The purpose of this experiment was to investigate immune function and energy metabolism, particularly the levels of antimicrobial peptides produced in Drosophila energetically compromised genotypes. This will provide the ability to investigate energetics of immunity without changing diet. Flies were infected with the bacteria P. rettgeri and the resulting immune response was investigated. Females did not mount as effective an immune response as males. All flies exhibited decreased survivorship from infection. Control flies survived at higher levels and showed no mitochondrial:nuclear interaction

Buchanan, Justin

Montooth, Kristi

Treu, Cassandra K

English

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnUCARE Research Products UCARE: Undergraduate Creative Activities &Research Experiences4-2016AMP Expression in Energetic Hybrid D.melanogaster Infected With P. rettgeriCassandra K. TreuUniversity of Nebraska-Lincoln, cassandra.treu@huskers.unl.eduJustin BuchananUniversity of Nebraska-Lincoln, justin.l.buchanan@gmail.comKristi MontoothUniversity of Nebraska-Lincoln, kmontooth2@unl.eduFollow this and additional works at: http://digitalcommons.unl.edu/ucareresearchPart of the Bacteriology Commons, Biology Commons, and the Immunity CommonsThis Poster is brought to you for free and open access by the UCARE: Undergraduate Creative Activities & Research Experiences atDigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in UCARE Research Products by an authorized administratorof DigitalCommons@University of Nebraska - Lincoln.Treu, Cassandra K.; Buchanan, Justin; and Montooth, Kristi, "AMP Expression in Energetic Hybrid D. melanogaster Infected With P.rettgeri" (2016). UCARE Research Products. 62.http://digitalcommons.unl.edu/ucareresearch/62AMP Expression in Energetic Hybrid D. melanogaster Infected With P. rettgeri Cassie Treu, Justin L. Buchanan, & Kristi L. MontoothSchool of Biological Science, University of Nebraska - Lincoln, Lincoln, NE 68588AbstractThe purpose of this experiment was to investigateimmune function and energy metabolism, particularlythe levels of antimicrobial peptides produced inDrosophila energetically compromised genotypes.This will provide the ability to investigate energetics ofimmunity without changing diet. Flies were infectedwith the bacteria P. rettgeri and the resulting immuneresponse was investigated. Females did not mount aseffective an immune response as males. All fliesexhibited decreased survivorship from infection.Control flies survived at higher levels and showed nomitochondrial:nuclear interaction.IntroductionLife-History TradeoffsNutrients from environment3,5Providencia Rettgeri• P. rettgeri, a natural cause of infection forDrosophila, is a gram-negative bacteria that ispathogenic to fruit flies.• The infection multiplies very quickly and causesmoderate survival rates, found to be ranging from40%-80%1.• Providencia has been found in humans as part ofnatural gut bacteria as well as gastric upset7.Innate Immunity• While adaptive and innate immunity is present inanimals, invertebrates, like Drosophila, only possessinnate immunity as a defense against pathogens.This form of defense results in production ofantimicrobial peptides (AMPs).Genotypes• The mitochondrial-nuclear incompatible genotype(simw501);OreR decreases the fitness and fecundityof fly larvae.• This is because the incompatibility causes incorrectsynthesis of certain proteins necessary fordevelopment, which delays it 2.• Five other genotypes were used in this experimentas compatible genotypic controls, with nointeractive effect between their mitochondrial andnuclear genotypes, their oxidative phosphorylationfunctions normally.ResultsMotivation Predictions1. The mitochondrial-nuclear incompatible genotype (simw501);OreR mount a less effective AMP response than the other genotypes due to its lower level of energy.  This will correspond with lower immune activation and decreased survival.2. Genotypes mount similar immune responses, but the incompatible genotype will have lower survival due to energy limitations. Continued Research• Two of four treatment blocks have been collectedand are in the stages of mRNA extraction and cDNAprocessing. qPCR primers will continue to beoptimized by changing temperature of the reaction.• Future experimentation on larvae will be done toinvestigate the differences in energy spent, sincelarvae don’t need an energy allotment forreproduction, just for growth. Bacterial load inadults will also be performed to test effectivenessof pathogen clearance.Works Cited1) Lazzaro, B.P. et al. 2008. Evolutionary Biology. 8:762) Meiklejohn, C.D. et al. PLoS Genetics 9: e1003238. 20133) Schwenke ,R. et al. Annual Review of Entomology. 20164) Short, S. et al. 2012. J Insect Physiol . 58 (9): 1192-12015) Zera, A.J. & Larsen, A. 2001. J Insect Physiol . 47(10):1147-11606) Rutschmann, S. et al. 2000. Nature Immunology. 1:342-3477) Gallac, M.R & Lazzaro, B.P. 2011. Microbes Infect. 13(7):673-83AcknowledgementsThanks to Justin Buchanan, Kristi Montooth, Katie, and Montooth Lab for help and guidance, Brian Lazzaro for P. rettgri and research, and the UCARE Program for funding. 051015202530-2 -1 0 1 2 3 4 5Cycles to critical thresholdlog2(dilution)Drosomycin Calibration0 Hour, y = -1.156x + 23.79, R² = 0.926424 Hour, y = -1.0942x + 23.399, R² = 0.94120510152025303540-2 -1 0 1 2 3 4 5Cycles to critical thresholdlog2(dilution)Diptericin Calibration0 Hour, y = -1.4922x + 29.92, R² = 0.321324 hour, y = -1.3852x + 28.172, R² = 0.75430510152025-2 0 2 4 6Cycles to critical thresholdlog2(dilution)RP49 Calibration0 hour, y = -1.055x + 19.767, R² = 0.936224 hour, y = -0.9651x + 18.234, R² = 0.9782Figure 2.  Females  have decreased survivorship in infection and under control conditions, with infection resulting in a greater decrease.  This may be due to differences in the life-history trade-off s.  Virgin females will lay unfertilized eggs. Figure 3.   The bacterial treatment reduced survivorship in all genotypes.  Control survivorship was lower then expected.  This may be due to desiccation as humidity was low during trials. Figure 4. Treatment decreases number of offspring ~15%.  Ore nuclear background has decreased fecundity.  (w501);Ore has further decreased fecundity.Fecundity Statisticsχ2 p-valueMito=             6.95         0.0016Nuc=              48.9        4.7*10-10Treatment=  12.1        7.9*10-4Methods and Materials (w501);Ore, (w501);Aut, (ore);Ore, (ore);AutVirgin males and females collected in Blocks4Flies infected by poking with 0.1 mm needle with P. rettgeri and sterile needle as control Samples flash frozen at 0 h, 5 h, 10 h, and 10 daysRNA extractionEtOH precipitation to RNA purificationRNA reverse transcribed to cDNAcDNA used for Quantitative PCRAMPs: Controls:dipterycin (IMD) RP49 (ribosome)drosomycin(Toll)   Actin5c(cytoskeleton)WO:OA:OOOA:OO:WAWA:OO:WOWA:OA:WOIMD Pathway      Toll Pathway6Survival Statisticsχ2 p-valueMito:nuc=     7.56        0.023Sex=              15.9        6.9*10-5Treatment=  59.3        1.4*10-14Figure 5. Diptericin is an AMP that is produced in reaction to Gram (-) and Fungi.  We expect to see an increase in treated genotypes.  This means that bacterial treatment will have fewer cycles to critical threshold.Figure 6. Drosomycinis an AMP that is produced from Gram (+) bacteria.  We do not expect a response from this AMP but will use it as a control.  The cycles to critical threshold between treatment and control should be zero.Figure 7. RP49 is a ribosomal subunit that will be used as a control. The cycles to critical threshold between treatment and control should be zero.Figure 1. The incompatible genotype shows decreased survivorship compared to genotypic controls under bacterial treatment.  Aut nuclear genotype has higher survivorship then Ore.  Survivorship of controls matches prior research on infection. The slopes in these plots are the efficiency of the QPCR and R2measure reproducibility between samples ideally, which should beone. A slope of one means that the efficiency of the primers predictthe concentration. Slopes under or over one will result in decreasedability to determine unknown concentrations.• Reproduction• Maintenance

AMP Expression in Energetic Hybrid D. melanogaster Infected With P. rettgeri

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University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
UCARE Research Products UCARE: Undergraduate Creative Activities &Research Experiences
4-2016
AMP Expression in Energetic Hybrid D.
melanogaster Infected With P. rettgeri
Cassandra K. Treu
University of Nebraska-Lincoln, cassandra.treu@huskers.unl.edu
Justin Buchanan
University of Nebraska-Lincoln, justin.l.buchanan@gmail.com
Kristi Montooth
University of Nebraska-Lincoln, kmontooth2@unl.edu
Follow this and additional works at: http://digitalcommons.unl.edu/ucareresearch
Part of the Bacteriology Commons, Biology Commons, and the Immunity Commons
This Poster is brought to you for free and open access by the UCARE: Undergraduate Creative Activities & Research Experiences at
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in UCARE Research Products by an authorized administrator
of DigitalCommons@University of Nebraska - Lincoln.
Treu, Cassandra K.; Buchanan, Justin; and Montooth, Kristi, "AMP Expression in Energetic Hybrid D. melanogaster Infected With P.
rettgeri" (2016). UCARE Research Products. 62.
http://digitalcommons.unl.edu/ucareresearch/62
AMP Expression in Energetic Hybrid D. melanogaster Infected With P. rettgeri 
Cassie Treu, Justin L. Buchanan, & Kristi L. Montooth
School of Biological Science, University of Nebraska - Lincoln, Lincoln, NE 68588
Abstract
The purpose of this experiment was to investigate
immune function and energy metabolism, particularly
the levels of antimicrobial peptides produced in
Drosophila energetically compromised genotypes.
This will provide the ability to investigate energetics of
immunity without changing diet. Flies were infected
with the bacteria P. rettgeri and the resulting immune
response was investigated. Females did not mount as
effective an immune response as males. All flies
exhibited decreased survivorship from infection.
Control flies survived at higher levels and showed no
mitochondrial:nuclear interaction.
Introduction
Life-History Tradeoffs
Nutrients from environment3,5
Providencia Rettgeri
• P. rettgeri, a natural cause of infection for
Drosophila, is a gram-negative bacteria that is
pathogenic to fruit flies.
• The infection multiplies very quickly and causes
moderate survival rates, found to be ranging from
40%-80%1.
• Providencia has been found in humans as part of
natural gut bacteria as well as gastric upset7.
Innate Immunity
• While adaptive and innate immunity is present in
animals, invertebrates, like Drosophila, only possess
innate immunity as a defense against pathogens.
This form of defense results in production of
antimicrobial peptides (AMPs).
Genotypes
• The mitochondrial-nuclear incompatible genotype
(simw501);OreR decreases the fitness and fecundity
of fly larvae.
• This is because the incompatibility causes incorrect
synthesis of certain proteins necessary for
development, which delays it 2.
• Five other genotypes were used in this experiment
as compatible genotypic controls, with no
interactive effect between their mitochondrial and
nuclear genotypes, their oxidative phosphorylation
functions normally.
Results
Motivation Predictions
1. The mitochondrial-nuclear incompatible genotype 
(simw501);OreR mount a less effective AMP response 
than the other genotypes due to its lower level of 
energy.  This will correspond with lower immune 
activation and decreased survival.
2. Genotypes mount similar immune responses, but 
the incompatible genotype will have lower survival due 
to energy limitations. 
Continued Research
• Two of four treatment blocks have been collected
and are in the stages of mRNA extraction and cDNA
processing. qPCR primers will continue to be
optimized by changing temperature of the reaction.
• Future experimentation on larvae will be done to
investigate the differences in energy spent, since
larvae don’t need an energy allotment for
reproduction, just for growth. Bacterial load in
adults will also be performed to test effectiveness
of pathogen clearance.
Works Cited
1) Lazzaro, B.P. et al. 2008. Evolutionary Biology. 8:76
2) Meiklejohn, C.D. et al. PLoS Genetics 9: e1003238. 2013
3) Schwenke ,R. et al. Annual Review of Entomology. 2016
4) Short, S. et al. 2012. J Insect Physiol . 58 (9): 1192-1201
5) Zera, A.J. & Larsen, A. 2001. J Insect Physiol . 47(10):1147-1160
6) Rutschmann, S. et al. 2000. Nature Immunology. 1:342-347
7) Gallac, M.R & Lazzaro, B.P. 2011. Microbes Infect. 13(7):673-83
Acknowledgements
Thanks to Justin Buchanan, Kristi Montooth, Katie, and 
Montooth Lab for help and guidance, Brian Lazzaro for P. 
rettgri and research, and the UCARE Program for funding. 
0
5
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-2 -1 0 1 2 3 4 5
C
yc
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s 
to
 c
ri
ti
ca
l t
h
re
sh
o
ld
log2(dilution)
Drosomycin Calibration
0 Hour, y = -1.156x + 23.79, R² = 0.9264
24 Hour, y = -1.0942x + 23.399, R² = 0.9412
0
5
10
15
20
25
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40
-2 -1 0 1 2 3 4 5
C
yc
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s 
to
 c
ri
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l t
h
re
sh
o
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log2(dilution)
Diptericin Calibration
0 Hour, y = -1.4922x + 29.92, R² = 0.3213
24 hour, y = -1.3852x + 28.172, R² = 0.7543
0
5
10
15
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25
-2 0 2 4 6
C
yc
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s 
to
 c
ri
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ca
l t
h
re
sh
o
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log2(dilution)
RP49 Calibration
0 hour, y = -1.055x + 19.767, R² = 0.9362
24 hour, y = -0.9651x + 18.234, R² = 0.9782
Figure 2.  Females  have 
decreased survivorship 
in infection and under 
control conditions, with 
infection resulting in a 
greater decrease.  This 
may be due to 
differences in the life-
history trade-off s.  
Virgin females will lay 
unfertilized eggs. 
Figure 3.   The bacterial 
treatment reduced 
survivorship in all 
genotypes.  Control 
survivorship was lower 
then expected.  This may 
be due to desiccation as 
humidity was low during 
trials. 
Figure 4. Treatment 
decreases number of 
offspring ~15%.  Ore 
nuclear background 
has decreased 
fecundity.  (w501);Ore 
has further decreased 
fecundity.
Fecundity Statistics
χ2 p-value
Mito=             6.95         0.0016
Nuc=              48.9        4.7*10-10
Treatment=  12.1        7.9*10-4
Methods and Materials 
(w501);Ore, (w501);Aut, (ore);Ore, (ore);Aut
Virgin males and females collected in Blocks4
Flies infected by poking with 0.1 mm needle with P. rettgeri and 
sterile needle as control 
Samples flash frozen at 0 h, 5 h, 10 h, and 10 days
RNA extraction
EtOH precipitation to RNA purification
RNA reverse transcribed to cDNA
cDNA used for Quantitative PCR
AMPs: Controls:
dipterycin (IMD) RP49 (ribosome)
drosomycin(Toll)   Actin5c(cytoskeleton)
WO:OA:OO
OA:OO:WA
WA:OO:WO
WA:OA:WO
IMD Pathway      Toll Pathway6
Survival Statistics
χ2 p-value
Mito:nuc=     7.56        0.023
Sex=              15.9        6.9*10-5
Treatment=  59.3        1.4*10-14
Figure 5. Diptericin is 
an AMP that is 
produced in reaction 
to Gram (-) and 
Fungi.  We expect to 
see an increase in 
treated genotypes.  
This means that 
bacterial treatment 
will have fewer cycles 
to critical threshold.
Figure 6. Drosomycin
is an AMP that is 
produced from Gram 
(+) bacteria.  We do 
not expect a response 
from this AMP but 
will use it as a 
control.  The cycles to 
critical threshold 
between treatment 
and control should be 
zero.
Figure 7. RP49 is a 
ribosomal subunit 
that will be used as 
a control. The 
cycles to critical 
threshold between 
treatment and 
control should be 
zero.
Figure 1. The 
incompatible genotype 
shows decreased 
survivorship compared 
to genotypic controls 
under bacterial 
treatment.  Aut nuclear 
genotype has higher 
survivorship then Ore.  
Survivorship of controls 
matches prior research 
on infection. The slopes in these plots are the efficiency of the QPCR and R2
measure reproducibility between samples ideally, which should be
one. A slope of one means that the efficiency of the primers predict
the concentration. Slopes under or over one will result in decreased
ability to determine unknown concentrations.
• Reproduction
• Maintenance


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AMP Expression in Energetic Hybrid D. melanogaster Infected With P. rettgeri

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