Endosymbiont-based immunity in Drosophila melanogaster against parasitic nematode infection

Abstract

Associations between endosymbiotic bacteria and their hosts represent a complex ecosystem within organisms ranging from humans to protozoa. Drosophila species are known to naturally harbor Wolbachia and Spiroplasma endosymbionts, which play a protective role against certain microbial infections. Here, we investigated whether the presence or absence of endosymbionts affects the immune response of Drosophila melanogaster larvae to infection by Steinernema carpocapsae nematodes carrying or lacking their mutualistic Gram-negative bacteria Xenorhabdus nematophila (symbiotic or axenic nematodes, respectively). We find that the presence of Wolbachia alone or together with Spiroplasma promotes the survival of larvae in response to infection with S. carpocapsae symbiotic nematodes, but not against axenic nematodes. We also find that Wolbachia numbers are reduced in Spiroplasma-free larvae infected with axenic compared to symbiotic nematodes, and they are also reduced in Spiroplasma-containing compared to Spiroplasma-free larvae infected with axenic nematodes. We further show that S. carpocapsae axenic nematode infection induces the Toll pathway in the absence of Wolbachia, and that symbiotic nematode infection leads to increased phenoloxidase activity in D. melanogaster larvae devoid of endosymbionts. Finally, infection with either type of nematode alters the metabolic status and the fat body lipid droplet size in D. melanogaster larvae containing only Wolbachia or both endosymbionts. Our results suggest an interaction between Wolbachia endosymbionts with the immune response of D. melanogaster against infection with the entomopathogenic nematodes S. carpocapsae. Results from this study indicate a complex interplay between insect hosts, endosymbiotic microbes and pathogenic organisms.

Fig 1. Survival of Drosophila melanogaster larvae carrying or lacking endosymbionts in response to nematode infection.

Survival of D. melanogaster third-instar larvae upon infection with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Sterile distilled water served as control (C) treatment. (A) Survival response of D. melanogaster strains lacking both Wolbachia and Spiroplasma (W-S-) and strains carrying both endosymbionts (W+S+), (B) Survival response of D. melanogaster strains lacking both endosymbionts (W-S-) and strains carrying Wolbachia only (W+S-). Survival was tracked every 12 h for 96 h and is represented as percent survival on the graph. Data were analyzed using the Log-Rank test (GraphPad Prism7 software). The experiment was repeated three times and bars represent standard errors (****P<0.001, ****P<0.0001).

Fig 2. Numbers for endosymbiotic and pathogenic bacteria in Drosophila melanogaster larvae responding to nematode infection.

D. melanogaster third instar larvae carrying no endosymbionts (W-S-), both endosymbionts (W+S+) or only Wolbachia (W+S-) were infected with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Relative number of cells for (A) Wolbachia at 12 and 36 h, and (B) Spiroplasma at 12, 36 and 60 h were determined using quantitative PCR. (C) Numbers of colony forming units of Xenorhabdus nematophila were estimated at 12, 36 and 60 h post infection using quantitative PCR. Data were analyzed using an unpaired two-tailed t-test. Means from three independent experiments are shown and standard deviations are represented by error bars (*P<0.05, **P<0.01).

Fig 3. Transcript levels of immune genes in Drosophila melanogaster larvae carrying or lacking endosymbionts upon nematode infection.

Gene transcript levels for (A, B and C) Diptericin, (D, E and F) Drosomycin, (G, H and I) Turandot-A (Tot-A), and (J, K and L) Puckered in D. melanogaster larvae containing no endosymbionts (W-S-), both Wolbachia and Spiroplasma (W+S+), or Wolbachia only (W+S-) at 12, 36 and 60 h after infection with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Sterile distilled water served as control (C) treatment. The experiment was repeated three times and error bars show standard deviations. Data were analyzed using one way analysis of variance with a Tukey post hoc test (*P<0.05).

Fig 4. Phenoloxidase activity and melanization response in uninfected and nematode-infected Drosophila melanogaster larvae carrying or lacking endosymbionts.

(A) Melanization response in D. melanogaster larvae containing no endosymbionts (W-S-), both Wolbachia or Spiroplasma (W+S+), or Wolbachia only (W+S-) following heat treatment. (B) Relative phenoloxidase (PO) activity was measured in the larval hemolymph of the three D. melanogaster strains at 24 h post-infection with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Sterile distilled water served as control (C) treatment. The experiment was repeated three times and error bars show standard deviations. Data analysis was performed using one way analysis of variance with a Tukey post hoc test (*P<0.05).

Fig 5. Metabolic activity in Drosophila melanogaster larvae carrying or lacking endosymbionts following nematode infection.

D. melanogaster third instar larvae lacking both endosymbionts (W-S-), carrying both Wolbachia and Spiroplasma (W+S+), or containing Wolbachia only (W+S-) were infected with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Application of sterile distilled water served as control (C) treatment. The relative amount of (A) Triglycerides, (B) Trehalose, (C) Glucose and (D) Glycogen was estimated 24 h post-infection. The experiment was repeated three times and error bars show standard deviations. Data were analyzed using one way analysis of variance with a Tukey post hoc test (*P<0.05, **P<0.01).

Fig 6. Lipid droplet size in Drosophila melanogaster larvae carrying or lacking endosymbionts upon nematode infection.

(A) Representative images of lipid droplets (LD) labeled with Nile Red (red) and DAPI (blue) in fat body tissues of D. melanogaster third instar larvae lacking both endosymbionts (W-S-), containing both Wolbachia and Spiroplasma (W+S+), or carrying Wolbachia only (W+S-) followed infection with Steinernema carpocapsae symbiotic (Sy) or axenic (Ax) nematodes. Sterile distilled water served as control (C) treatment. Magnification: 40X. (B) Quantification of LD area in the fat body tissues obtained from 10 D. melanogaster larvae per treatment using ImageJ. Values show the means from three independent experiments and error bars show standard deviations. Data were analyzed using one way analysis of variance with a Tukey post hoc test (*P<0.05, **P<0.01, ****P<0.0001).