List of mutants used in this study.

“-” indicates the deleted immune module.

Each immune module functions largely independently from other immune modules.

A) Activation of the Imd pathway, as revealed by DptA expression following infection with a mixture of heat-killed E. coli and M. luteus, is broadly wild-type (WT) in single module mutants other than Imd. In the case of ΔPhag flies, we observed a lesser induction at 6h compared to wild-type. B) Activation of the Toll pathway as revealed by the expression of Drs is broadly wild-type in single module mutants other than ΔTOLL flies. Note that Drs receives a minor input from Imd signaling (Leulier et al., 2000), explaining minor induction of Drs in ΔTOLL flies at early time points. In the case of ΔMel, we observed a slightly greater Toll pathway activity. C) The ability of plasmatocytes to phagocytose bacterial particles is not negatively affected in single module mutants except ΔPhag (also see Figure 1supp1) D) Cuticle blackening after clean injury is not impaired except in ΔMel flies. E) Activation of the Imd pathway remains strongly inducible in compound mutants except when deficient for the Imd pathway. F) Activation of the Toll pathway remains strongly inducible in compound mutants except when flies were deficient of the Toll pathway. G) The ability of plasmatocytes to phagocytose bacterial particles is not significantly affected in compound mutants except those including ΔPhag. H) Cuticle blackening after clean injury is not impaired in compound mutants except in those including ΔMel.

Lifespans of mutants used in this study in unchallenged flies and upon clean injury conditions at 25°C.

A-B) Single module mutants; C-D) compound module mutants. See Figure 2supp1 for 29°C comparisons.

Heatmap of lifespans of immune module-deficient flies upon infection by various pathogens..

Darker blue indicates lower survival, while white indicates maximum survival. Experiments used either 7 or 10 days as a maximum lifespan/time course, and the heatmap is adjusted accordingly per row to have white fill for the maximum possible lifespan of that row. Small text to right of mean lifespan indicates total flies. Survival curves are presented in Supplementary file 1, and a summary of susceptibilities is provided in Figure 3supp1.

Growth kinetics of P. rettgeri (A), S. aureus (B), and C. albicans (C) in wild-type, single module mutant and ΔITPM flies..

Survival curves from Supplementary file 1 underlying data in Figure 3 are shown for context. Each data point reflects a pooled sample of 5 flies.

Measurement of PLUD upon infection with P. rettgeri, S. aureus, and C. albicans.

A) The PLUD of P. rettgeri in individual module mutant flies is not significantly different from wild-type. The PLUD of ΔITPM flies was not significantly different, although censoring of a single low-PLUD outlier in the wild-type would result in a significant difference between wild-type and ΔITPM flies (p < .01). B) The PLUD of S. aureus-infected flies is not different across any genotype. C) The PLUD of C. albicans-infected flies was significantly lower in ΔPhag flies with a notably lower mean PLUD. This difference was robust to use of different ΔPhag genetic backgrounds (merged data shown here). ΔMel flies and ΔITPM flies also had significantly lower PLUD. *, P < 0.05; **, P < 0.01

Microbe characteristics and growth conditions.

We used ΔPhag mutants in the genetic background of Melcarne et al. (2019) (+; ΔPhag) in some experiments, and confirm here that these mutants are equally deficient in phagocytic capacity to ΔPhag flies in the iso DrosDel genetic background (ΔPhag).

Lifespans of mutants used in this study in unchallenged flies and upon clean injury conditions at 29°C.

A-B) Single module mutants; C-D) compound module mutants.

Summary table of susceptibilities per the three conditions outlined in section “Systemic infections and survival.”

✓ indicates that the pathway contributes to defense against a given pathogen. In the event that a single module mutant is not more susceptible to the infection, but contributes to increased mortality in a double mutant line, a ✓ is given and the co-deleted relevant pathways highlighted by their first letter. Black boxes indicate no difference to wild-type or to clean injury. NI, natural infection; SI, septic infection.

Survival curves for all data summarized in Figure 3.