Notably, a small level of Drs expression is retained in ΔTOLL mutant flies, but was fully abolished in ΔIMD, ΔTOLL flies (Figure 1A vs 1E), confirming observations that the Drs gene receives a small input from the Imd pathway (Leulier et al., 2000). The ΔTOLL, ΔMel^PPS and, ΔTOLL, ΔMel^HP flies display Drs induction at 6h that is resolved to unchallenged WT levels by 12h, an expression pattern consistent with mediation of this induction by early input from the IMD pathway in these backgrounds. Taken together, all the compound mutant flies behave largely as expected given the sum effects of their individual mutations, confirming that these modules largely function independently.

Both the Melanization and TOLL modules contribute to lifespan and survival to wounding

Before assessing resistance to infection, we monitored fly survival at 25°C and 29°C to determine if module mutations impact viability in the absence of challenge. At 25°C, no single module mutants died significantly more than wild-type in the absence of challenge within the ten-day window (Figure 2A). However, even in the absence of challenge all compound mutants other than ΔIMD, ΔTOLL suffered at least 25% cumulative mortality by 10 days post-eclosion, while ΔTOLL, ΔMel^PPS and ΔITPM flies suffered even greater cumulative mortality. Similar trends were observed at 29°C with slightly elevated mortality (Figure 2supp1). The observation that ΔTOLL, ΔMel^PPS (PPO1¹,PPO2¹, spz^rm7) flies have shorter lifespan compared to ΔTOLL, ΔMel^HP (Hayan-psh^Def) could be explained by residual Toll and/or PO activity in Hayan-psh^Def or additional indirect effects in ΔTOLL, ΔMel^PPS flies. As such, the combined effect of Toll and Melanization deficiencies in unchallenged and clean injury survival is best assessed by considering signals from both genotypes. ΔITPM flies (Hayan-psh^Def; NimC1¹; Eater¹, Rel^E20) have the lowest unchallenged survival rate of genotypes included in this study. Taken together, the Imd, Toll, Melanization, and Phagocytosis modules contribute little to lifespan maintenance individually, but are crucial to maintain lifespan collectively (Figure 2B).

Figure 2:

Systemic infections were performed by septic injury (pricking with a contaminated needle). We therefore monitored the survival of single and double mutant flies after a clean injury to disentangle the effects of injury from those of infection. We found a cumulative mortality of ∼50% for ΔMel flies by day 10 (Figure 2C), while other single module mutants displayed relatively little mortality over the 10-day window; ΔIMD had notable late-onset mortality in a minority of experimental replicates, which may be due to stochastic dysbiosis effects during aging (Hanson and Lemaitre, 2023; Marra et al., 2021). Double mutant ΔIMD, ΔTOLL and ΔIMD, ΔPhag flies retained a high survival rate. Consistent with an importance of melanization in the injury response (Binggeli et al., 2014), all the compound mutants affected in melanization were susceptible to clean injury, with similar mortality seen in ΔPhag, ΔMel, ΔIMD, ΔMel, ΔTOLL, ΔMel^PPS, ΔTOLL, ΔMel^HP, and ΔITPM flies. Thus, while these genotypes differ in their base lifespan, their viability upon injury is remarkably similar. These results emphasize the interconnectedness of the melanization response with every other immune module for maintaining health after injury.

Similar results were obtained for both unchallenged and clean injury lifespans at 29°C. Of note, ΔTOLL, ΔMel^PPS suffered significantly greater mortality in unchallenged conditions than other genotypes, even including ΔITPM flies (Figure 2supp1). Other compound mutant genotypes displayed a continuum of mortality in unchallenged conditions between ΔITPM (∼50% cumulative mortality) and ΔTOLL, ΔMel^HP (∼15% cumulative mortality). Upon clean injury, results were broadly consistent with 25°C trends, with a more uniform mortality across all genotypes.

Collectively, our study highlights that all these immune modules contribute somewhat to the survival of flies upon clean injury, with a major role of the melanization response in maintaining lifespan and survival to injury. In the next steps of our study, we compared survival upon infection to both unchallenged and clean injured flies to distinguish the impact of microbial infection from the injury itself.

Multiple mechanisms contribute to resistance to microbial infection

To compare the contribution of each module to host defense, we performed infection experiments of wild-type and single- and multiple-module flies following septic infection with five viruses, eight Gram-positive bacteria, eight Gram-negative bacteria, septic injury for two fungal species, and natural infection (i.e. spore deposition on the cuticle) for two fungal species. Temperature, dose, and days monitored are shown in Figure 3, and see Table S1 for other key parameters. Survival curves are shown in Supplementary file 1 for each pathogen and results of these survivals are summarized in Figure 3.

Figure 3:

We performed infection experiments with 24 viruses and microbial species: DCV, FHV, DXV, IIV-6, SINV, Aspergillus fumigatus, Beauveria bassiana, Candida albicans, Mycobacterium marinum, Corynebacterium diphtheriae, Micrococcus luteus, Streptococcus pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, Vibrio parahemolyticus, Providencia rettgeri, Providencia burhodogranariea, Pectobacterium carotovorum Ecc15, Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, and Salmonella enterica ser. typhimurium. Collectively, this represents infections with 24 pathogens across 14 genotypes and two infection routes (∼350 genotype-by-pathogen interactions). When combined with comparisons to clean injury and unchallenged controls to validate if a lifespan difference is meaningful, and to wild-type or ΔITPM treatments, this inflates to 1000+ interactions. We therefore focused our survival analysis on major effects. We report the core summary statistics and overt trends, and comment only where difference in mean lifespan between treatments and reference genotypes or controls was a minimum of 1 day.

Our study confirms that the Imd pathway plays a major role against all tested Gram-negative bacteria, as ΔIMD flies succumb more rapidly than wild-type flies to all of them. This pathway also contributes, albeit to a lesser extent, to survival against the Gram-positive bacteria B. subtilis and L. monocytogenes, which bear DAP-type peptidoglycan at their membrane that triggers Imd activation (Kaneko et al., 2004; Leulier et al., 2003). We were interested to see the impact of our ΔIMD flies in host defense to viruses as the specific mutation we used (Rel^E20) deletes a transcription factor common to both the canonical Imd pathway and the recently-appreciated cGLR-Sting-Relish pathway with roles in viral defense (Goto et al., 2018). We found that ΔIMD mutant flies were somewhat susceptible to the four viruses DCV, FHV, DXV, and IIV6, but not SINV, agreeing with previous studies showing a reduction in lifespan of 1-2 days for Rel mutant flies after viral infection (Costa et al., 2009; Goto et al., 2018; Sansone et al., 2015).

This study also confirms a prominent role of the Toll pathway in survival upon infection by all virulent Gram-positive bacteria and fungi upon septic injury. Less virulent microbes and infectious routes (such as A. fumigatus by natural infection) did not result in increased mortality in individual ΔTOLL mutant flies. A contribution of the Toll pathway to survival was also observed for some Gram-negative bacteria, including a minor contribution to survival upon P. rettgeri infection, and a major contribution after V. parahemolyticus infection. In addition, we found a susceptibility of ΔTOLL flies to FHV, DXV, and IIV6, suggesting Toll-regulated effectors mediate defense against some viral infections.

Use of ΔPhag mutants reveals a role of the cellular response in survival against the fungus B. bassiana. ΔPhag flies further show a minor susceptibility to S. aureus, and a subset of Gram-negative bacteria (P. rettgeri, Ecc15, and E. coli). Susceptibilities to Gram-negative bacteria could result from the observed lag in Imd pathway activation (Figure 1A); indeed, previous studies have shown a full DptA response is pivotal for defense against P. rettgeri (Hanson et al., 2023, 2019; Unckless et al., 2016), and lifespans are greatly impacted by subtle difference for pathogens with intermediate levels of virulence (Duneau et al., 2017). A role for Imd in explaining ΔPhag susceptibilities does not detract from the importance of the cellular response, but instead offers a route to explore mechanism for these phenotypes.

Surprisingly, our study reveals a relatively consistent role of melanization against all the tested viruses, notably so for DXV, IIV-6, and SINV. ΔMel flies also display a minor increased susceptibility to B. bassiana natural infection and septic injury. As previously described (Dudzic et al., 2019), ΔMel flies are highly susceptible to the Gram-positive bacterium S. aureus, rivaling the susceptibility of ΔITPM flies. ΔMel flies further show a strong susceptibility to P. rettgeri rivalling ΔIMD flies, and also a minor susceptibility to infection by other Gram-negative bacteria (P. burhodogranariea, E. coli).

Collectively, the use of single module mutants confirms the major role of the Imd pathway against Gram-negative bacteria and Toll against virulent Gram-positive bacteria and fungi. Interestingly, melanization was consistently important to survive viral infection, and plays a prominent and important role in defense against specific bacterial species. While it was almost never the most critical module, phagocytosis and the cellular response contributes to survival against various germs. Use of single module deficient flies alongside ΔITPM flies reveals that survival to some of the microbes tested relies almost entirely on one pathway (Imd – Ecc15, K. pneumoniae, E. cloacae, P. burhodogranariea; Toll – E. faecalis), or can receive notable contributions from two pathways (Toll and IMD – L. monocytogenes, B. subtilis, V. parahemolyticus; Toll and Phagocytosis – A. fumigatus and B. bassiana natural infection; Imd and Melanization - DCV). Strikingly, many pathways contribute to host defense to S. aureus and P. rettgeri.

Contribution of AMP and Bomanins to Toll and Imd activities

Recent studies have described a prime importance of antimicrobial peptides (AMPs) and host defense peptides (HDPs) in responding to infection (Clemmons et al., 2015; Hanson, 2024; Hanson et al., 2019). These peptides are the principal effectors of the Toll and Imd pathways, yet mutations for AMPs and HDPs typically have not been tested alongside mutations for both the Imd and the Toll pathways across pathogens, as studies have typically included just the single relevant pathway mutation as a positive control. Thus, the importance of AMPs and HDPs relative to the pathways that regulate them has been only partially investigated. We used ΔAMP14 and ΔBom flies to assess the extent to which AMPs and Bomanins mediate Toll and Imd contributions to defense (Figure 3). Here we found that AMPs are critical for survival to infection against all Gram-negative bacteria tested, often rivaling the susceptibility of ΔIMD flies. Notably, ΔAMP14 flies were also somewhat susceptible to the yeast C. albicans, and the Gram-positive bacteria B. subtilis and M. luteus, reinforcing minor susceptibilities seen previously (Carboni et al., 2022; Hanson et al., 2019). Interestingly, we found that ΔAMP14 survivals to DCV, FHV, DXV, and IIV6, largely paralleled those of ΔIMD flies (Figure 3, Supplemental File 1), suggesting Imd-mediated survival phenotypes rely greatly on the presence of the seven classical AMP families.

On the other hand, ΔBom flies were susceptible to septic injury by B. bassiana and also somewhat to C. albicans, consistent with previous studies (Supplementary file 1), as well as all virulent Gram-positive bacteria, consistent with their important role downstream of the Toll pathway (Clemmons et al., 2015; Lindsay et al., 2018; Xu et al., 2023). In addition, ΔBom flies paralleled the ΔTOLL susceptibility to DCV, FHV, and DXV, but were not susceptible to IIV6 like ΔTOLL.

Collectively, our study confirms the key role of antimicrobial peptides downstream of the Imd pathway in the defense against Gram-negative bacteria and a primary role of Bomanins downstream of Toll in defense against fungi and Gram-positive bacteria, with little input to survival against Gram-negative bacteria. We additionally find these secreted peptides can contribute to survival after certain viral infections.

Immune modules mostly contribute additively to host defense

The results described above reveal the key contribution of single modules and immune effectors to host defense. However, they did not assess possible additive, synergistic, or antagonistic contribution of these four modules. Moreover, they did not monitor to what extent these modules are important in the absence of other modules. To address this, we compared the survival of single-module deficient flies to flies lacking two modules, and ΔITPM flies lacking all four modules (Figure 3).

We first observed that ΔITPM flies, which lack the four modules, were always as or more susceptible than single- and double-module deficient flies. In multiple microbe-specific cases, single or double mutants rivalled the susceptibility of ΔITPM flies. In particular, the susceptibility of ΔIMD-flies to most Gram-negative bacteria rivalled ΔITPM, and for some germs ΔTOLL (L. monocytogenes, E. faecalis, B. bassiana injury) or ΔMel (S. aureus) alone was comparable to ΔITPM. In other cases, however, combined loss of two pathways was necessary to rival ΔITPM susceptibilities. For instance, ΔIMD, ΔTOLL deletion causes complete ΔITPM-like susceptibility to B. subtilis, M. luteus, and E. coli, and near-complete susceptibility to S. typhimurium. ΔIMD, ΔTOLL further causes increased susceptibility to most viruses compared to its single module counterparts. In these cases, there is clear synergy or additivity of the contributions of the two modules, as either pathway alone results in only minor or no susceptibility. We also observed intriguing differences between our two versions of ΔTOLL, ΔMel flies, as ΔTOLL, ΔMel^PPS tended to succumb to infection to a greater extent than ΔTOLL, ΔMel^HP, consistent with the differences observed upon clean injury (Figure 2). The ΔTOLL, ΔMel^PPS displayed a minor increase in susceptibility compared to ΔTOLL, ΔMel^HP to A. fumigatus natural infection, and more prominent susceptibilities to the yeast C. albicans, the Gram-positive bacteria M. marinum, M. luteus and also to Gram-negative bacteria K. pneumoniae, E. coli, P. rettgeri and P. burhodogranariea.

Collectively, use of double-module deficient flies uncovers contributions of immune modules to host defense that were masked by other modules. While ΔIMD and ΔTOLL deficiencies sometimes displayed a synergistic effect (i.e. little mortality of single mutants, but ΔITPM-like mortality of double mutants), for combinations of other pathways the results were more often minor with a cumulative effect on susceptibility, suggesting most modules contribute to defense independently. The use of two variations of ΔTOLL, ΔMel, with ΔTOLL, ΔMel^PPShaving a stronger impact than ΔTOLL, ΔMel^HP, also highlights how the level of pathway disruption can affect infection outcomes, particularly for certain fungal or bacterial pathogens like Providencia species that may cleave host immune proteases (suggested by Duneau et al., 2017; Issa et al., 2018).

Timing of Toll, Imd, Phagocytosis, and Melanization contribution to host defense

Host defense programs can rely on resistance mechanisms that directly target or limit growth of pathogens (Howick and Lazzaro, 2017; Lafont et al., 2021; Medzhitov et al., 2012). When effectors reach a critical concentration threshold that promotes resistance, pathogen growth is inhibited; for instance, the time to pathogen control of P. rettgeri is ∼7 hours in Drosophila, relying heavily on the expression and production of DptA (Duneau et al., 2017; Hanson et al., 2023). Phagocytosis and the melanization response are often described as providing near-immediate immune protection (Haine et al., 2008), with reactions ex vivo progressing within minutes. Meanwhile activation of the Imd and Toll pathways takes hours to reach peak concentrations of host defense peptides (Uttenweiler-Joseph et al., 1998). Thus, we were curious if different mutations would alter time of action to control microbial growth. We therefore measured the microbial growth rate for P. rettgeri, S. aureus, and C. albicans at different time points post-infection in wild-type and single module mutant flies as these pathogens were combatted by several immune modules in our survival data.

Agreeing with contributions to survival, flies singly deficient for IMD, Phagocytosis, or Melanization modules display a higher bacterial load than wild-type when infected with P. rettgeri (Figure 4A), which was apparent at 12 hours post-infection. Our observation that ΔPhag showed impaired IMD signaling at 6h (Figure 1A) may explain the contribution of phagocytosis to P. rettgeri microbial growth control with the same timing as ΔIMD flies. We observed that ΔIMD flies, ΔMel flies, and ΔITPM flies, each of which succumb completely to P. rettgeri infection, also show high and consistent microbe loads at 24h that are indicative of sepsis-induced death. On the other hand, wild-type, ΔTOLL, and ΔPhag flies show a greater stochasticity in bacterial load at 24h that is consistent with a fraction of individuals surviving the infection by controlling the pathogen. This indicates deletion of the Toll pathway and phagocytosis does not fully ablate resistance mechanisms essential for combatting P. rettgeri.

Figure 4:

In the case of S. aureus (Figure 4B), we observed some individuals with higher bacterial loads in ΔPhag, and ΔIMD flies at 2h and 6h, which was not seen in wild-type, ΔTOLL, or ΔMel flies. This suggests that the Imd pathway can suppress early S. aureus growth, and possibly also phagocytosis, although this difference in ΔPhag could ultimately stem from reduced IMD induction. Interestingly, flies lacking melanization displayed the highest susceptibility, but did not depart from wild-type or other modules in pathogen load until 12h post-infection. While the melanization reaction is a rapid response, these results suggest that the killing activity of the melanization response acts with slower kinetics. In ΔITPM flies, we observed earlier S. aureus growth at 2h and 6h as in ΔIMD and ΔPhag flies. However, S. aureus loads at 12-24h were more comparable in ΔITPM flies to ΔMel flies. These trends agree both with survival data kinetics, and an independent action of each of these pathways in their resistance effects against S. aureus. This suggests that ΔITPM flies succumb even more quickly than individual module mutants due to loss of multiple resistance mechanisms with different kinetics of activation that independently contribute to defense.

For C. albicans (Figure 4C), mortality begins 2-3 days post-infection in ΔTOLL mutants, but takes place at later time points for ΔIMD, ΔPhag, and ΔMel flies. Microbial growth kinetics show high C. albicans loads at 24h in ΔTOLL, consistent with a previous study (Hanson et al., 2019) and observations done with Candida glabrata (Quintin et al., 2013). Interestingly, a few ΔIMD flies also showed elevated growth of C. albicans at early time points. This suggests Imd-regulated effectors may help to suppress initial C. albicans growth, while Toll-regulated genes (e.g. Bomanins, Drs) contribute to this pathogen suppression to a greater extent at later time points. On the other hand, ΔPhag and ΔMel flies did not show any increased C. albicans load within the 24h time window, despite onset of mortality at later time points. Finally, ΔITPM flies displayed more consistent and rapid growth of C. albicans than ΔTOLL or ΔIMD alone, particularly visible at 12-24h post-infection, consistent with independent contributions of both Toll and Imd in resistance to this yeast.

Collectively, we observed that microbial growth in each module mutant parallels susceptibility, indicating that they contribute to resistance. Surprisingly we did not observe an early role in pathogen killing for melanization despite the rapid blackening response mediated by melanization in ex vivo assays (Nakhleh et al., 2017). We further recover stochasticity in microbe loads across genotypes, as expected when only a subset of individuals suppress the pathogen and survive the infection, resulting in bimodal outcomes (Duneau et al., 2017). The more rapid and consistent microbial growth kinetics of ΔITPM flies, particularly visible at 18h, further demonstrate that the modules contribute to resistance collectively.

Contributions of individual modules to disease tolerance

A metric increasingly used to delineate roles of resistance and tolerance is the Pathogen Load Upon Death (PLUD) (Duneau et al., 2017; Duneau et al., 2025). PLUD is affected by both the virulence of the pathogen and tolerance of the host to survive to a given pathogen burden before succumbing to infection. We monitored PLUD for the same pathogens used to determine the timing of defense modules: P. rettgeri, S. aureus, and C. albicans. The PLUD of both P. rettgeri and S. aureus was overall similar in wild-type and single-module mutant flies (Figure 5A-B). Notably, there was a greater stochasticity in P. rettgeri infections trending towards lower PLUD in ΔITPM flies, suggesting a stochastic reduction in disease tolerance of ΔITPM flies to this bacterium, although this was not significant compared to wild-type (P > .05).

Figure 5:

For C. albicans, the distribution of PLUD values for ΔPhag flies was markedly different from wild-type, also seen to some extent for ΔMel. Interestingly, flies with these genotypes suppress C. albicans growth well (Figure 4C). Yet here we recovered many individuals from both of these module-deficient lines that died with a far lower PLUD (Figure 5C). Taken together, this suggests that the humoral immune pathways, particularly Toll, contribute to resistance against C. albicans. However, the cellular response and melanization reaction instead regulate tolerance to C. albicans infection. The use of ΔITPM flies further emphasizes that when both resistance (Toll, Imd) and tolerance (Phagocytosis, Melanization) mechanisms are deficient, the loss of resistance plays a primary importance, and can mask observations of reduced tolerance.

Collectively, monitoring of PLUD suggests that some modules, specifically phagocytosis and melanization, contribute to tolerance of certain infections. Importantly, this tolerance effect was only seen in genotypes that are able to resist infection. These findings demonstrate the collective contribution to resistance and/or tolerance of the four immune modules studied here.

Discussion

In this article, we have generated a set of single and compound immune module deficient fly lines in a controlled genetic background. Using various assays, we confirmed the validity of our lines, revealing that each module can be activated independently. We did, however, observe higher Toll activation in ΔMel flies upon systemic infection with dead bacteria. Future studies may reveal that this higher Toll activity in ΔMel flies is due to interaction with a regulatory pathway, or reflects higher persistence of microbial elicitors (e.g. peptidoglycan) that activate the Toll pathway in melanization-deficient flies. We also observed reduced Imd activation in ΔPhag flies at 6 hours post-infection, which could reflect a role for hemocytes in stimulation of the Imd systemic response. In this study, we used NimC1¹; eater¹ double mutants to assess the cellular response. These flies have defects in phagocytosis, but also hemocyte adhesion and sessility (Melcarne et al., 2019). NimC1¹; eater¹ larvae also have increased hemocyte number at the larval stage, but Hml positive cells are rapidly lost in adults (Melcarne, 2020). Despite these limitations, we believe that the mutations we have used here are among the best available to assess the roles of these four modules to host defense. Surprisingly, we were able to produce a fly line lacking all four main defense mechanisms of the systemic immune response, indicating that none of these modules is essential for survival. Theoretically, ΔITPM flies are almost completely immune deficient, but they are still able to clot wounds, activate the JNK and JAK-STAT pathways that mediate the wound healing response, and retain constitutive immune defense molecules that could provide a certain degree of protection. Consistent with several studies (Capilla et al., 2017; Carvalho et al., 2014; Rämet et al., 2002), we show that Toll and melanization contribute synergistically to wound healing in adults. However, the observation that ΔPhag, ΔMel or ΔIMD, ΔMel double mutant flies are also more susceptible to clean injury than ΔMel flies indicates that the Imd pathway and phagocytosis also contribute to wound healing. Thus, although immune deficient fly lines for the four modules are viable, the use of compound module mutants and ΔITPM flies reveals a clear role of the immune system in response to wounding and lifespan maintenance.

Use of single- and double module-deficient flies provides key insights on the mechanisms used by Drosophila to combat infection. We confirm previous studies revealing the role of Imd against Gram-negative bacteria, Toll against Gram positive bacteria and virulent fungi, and an importance of Melanization and Phagocytosis against specific pathogens (Charroux and Royet, 2009; Defaye et al., 2009; Garg and Wu, 2014; Nehme et al., 2011). Surprisingly, the Melanization module was consistently important in survival to viral infection. It seems unlikely that prophenoloxidase activity in the hemolymph can combat intracellular viral agents. We instead speculate this contribution of melanization to viral defense could be due to its role in wound healing, or perhaps autotoxic contributions of melanization reaction intermediates that fail to convert in phenoloxidase-deficient flies. We additionally recovered a role of Imd signaling (i.e. Relish) in antiviral defense, which was expected given recent characterizations of cGLR-Sting-Relish antiviral immunity (Ai et al., 2024; Cai et al., 2022; Goto et al., 2018). However, susceptibilities of Rel^E20 flies were paralleled by ΔAMP14 in all cases, suggesting AMPs largely mediate this susceptibility. While Sting regulates a number of genes likely important for antiviral defense (Goto et al., 2018), the susceptibility we observe here could be a direct action of AMPs on enveloped viruses as described in some studies (Feng et al., 2020; Huang et al., 2013; Yasin et al., 2004), or an indirect effect, such as a need to regulate gut microbes after the damage induced by viral replication (Marra et al., 2021). We additionally used dual modes of infection for the fungus B. bassiana, finding that both Melanization and Toll modules have a role in both infection modes. However, our study reveals that Bomanin effectors explain most of the Toll contribution upon septic injury but not natural infection by B. bassiana. This observation is in line with previous observations showing differential importance of effectors or modules according to infection route (Martins et al., 2013).

Our double module mutant analysis reveals that most modules contribute additively to host defense. This is consistent with largely independent function of these modules. However, we noted some instances of synergy between two pathways, notably Toll and Imd. Synergy between Toll and Imd can be explained by the fact that many immune-inducible regulated genes receive input of both the Imd or Toll pathways; some like Metchnikowin, Drosomycin and Transferrin1 that can be induced through either Toll or Imd (De Gregorio et al., 2002). Finally, we observed rarer cases of synergy between Toll and Melanization, or Imd and Melanization.

Our study confirms that the Toll and Imd humoral modules have a defined roles against broad classes of pathogens, Imd for Gram-negative bacteria and DAP-type containing Gram-positive bacteria, and Toll for fungi and Gram-positive bacteria. Use of AMP and Bomanin mutants revealed that this can be largely explained by the effectors they control. In contrast, the contributions of Phagocytosis and Melanization appear to be critical to more specific and diverse sets of pathogens. We speculate that Phagocytosis and/or Melanization have critical roles in defense against bacteria that resist host defense peptides (Hanson et al., 2019), or can hide from them (Touré et al., 2023). The melanization reaction is a source of ROS that is a potent defense against pathogens such as S. aureus resistant to Toll and Imd defense (Dudzic et al., 2019; Ford and King, 2021; Needham et al., 2004; Ramond et al., 2021). These two modules play important roles in immune-related processes such as encapsulation (Melanization), the uptake of bacteria escaping from the gut, or tissue homeostasis (Phagocytosis) (Braun et al., 1998; C. Melcarne et al., 2019; Nehme et al., 2007) that were not assessed here. Collectively, our study validates, with minor discrepancies, many studies that have assessed the individual contributions of these modules (e.g. Apidianakis et al., 2005; Binggeli et al., 2014; Lamiable et al., 2016; Lemaitre et al., 1996, 1995; Nehme et al., 2011). Our mutant lines can now be used to analyze the contribution of these immune modules in resistance to other pathogens, notably wasps, nematodes, microsporidia and protozoans, or in other contexts such as mating and local infection.

While defense against some pathogens is largely mediated by single immune modules, other pathogens are handled by multiple modules. We hypothesize that pathogens that have intermediate virulence levels (kill only a fraction of wild-type flies), may better reveal the roles of multiple modules. Indeed, stochasticity in survival analyses partly stems from the arms-race occurring between the pathogen and host immunity, as shown for P. rettgeri (Duneau et al., 2017). In this scenario, any small change in immune competence may tip the outcome of the arms-race toward lethality or survival. Here, it is notable that multiple modules contribute to survival in P. rettgeri infection. Previous studies have revealed a major role of the Imd pathway AMP Diptericin against this bacterium (Hanson et al., 2023, 2019; Unckless et al., 2016). However, Duneau et al. (Duneau et al., 2017) showed that survival patterns to P. rettgeri bifurcate into two outcomes based on time taken to fully activate systemic defenses, and showed a role for a Toll-PO SP cascade regulating serine proteases in defense against this microbe (Duneau et al., 2017). We may speculate that Melanization, although not as great a contributor as Diptericin, may be a factor that tips the balance of this precarious arms race towards host lethality. The observation that ΔPhag flies expressed less DptA (Figure 1) can explain their susceptibility to P. rettgeri. Future studies should investigate how multiple modules can contribute to host survival according to additive or Achilles dynamics – the concept that microbes have specific weaknesses available for host effectors to target (Hanson, 2024). It will also be important to consider the distinct roles of resistance, tolerance, and resilience in host defense (Howick and Lazzaro, 2017; Wukitch et al., 2023).

We observed a good correlation between survival analysis and pathogen growth in single module flies for P. rettgeri, S. aureus, and C. albicans. This indicates that these immune modules mostly contribute to resistance mechanisms that suppress pathogen growth. Our study did not reveal key early contributions of phagocytosis or melanization in pathogen growth control, despite quasi-immediate activation of these modules; higher S. aureus growth at 2 hours in ΔPhag flies was paralleled by ΔIMD flies, and we observed reduced Imd activation in ΔPhag flies. Melanization, while critical to surviving S. aureus infection, impacts bacterial growth beginning only at the 12-hour time point. This indicates that the microbicidal activity associated with Melanization in vivo acts more slowly than the blackening reaction observed in bled hemolymph. Interestingly, ΔPhag and also ΔMel flies suppress C. albicans yeast growth, but some individuals ultimately succumb to infection with lower PLUD levels. We confirmed ΔPhag PLUD results using both an isogenic and a second wild-type genetic background, suggesting that this lower PLUD is genuine. Susceptibility to fungal infection independent of fungal proliferation has also been reported using an A. fumigatus septic infection model, and relies on the protection offered by Bomanins from pathogen-secreted toxins (Xu et al., 2023). It is tempting to speculate, based on the modules involved, that the loss of tolerance to C. albicans we observe is related to wound repair or accumulating damage, similar to reports of renal failure in beetles (Khan et al., 2017; Li et al., 2020), or flies with autotoxic trachea degradation upon stress pathway disruption (Rommelaere et al., 2024). Thus, this tolerance effect could rely on pathogen-mediated or autotoxic damage, which may be elucidated in a future study.

Here we have provided a single and double mutant analysis of Drosophila immune module functions. Our study provides several insights showing which modules are most important to survive infection by defined pathogens. However, we also highlight collective contribution of modules to defense, even when one module is of outsized importance. We extend our comprehension of innate immune responses by revealing higher complexity in defense, implicating multiple host immune modules in survival to various germs, including some with more cryptic contributions. As illustrated by our previous characterizations of AMP function (Carboni et al., 2022; Hanson et al., 2019), the melanization response (Dudzic et al., 2015), and stress-induced Turandot proteins (Rommelaere et al., 2024), a combinatorial mutation approach to deciphering immune function can be extended even to the broad level of whole immune modules. Together with these previous studies and others, our study provides an immunity toolkit, including flies simultaneously deficient for four immune modules, which can be used to better characterize the contributions of immune processes to host defense. Our tools can also be of use to study various questions at the cutting edge of aging, memory, neurodegeneration, cancer, and more, where immune genes are repeatedly implicated. We hope that this set of lines will be useful to the community to better characterize the Drosophila host defense.

Acknowledgements

We thank Mélanie Blokesch (EPFL), Jan-Willem Veening (Unil), Vivek Thacker (Heidelberg University), Carla Saleh (Pasteur Institut) and Ronald P van Rij (Radboud University Medical Center) for key reagents. We thank Luis Teixeira for iso Drosdel wild-type, rel and spz flies. We thank Koenig E, Prince Kumar Sah for experimental help and Hannah Westlake for editing. The AMP, Bom and single and quadruple module mutants were deposited at the Vienna Drosophila Research Center. This project was supported by the SNSF grant 310030_215073 awarded to B.L. and Wellcome Trust grant 227559/Z/23/Z awarded to M.H.

Additional information

Author contributions

Conceptualization: FR, MAH, BL

Data curation: FR, MAH

Funding acquisition: BL, MAH

Investigation: FR, YT, MR, FS

Methodology: FR

Project administration: BL

Validation: YT, MAH

Resources: FR, MAH

Supervision: MAH, BL

Writing – original draft: FR, BL

Writing – review & editing: MAH, BL