Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

et al. (2010) Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains. PLoS ONE 5(9): e12504. doi:10.1371/journal.pone.0012504 Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains Marika H. Olcott 0 Marcella D. Henkels 0 Kise L. Rosen 0 Francesca L. Walker 0 Baruch Sneh 0 Joyce E. 0 Loper 0 Barbara J. Taylor 0 Francois Leulier, CNRS - Universite Aix-Marseille, France 0 1 Department of Zoology, Oregon State University , Corvallis, Oregon , United States of America, 2 Horticultural Crops Research Laboratory, United States Department of Agriculture-Agricultural Research Station , Corvallis, Oregon , United States of America, 3 Department of Botany and Plant Pathology, Oregon State University , Corvallis, Oregon , United States of America, 4 Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Sciences, Tel Aviv University , Tel Aviv , Israel Background: The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development. Methodology/Principal Findings: We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria. Conclusions/Significance: These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled. - Funding: This project was supported by National Research Initiative Competitive Grants 2006-35319-17427 and 2008-35600-18770 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service to JEL and by National Institutes of Health Grant 9RO1 GM085818-14A2 to BJT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The fruit fly, Drosophila melanogaster, is a well-established model organism for the study of host response to microbial infection and animal development. To protect against infection, D. melanogaster employs a suite of cellular and humoral defense mechanisms, making it a good reference organism in which to dissect host responses (reviewed in [1,2,3,4,5,6]). For example, D. melanogaster employ mechanical barriers (such as the cuticle and tightly connected epithelial cells) to reduce the entry of environmental pathogens. In addition, a rapid defense is mounted through the secretion of a battery of inducible effector molecules (i.e. antimicrobial peptides such as Diptericin and reactive oxygen species), activation of phenoloxidase through a complement-like protease cascade (which leads to the production of reactive compounds and melanin), clotting of the hemolymph, and phagocytosis and encapsulation of foreign objects by blood cells (hemocytes), all of which are tightly regulated mechanisms for neutralizing intruders while maintaining a balanced microbiota. D. melanogaster larvae and adults are exposed to a variety of environmental bacteria in their natural habitats. Among these are species of Pseudomonas, a diverse genus of gamma proteobacteria commonly found in soil, water, or in association with plants or animals. Certain strains of Pseudomonas entomophila [7] and Pseudomonas aeruginosa [8,9] are known pathogens of D. melanogaster, but this pathogenicity is thought to be uncommon for the genus. Of 28 strains representing a spectrum of Pseudomonas spp. that were tested by Vodovar et al. [7], only P. entomophila exhibited pathogenicity against D. melanogaster. Nevertheless, genes with predicted functions in insect toxicity are present in genomic sequences of other Pseudomonas spp., including fitD in Pseudomonas fluorescens strains Pf-5 and CHA0 [10], and tc-like toxins in Pseudomonas syringae pv. syringae strain B728a and P. fluorescens Pf0-1 [11]. A recent study demonstrated that fitD confers strong insecticidal activity, which was exhibited when Pf-5 or the closelyrelated strain CHA0 was injected into larvae of two insect species, the tobacco hornfworm Manduca sexta and the greater wax moth Galleria mellonella [10]. These results highlighted the exciting possibility that strains of P. fluorescens have additional previously-unappreciated insecticidal activities. This study was initiated to determine the oral toxicity of P. fluorescens against the model organism D. melanogaster, and to characterize the effect of the microbial infection throughout host development. The immune response to high doses of pathogenic and non-pathogenic bacteria has been studied extensively in adult D. melanogaster and late stage larvae. Very few experiments have examined the impact of exposure to bacteria at earlier developmental stages when the animals are undergoing rapid growth of both body tissues and rapid divisions of the imaginal disc cells, which will generate the adult body. We developed a new protocol to feed timed cultures of D. melanogaster larvae with bacterial strains without handling the larvae, hence reducing the risk of injuries and stress prior to infection. Using this non-invasive protocol, we observed three distinct and strain-specific responses of D. melanogaster to infection by three strains of P. fluorescens (Pf0-1, SBW25 and Pf-5) with fully sequenced genomes [12,13]. Oral ingestion of strain Pf0-1 had little effect on larvae. In contrast, strains SBW25 and Pf-5 were toxic to D. melanogaster, with SBW25 causing a systemic melanization response and Pf-5 causing a dosedependent lethality and delayed metamorphosis coupled with morphological defects in adults. Indu (...truncated)