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Pre-exposure to non-pathogenic bacteria does not protect Drosophila against the entomopathogenic bacterium Photorhabdus - PubMed

  • ️Mon Jan 01 2018

Pre-exposure to non-pathogenic bacteria does not protect Drosophila against the entomopathogenic bacterium Photorhabdus

Jelena Patrnogic et al. PLoS One. 2018.

Abstract

Immune priming in insects involves an initial challenge with a non-pathogenic microbe or exposure to a low dose of pathogenic microorganisms, which provides a certain degree of protection against a subsequent pathogenic infection. The protective effect of insect immune priming has been linked to the activation of humoral or cellular features of the innate immune response during the preliminary challenge, and these effects might last long enough to promote the survival of the infected animal. The fruit fly Drosophila melanogaster is a superb model to dissect immune priming processes in insects due to the availability of molecular and genetic tools, and the comprehensive understanding of the innate immune response in this organism. Previous investigations have indicated that the D. melanogaster immune system can be primed efficiently. Here we have extended these studies by examining the result of immune priming against two potent entomopathogenic bacteria, Photorhabdus luminescens and P. asymbiotica. We have found that rearing D. melanogaster on diet containing a non-pathogenic strain of Escherichia coli alone or in combination with Micrococcus luteus upregulates the antibacterial peptide immune response in young adult flies, but it does not prolong fly life span. Also, subsequent intrathoracic injection with P. luminescens or P. asymbiotica triggers the Immune deficiency and Toll signaling pathways in flies previously exposed to a live or heat-killed mix of the non-pathogenic bacteria, but the immune activation fails to promote fly survival against the pathogens. These findings suggest that immune priming in D. melanogaster, and probably in other insects, is determined by the type of microbes involved as well as the mode of microbial exposure, and possibly requires a comprehensive and precise alteration of immune signaling and function to provide efficient protection against pathogenic infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Drosophila melanogaster bacterial pre-exposure.

(A) Experimental scheme. Oregon-R wild-type flies (P, Parental generation) were reared and allowed to oviposit on normal untreated diet. After parent removal, the progeny (F1 generation) completed their development on diet supplemented with Luria-Bertani media (LB), a mix of live Escherichia coli and Micrococcus luteus (Ec+Ml), or a mix of heat-killed bacteria (HK Ec+Ml) (Pre-exposure). (B) Effect of pre-exposure to live (Ec+Ml) or dead (HK Ec+Ml) bacteria on the transcript levels of the antimicrobial peptide genes Diptericin-A (B) and Drosomycin (C) in larvae, pupae and young adult flies (1–3 day-old) of D. melanogaster. All values were normalized to LB-containing media controls and analyzed using unpaired t-test. Error bars represent standard error of the mean. (D) Longevity of D. melanogaster following pre-exposure to live (Ec+Ml) or dead (HK Ec+Ml) bacteria. Fly mortality for each treatment is shown over a 70-day period. A log-rank (Mantel Cox) test performed using GraphPad Prism software did not detect significant differences between the survival curves (P = 0.3873), indicating that any effect on longevity produced by pre-exposure is relatively slight and inconsequential.

Fig 2
Fig 2. Experimental set up for Drosophila melanogaster bacterial pre-exposure, subsequent infection, and incubation.

Oregon-R wild-type flies (P, Parental generation) were raised and allowed to lay eggs on normal untreated food. Parent flies were then removed from the vials and the progeny (F1 generation) were fed on food containing a mix of live Escherichia coli and Micrococcus luteus (Ec+Ml), a mix of heat-killed bacteria (HK Ec+Ml) or on control media supplemented with Luria-Bertani (LB) (Pre-exposure). Pre-exposed flies were then injected intrathoracically with phosphate-buffered saline (PBS), a non-pathogenic strain of E. coli, or the insect pathogenic bacteria Photorhabdus luminescens and P. asymbiotica. After injection, adult flies were incubated in vials containing normal untreated media.

Fig 3
Fig 3. Antimicrobial peptide gene expression in Drosophila melanogaster pre-exposed to non-pathogenic bacteria and subsequently injected with pathogenic bacteria.

Transcript levels of Diptericin A (A-C) and Drosomycin (D-F) at 0, 6, and 24 hours following intrathoracic injection with phosphate-buffered saline (PBS), Escherichia coli, Photorhabdus luminescens, or P. asymbiotica. Prior to injection, D. melanogaster flies were grown on fly food mixed with Luria-Bertani (LB), E. coli and M. luteus mix (Ec+Ml), or heat-killed E. coli and M. luteus mix (HK Ec+Ml). All values were normalized to the PBS-injected control flies at the corresponding time points and analyzed using two-way ANOVA. Error bars represent standard error of the mean (*P≤0.05, ***P≤0.001, ****P ≤0.0001).

Fig 4
Fig 4. Survival of Drosophila melanogaster flies pre-exposed to non-pathogenic bacteria and subsequently injected with pathogenic bacteria.

Emerged flies pre-exposed to Luria-Bertani (LB) broth, a non-pathogenic strain of Escherichia coli (Ec), or heat-killed E. coli (HK Ec) were transferred to fresh untreated diet and then injected with (A) phosphate-buffered saline (PBS), (B) E. coli, (C) Photorhabdus luminescens, or (D) P. asymbiotica. Survival was monitored for 72 hours post injection. Survival experiments were replicated three times and results were analyzed using Log-rank (Mantel-Cox) test in GraphPad Prism software.

Fig 5
Fig 5. Survival of Drosophila melanogaster flies pre-exposed to a non-pathogenic bacterial mix and subsequently injected with pathogenic bacteria.

Emerged flies pre-exposed to Luria-Bertani (LB) broth, a mix of live Escherichia coli and Micrococcus luteus (Ec+Ml), or a mix of heat-killed E. coli and M. luteus (HK Ec+Ml) were transferred to fresh untreated diet and then injected with (A) phosphate-buffered saline (PBS), (B) E. coli, (C) Photorhabdus luminescens, or (D) P. asymbiotica. Survival was monitored for 72 hours post injection. Survival experiments were replicated three times and results were analyzed using Log-rank (Mantel-Cox) test in GraphPad Prism software.

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References

    1. Cooper D, Eleftherianos I (2017) Memory and Specificity in the Insect Immune System: Current Perspectives and Future Challenges. Front Immunol 8: 539 10.3389/fimmu.2017.00539 - DOI - PMC - PubMed
    1. Eleftherianos I, Marokhazi J, Millichap PJ, Hodgkinson AJ, Sriboonlert A, ffrench-Constant RH, et al. (2006) Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference. Insect Biochem Mol Biol 36: 517–525. 10.1016/j.ibmb.2006.04.001 - DOI - PubMed
    1. Freitak D, Wheat CW, Heckel DG, Vogel H (2007) Immune system responses and fitness costs associated with consumption of bacteria in larvae of Trichoplusia ni. BMC Biol 5: 56 10.1186/1741-7007-5-56 - DOI - PMC - PubMed
    1. Evans JD, Lopez DL (2004) Bacterial probiotics induce an immune response in the honey bee (Hymenoptera: Apidae). J Econ Entomol 97: 752–756. - PubMed
    1. Miyashita A, Takahashi S, Ishii K, Sekimizu K, Kaito C (2015) Primed Immune Responses Triggered by Ingested Bacteria Lead to Systemic Infection Tolerance in Silkworms. PLoS One 10: e0130486 10.1371/journal.pone.0130486 - DOI - PMC - PubMed

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