Bacterial Colonization of
Drosophila Larvae
Helen Hobbs
Taylor Laboratory
HHMI-Summer 2011
Background
McLaughlin, Erik. Traveler's Diarrhea: Getting it, fixing it, and
preventing it. Gadling, 2008. Print.
• Wasting
- Causes
• poor nutrition
• immune response
- 1.5 million children1
• Staphylococcus
• E. coli
K., Erik, and Kevin P. "Rivers and Streams." Biomesfirst09.
Web. 14 Sept. 2011.
Taylor Laboratory’s Research
• Drosophila melanogaster
– First line of defense against bacteria
• Innate immune system
• In Drosophila…
– Hemocytes
– Anti-microbial peptides
– Crystal Cells
• Pseudomonas fluorescens
– Cause symptoms of wasting
– Sequenced genome
Wood, Will. "Will Wood - Research Profile - Department of Biology &
Biochemistry." University of Bath. Web. 14 Sept. 2011.
Drosophila
Genotypes
Pseudomonas
Strains
• CSA
– Wild Type
– control
• Pf-5
– pathogenic
– developmental defects
• Black Cell (Bc)
– mutant crystal cells
• Pf-5 gac A
– mutant
– control
• SBW25
– pathogenic
– systemic melanization
Experimental Protocol
nd Instars
–
Early
2
• Dissection of 3rd Instar larvae
– Bacteria
• Killed yeast
• Antibody
Staining
– Anti Feeding
BrdU
• Second
ddH2O
• –Slides
• Yeast or Sucrose
– DAPI and Phalloidin
• Bromodeoxyuridine
(BrdU)
– Nucleoside
 Marks actively dividing cells
Katrin Weigmann, Robert Klapper, Thomas Strasser, Christof Rickert, Gerd Technau,
Herbert Jäckle, Wilfried Janning and Christian Klämbt:FlyMove – a new way to look at
development of Drosophila.Trends Genet (2003) 19, 310.
• Egg lay and transfer
Data Collection
• •Initial
Feeding
General Experimental Design
Genotype
ddH2O
Yeast
Sucrose
Pf-5
Yeast
Sucrose
Pf-5 gac A
Yeast
Sucrose
SBW25
Yeast
Sucrose
Developmental Delay
Average Percent Pupariated
120
100
Percentage
80
ddH2O Yeast
ddH2O Sucrose
60
Pf-5 Yeast
Pf-5 Sucrose
SBW25 Yeast
40
SBW25 Sucrose
Pf-5 gac A Yeast
20
Pf-5 gac A Sucrose
0
0
50
100
150
200
-20
Time (Hours)
250
300
350
BrdU Staining
• Anti-BrdU primary antibody
– Dissected 3rd instar larvae
• Problems
– Signal Strength
– Fading
– Successful Incorporation
Results
• Definite developmental delay in Pf-5 treated
larvae
– Approximately 72 hours (3 days)
• Starvation appears to have greatest effect on
SBW25-treated larvae
– 29% vs. 68%
• Disruption of organ morphology in Pf-5 and
SBW25 treated larvae
– Fat body cells
– Intestinal cell layer
What’s next?
• More starvation experiments
– Confirm SBW25 results
• BrdU
– Quantitative results for cell division at imaginal
discs
• New Drosophila genotypes
– Relish
• Mutant in innate immunity pathway
• New Strains of Pseudomonas
Acknowledgements
•
•
•
•
•
•
•
•
•
HHMI
Cripps
Dr. Barbara Taylor
Francesca Walker
Kise Rosen
Dr. Joyce Loper
Marcella Henkels
Dr. Kevin Ahern
The fruit flies!
References
• World Health Organization. Diarrhoeal disease.
Available from: http://www.who.int/en/
• Olcott, MH, MD Henkels, KL Rosen, FLWalker, B
Sneh, JE Loper, BJ Taylor. Lethality and
Developmental Delay in Drosophila melanogaster
larvae after ingestion of selected Pseudomonas
fluorescens strains. PloS One. Sept 13;5(9):e12504
http://www.plosone.org/article/info%3Adoi%2F10.13
71%2Fjournal.pone.0012504