A Specific Primed Immune Response
in Drosophila Is Dependent on Phagocytes
Linh N. Pham, Marc S. Dionne, Mimi Shirasu-Hiza, David S. Schneider*
Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America
Drosophila melanogaster, like other invertebrates, relies solely on its innate immune response to fight invading
microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming
Drosophila with a sublethal dose of Streptococcus pneumoniae protects against an otherwise-lethal second challenge of
S. pneumoniae. This protective effect exhibits coarse specificity for S. pneumoniae and persists for the life of the fly.
Although not all microbial challenges induced this specific primed response, we find that a similar specific protection
can be elicited by Beauveria bassiana, a natural fly pathogen. To characterize this primed response, we focused on
S. pneumoniae–induced protection. The mechanism underlying this protective effect requires phagocytes and the Toll
pathway. However, activation of the Toll pathway is not sufficient for priming-induced protection. This work
contradicts the paradigm that insect immune responses cannot adapt and will promote the search for similar responses
overlooked in organisms with an adaptive immune response.
Citation: Pham LN, Dionne MS, Shirasu-Hiza M, Schneider DS (2007) A specific primed immune response in Drosophila is dependent on phagocytes. PLoS Pathog 3(3): e26.
Immune responses are typically characterized as being
either adaptive or innate. Adaptive immunity, which requires
T and B cells, is specific, has memory, and is generally
considered to be restricted to vertebrates. In contrast, the
innate immune response is thought to act naı ¨vely to each
encounter with a pathogen [1,2]. Innate immunity depends on
the recognition of broadly conserved molecular moieties and
exhibits only weak specificity, such as the ability to distinguish
between different structural classes of peptidoglycan .
However, recent work suggests that the invertebrate innate
immune response may exhibit adaptive characteristics
(reviewed in  and ).
Functional immune adaptation can be defined most
broadly as any case where an immune response differs
between a first and second challenge. The simplest form
involves the immune system remaining activated after an
initial challenge. This sort of response has long been known
in invertebrates as shown by Hans Boman and coworkers .
They found that antibacterial activity in Drosophila hemo-
lymph persists after bacterial challenge and can provide
protection against subsequent challenges. More recently,
Moret et al.  found a similar persistence of humoral
antibacterial activity in mealworms.
More complex adaptive phenomena have also been
observed in arthropods; for example, flour moths  and
Daphnia  possess strain-specific immunity that is passed
from a mother to her offspring. The molecular mechanisms
underlying the maternal transfer of strain-specific protection
have not been characterized. Specific memory has also been
examined in cockroaches  and bumblebees . In both
insects, the initial immune activation is nonspecific and
confers protection against many types of challenges. How-
ever, both cockroaches and bees are also able to mount long-
term specific protection: a priming dose of a particular
species of bacteria only protects against that species (or class
of species in the case of bumblebees). From this work, it seems
that innate immunity possesses the adaptive characteristics of
specificity and memory; unfortunately, the animals used in
past studies have not been amenable to deeper analysis. We
examined the well-understood Drosophila innate immune
response for specificity and memory because this model
organism would give us genetic and physiological assays to
dissect adaptive aspects of innate immunity.
Drosophila has been proved to be a powerful model
organism to study innate immunity [1,2]. The Drosophila
innate immune response has three effector mechanisms: the
humoral response, melanization, and the cellular response
[1,2]. The humoral immune response involves the secretion of
soluble factors, such as antimicrobial peptides (AMPs), into
the hemolymph following immune activation. Melanization is
the process whereby melanin is deposited at wound sites and
parasite surfaces, resulting in the release of toxic reactive
oxygen species. The cellular immune response consists of
hemocytes that phagocytose, encapsulate, and kill invading
microbes, much like vertebrate macrophages. These mecha-
nisms depend in various ways on pathogen detection via the
Toll or imd signaling pathways [1,2,12,13].
We found that S. pneumoniae–primed flies are protected
against a subsequent lethal challenge with S. pneumoniae. This
response is specific for S. pneumoniae and persists for the life
of the fly. In this paper, we demonstrate that the Toll
pathway, but not the imd pathway, is required for this
protective effect. Notably, activation of the Toll pathway is
Editor: Kenneth Vernick, University of Minnesota, United States of America
Received November 5, 2006; Accepted January 12, 2007; Published March 9, 2007
Copyright: ? 2007 Pham et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: AMP, antimicrobial peptide; CFU, colony-forming units; PBS,
phosphate-buffered saline; qRT, quantitative real-time reverse transcription
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
PLoS Pathogens | www.plospathogens.org March 2007 | Volume 3 | Issue 3 | e260001
not sufficient to elicit a primed response. We show that AMPs
are not involved and that phagocytes are the critical effectors
of the primed response. Taken together, we demonstrate that
the Drosophila primed response is specific and persists for the
life of the fly. We identified a signaling pathway required for
the process and ascertained which branch of the fly immune
response is responsible for the primed response.
The Drosophila Immune Response Can Adapt
We found that previous exposure to S. pneumoniae perma-
nently alters the fly’s response to this bacterium. S. pneumoniae
is a Gram-positive encapsulated bacterium that is the
causative agent of otitis media, pneumonia, and meningitis
. The injection of 3,000 colony-forming units (CFU)
directly into Drosophila hemolymph is normally lethal, killing
the fly within 2 d (Figure 1A), and death is correlated with
bacterial proliferation (Figure 1B). However, flies primed
with a sublethal dose of bacteria were protected against a
lethal challenge of S. pneumoniae administered 1 wk later
(Figure 1C). Flies primed with S. pneumoniae and challenged
with a lethal dose died at the same rate or slower than
wounded controls. A priming dose of dead heat-killed
S. pneumoniae was also sufficient to protect flies against a
subsequent lethal challenge. Priming flies with S. pneumoniae
thus induces long-term changes in the fly immune response,
conferring protection against an otherwise-lethal challenge.
We considered two scenarios that could explain enhanced
survival in S. pneumoniae–primed flies. First, bacterial numbers
may not differ between naı ¨ve versus primed flies, and primed
flies might survive the stress of the infection better than naı ¨ve
flies. Second, S. pneumoniae–primed flies could kill the bacteria
faster, and bacterial clearance would correlate with enhanced
survival. To distinguish between these two possibilities, we
examined bacterial load in naı ¨ve versus primed flies. Flies
were injected with a priming dose of either dead S. pneumoniae
or phosphate-buffered saline (PBS) 1 wk prior to a challenge
of 400 CFU S. pneumoniae. This dose was chosen to emphasize
the difference between naı ¨ve and primed flies; 400 CFU is the
lowest dose that is lethal to naı ¨ve flies but not S. pneumoniae–
primed flies (unpublished data). Within 1 d, S. pneumoniae–
primed flies had killed almost all of the S. pneumoniae, whereas
naı ¨ve flies still contained bacteria (Figure 1D). This result
indicates that the survival difference between naı ¨ve and
S. pneumoniae–primed flies results from different rates of
S. pneumoniae killing.
Functionally, immunological memory is characterized by a
more effective immune response upon repeat exposure that
persists for the life of the animal. Although the best-described
model for immune memory involves T and B cells and
recombination-derived variation of receptors, we note that
the definition of memory is independent of mechanism and
immune memory could arise in a variety of ways. We
demonstrated that preinoculation with dead S. pneumoniae
alters the fly immune response such that it is more effective
against subsequent challenges with S. pneumoniae. To deter-
mine how long these immune changes persist in the fly, we
next varied the length of time between the priming and
challenge dose. Flies primed with dead S. pneumoniae on day 0
were challenged between 1 and 14 d later with a lethal dose of
S. pneumoniae. S. pneumoniae–primed flies always died signifi-
cantly more slowly than naı ¨ve PBS-injected flies (Figures 1E
and S1). An interval of 2 wk between the priming dose and
challenge dose was the longest time we could assay; at 3 wk
postpriming, the flies are actually 5 wk old and die from the
stress of wounding alone (unpublished data). In summary,
protection due to a priming dose of S. pneumoniae was
detectable within 24 h and persisted for the life of the fly, or
as long as we could assay survival differences.
The Drosophila Immune Response Possesses a Surprising
Degree of Specificity
To explore the specificity of this immune response, we
asked whether other pathogens can induce a protective
response against themselves. We chose a broad range of
microbes pathogenic to wild-type Drosophila, including a
Gram-negative bacterium, a Gram-positive bacterium, a
Mycobacterium, and a natural fungal pathogen [15–18].
Priming doses of heat-killed Salmonella typhimurium, Listeria
monocytogenes, and Mycobacterium marinum did not elicit a
protective effect against a subsequent lethal challenge of
the same bacteria used in the priming dose (Figures 2A and
S2). Dead bacteria were used as priming doses in these
experiments because all of these bacteria have an LD50of one
bacterium. Protection by priming is thus not a general
characteristic of all microbial challenges in the fly. However, a
priming dose of the natural fungal pathogen, Beauveria
bassiana , conferred a protective effect against a subse-
quent lethal challenge (Figures 2A and S2).
Perhaps S. pneumoniae is a uniquely powerful immune
activator; priming with S. pneumoniae might protect against
challenges with other pathogens. To test this hypothesis, we
challenged S. pneumoniae–primed flies with lethal doses of the
same panel of microbes as above. S. pneumoniae–primed flies
were not protected against lethal challenges of other
pathogens (Figures 2A and S2). The protective effect of the
primed response thus is not due to general activation of the
Drosophila immune response.
Having seen that the priming-induced protective response
was specific in the sense that S. pneumoniae was incapable of
protecting against other immune challenges, we next deter-
mined whether other immune activators were capable of
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Priming in the Drosophila Immune Response
Due to the common practice of vaccination and prominence of
AIDS, people are already aware of the distinction between adaptive
and innate immunity without realizing it. All organisms have an
innate immune response, but only vertebrates possess T cells and
the ability to produce antibodies. It has been a long-standing
assumption that invertebrate immune systems are not adaptive and
respond identically to multiple challenges. In this study, we
demonstrate that the fly innate immune response adapts to
repeated challenges; flies preinoculated with dead Streptococcus
pneumoniae are protected against a second, otherwise-lethal dose.
Although the underlying mechanisms are likely to be very different,
this primed response is reminiscent to vaccine-induced protection in
that it exhibits coarse specificity (dead S. pneumoniae only protects
against itself), persists for the life of the fly and is dependent on
phagocytic cells. This result prompts the obvious question of
whether the innate immune system of vertebrates shares a similar
biology. Such a finding is of particular interest since immunocom-
promised individuals only possess an innate immune system.
inducing a protective response against S. pneumoniae. To test
this, we primed flies with a mixture of strong immune
activators (dead Escherichia coli, dead Micrococcus luteus, and
dead Beauveria bassiana) [17,19–21]. Flies injected with this
mixture were not protected against a lethal challenge of
S. pneumoniae (Figure 2B). Furthermore, injection with this
mixture does not interfere with the ability to induce a
protective response because the addition of dead S. pneumo-
niae to the mixture protected the flies from a second lethal
challenge (Figure 2B). These results also demonstrate that a
priming dose of B. bassiana does not protect against a lethal
dose of S. pneumoniae. We conclude that protection conferred
by a priming dose of S. pneumoniae specifically protects against
lethal doses of S. pneumoniae and persists for the life of the fly.
The Toll Pathway, but Not the imd Pathway, Is Required
for the Primed Response to S. pneumoniae
Toll and imd are the best-characterized fly immunity
pathways that control the majority of genes found to be
induced by bacterial and fungal infections, including AMPs
[1,2,17,19–21]. The mixture of microbes used in Figure 2B was
chosen to strongly activate both the Toll and imd pathways
Figure 1. Protection from a Priming Dose of S. pneumoniae Persists for the Life of the Fly
(A) Survival curves of flies injected with PBS (triangles, n¼60) or 250 (circles, n¼60) or 3,500 (squares, n¼65) CFU S. pneumoniae. p , 0.001, comparing
3,500 CFU to the other treatments (log-rank analysis).
(B) Flies were injected with 250 (circles) or 3,500 (squares) CFU of S. pneumoniae. Bars represent geometric means of bacterial load with 95% confidence
(C) Flies were primed on day 0 with PBS (circles), 250 CFU of S. pneumoniae (triangles), or dead S. pneumoniae (squares). One week later, flies were
injected again with either PBS (open shapes) or 3,000 CFU S. pneumoniae (filled shapes). Naı ¨ve PBS-injected flies challenged with 3,000 CFU of
S. pneumoniae die significantly faster than flies primed with 250 CFU of S. pneumoniae or dead S. pneumoniae (p , 0.0001, log-rank test). Dotted lines
correspond to double injection controls. n ¼ 158 to 228 for each condition.
(D) Flies were injected with PBS (circles) or dead S. pneumoniae (squares) on day 0 and challenged 1 wk later with 400 CFU of S. pneumoniae. Bars
represent geometric means of bacterial load with 95% confidence intervals.
(E) Flies were primed on day 0 with PBS (open bars) or dead S. pneumoniae (filled bars) and challenged with 5,000 CFU of S. pneumoniae on the
indicated days. Mean survival with standard error values are plotted. n ¼ 37 to 49 for each condition. At each time point, the survival curves differ
significantly. See Figure S1 for log-rank analysis and individual survival curves.
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Priming in the Drosophila Immune Response
[19,20]. Although activation of Toll and imd signaling is
insufficient to induce a protective response, it remained
possible that the pathways are necessary for protection. We
tested loss-of-function mutants in each pathway to determine
whether they were necessary for a protective response.
Because both Toll and imd pathway mutants are immuno-
compromised with respect to S. pneumoniae (Figure S3), we
reduced the lethal challenge dose of S. pneumoniae (20 CFU for
Toll mutants, 100 CFU for imd mutants). These doses are
normally sublethal to wild-type flies, but higher doses killed
the mutant flies too quickly to detect a difference in survival.
Loss-of-function mutants of imd (Figure 3) and dTak1 
(unpublished data) were protected by a priming dose of dead
S. pneumoniae. Thus, although the imd pathway normally
contributes to killing S. pneumoniae, it is not necessary to elicit
a protective response. In contrast, flies homozygous for
Figure 3. The Toll Pathway Is Required for the Primed Response
Partial loss-of-function alleles of PGRP-SA (circles) and imd (triangles) were injected with PBS (open shapes) or dead S. pneumoniae (filled shapes) on day
0 and challenged 1 wk later with a lethal dose of S. pneumoniae (20 CFU for PGRP-SAsemland 100 CFU for imd10191). Log-rank analysis of the survival
curves indicates that naı ¨ve and S. pneumoniae–primed PGRP-SAsemlflies die at the same rate, whereas the curves for naı ¨ve and S. pneumoniae–primed
imd10191flies are significantly different (p , 0.0001). n ¼ 154 to 245 for each condition. Molecular information for imd10191is given in Materials and
Figure 2. The S. pneumoniae–Induced Primed Response Is Specific for S. pneumoniae
(A) Flies were injected with a priming dose of PBS (white bars), the same bacteria used for the lethal challenge (gray bars), or S. pneumoniae (black bars).
Bacteria used for lethal challenges are indicated below the graph. Mean survival with standard error values are plotted. n¼57 to 128 for each condition.
p , 0.001 for the indicated set of bars (log-rank test). See Figure S2 for individual survival curves and log-rank analysis.
(B) Flies were primed on day 0 with PBS (circles, n¼59), dead S. pneumoniae (squares, n¼60), mixture 1 (dead E. coli, M. luteus, and B. bassiana; triangles,
n¼59), or mixture 2 (dead E. coli, M. luteus, B. bassiana, and S. pneumoniae; triangles, n¼60) and challenged 1 wk later with 3,000 CFU of S. pneumoniae.
Log-rank analysis indicates that curves corresponding to PBS and mixture 1 (dotted lines) are significantly different from those for flies primed with
S. pneumoniae or mixture 2 (solid lines) (p , 0.001).
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Priming in the Drosophila Immune Response
partial loss-of-function mutations that disrupt the Toll
pathway, PGRP-SA [23,24] (Figure 3) and Dif  (unpub-
lished data), were not protected by a priming dose of dead
S. pneumoniae. The Toll pathway is therefore necessary for the
primed response, but the imd pathway is not required.
Phagocytes Are the Critical Effectors of the Primed
Next, we wanted to determine the contribution of melani-
zation, the humoral response, and the cellular response to
priming (Figure 4A) [1,2]. We do not observe melanization in
Figure 4. S. pneumoniae–Primed Flies Exhibit an Enhanced Phagocytic Response That Is Specific to S. pneumoniae
(A) Model for S. pneumoniae–induced primed response.
(B and C) RNA was extracted from whole flies. Defensin transcript levels were quantified using qRT-PCR and normalized to 0-h media injection. Bars
represent mean values with standard deviation. See Figures S4 and S5 for diptericin and attacin transcript levels. (B) Flies were injected with media
(open bars), 250 CFU of S. pneumoniae (gray bars), or 250 CFU of E. coli (black bars). (C) Flies were primed on day 0 with media (white bars) or dead
S. pneumoniae (gray bars) and challenged 1 wk later with media, 3,500 CFU of S. pneumoniae, or 3,500 CFU of E. coli (indicated above the graph).
(D) Flies were injected with polystyrene beads (triangles, n¼122) or water (circles, n¼115) 3 d prior to day 0 to fully inhibit phagocytosis. On day 0, flies
were injected with 20 CFU of S. pneumoniae. Bead-inhibited flies die significantly faster than do mock-injected flies (p , 0.0001, log-rank analysis of
(E) Flies were injected with polystyrene beads (circles) or water (squares) on day 0, injected with a priming dose of PBS (open shapes) or S. pneumoniae
(filled shapes) on day 3, and injected with a lethal dose of 1,000 CFU on day 10. Log-rank analysis of the survival curves indicates that all flies died
significantly faster than did mock-treated S. pneumoniae–primed flies (p , 0.0001). n ¼ 51 to 62 for each condition.
(F) Flies were injected with PBS (circles) or dead S. pneumoniae (squares) on day 0 and challenged 1 wk later with 6,000 CFU of E. coli. Bars represent
geometric means of bacterial load with 95% confidence intervals.
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Priming in the Drosophila Immune Response
response to S. pneumoniae infections and therefore do not
expect that it plays a strong role in the protective response
(unpublished data) . Because the Toll pathway is required
for the priming-induced protection, we first examined the
contribution of the inducible humoral response.
We found four lines of evidence suggesting that AMPs are
not responsible for protecting flies against a second lethal
challenge of S. pneumoniae. First, S. pneumoniae was not a strong
AMP inducer because the peak of AMP transcription in
response to S. pneumoniae was not as high as the peak induced
by the positive control elicitor E. coli . Flies were injected
with a priming dose of dead S. pneumoniae, media (wounding
control), or E. coli (positive control), and quantitative real-time
reverse transcription–PCR (qRT-PCR) was used to assess
transcript levels of three different AMPs: defensin (Figure
4B), attacin (Figure S4A), and diptericin (Figure S4B) .
These AMPs were chosen because they are strongly induced by
Gram-positive bacterial infections. Second, we found that not
only was S. pneumoniae a poor inducer of AMPs but also AMP
transcript levels did not remain elevated 1 wk later (Figure 4B).
Thus, AMP transcription should be back to the ground state by
the time the challenge dose is administered, 1 wk after the
priming dose. It has been reported that AMPs can persist in the
hemolymph . However, we have shown that simultaneous
activation of Toll and imd, and thus AMP induction, is not
sufficient to protect the fly (Figure 2B). Finally, we asked if this
ground state is ‘‘sensitized’’—that is, whether AMP induction is
enhanced in flies that have been primed with S. pneumoniae
compared to media. Using qRT-PCR, we measured defensin
(Figure 4C), attacin (Figure S5A), and diptericin (Figure S5B)
transcript levelsaftera secondchallenge ofS.pneumoniae, media
(wounding control), or E. coli (positive control). None of the
AMPs were differentially induced in S. pneumoniae–primed flies
compared to naı ¨ve media-injected flies. Thus, we find no
evidence to support the involvement of AMP induction in the
primed immune response.
In light of these data, and the fact that hemocytes are
altered in Toll pathway mutants [12,13], we examined
whether the cellular immune response is the main effector
of the primed response—that is, a priming dose of
S. pneumoniae might specifically increase S. pneumoniae clear-
ance by phagocytes upon a second exposure. We first assessed
the contribution of phagocytosis to S. pneumoniae killing in
naı ¨ve flies by injecting flies with polystyrene beads to block
phagocytosis prior to infection . Bead-inhibited flies were
extremely sensitive to S. pneumoniae; 3,000 bacteria were
required to kill a wild-type fly, whereas 20 CFU was sufficient
to kill a wild-type fly that lacks phagocytosis (Figure 4D). We
demonstrated above that the survival difference between
S. pneumoniae–primed flies and naı ¨ve flies is linked to
enhanced clearance (Figure 1D); here we show that phag-
ocytosis is required to kill S. pneumoniae in naı ¨ve flies.
We then asked if the enhanced clearance in primed flies is
due to increased killing by phagocytes and not a second,
mechanistically, different method of killing. To test this, we
inhibited phagocytosis in both primed and naı ¨ve flies and then
challenged with a lethal dose of S. pneumoniae 1 wk later, at
which time point phagocytosis remained inhibited . Primed
flies died at the same time as naı ¨ve flies and were therefore not
protected by a priming dose of S. pneumoniae, regardless of
whether they were injected with beads before (Figure 4E) or
after (unpublished data) the priming dose. Fly phagocytes are
therefore an essential effector of the primed response.
These data suggested that a priming dose of S. pneumoniae
activates fly phagocytes to kill S. pneumoniae more efficiently. Is
this enhanced killing specific to S. pneumoniae, or does a
priming dose of S. pneumoniae simply cause general phagocyte
activation? If phagocytes are generally more activated in
S. pneumoniae–primed flies, these flies should be able to clear
other bacteria more rapidly. To test this hypothesis,
S. pneumoniae–primed flies were tested for their ability to kill
E. coli (Figure 4F). There was no difference between naı ¨ve PBS-
injected and S. pneumoniae–primed flies in their ability to clear
E. coli. Combined with the fact that a priming dose of
S. pneumoniae does not offer protection against any other
lethal challenges of bacteria (Figure 2A) and the fact that the
primed response is specific for S. pneumoniae (Figure 2), we
conclude that a priming dose of S. pneumoniae alters the fly
immune system in a persistent manner that specifically allows
phagocytes to recognize and kill S. pneumoniae more efficiently.
We have presented evidence that the fly modulates its
immune response as a result of multiple challenges: a priming
dose of S. pneumoniae is sufficient to protect the fly against a
subsequent lethal dose of S. pneumoniae. Using a functional
immune assay, we have shown that the fly immune system
exhibits the adaptive characteristics of specificity and
persistence. The mechanism underlying this protective
response requires the Toll pathway, although its contribution
is not via activation of AMPs. We have eliminated contribu-
tions from the imd pathway and AMPs and identified
phagocytes as the critical effectors of the primed response.
This system is uniquely positioned to further characterize the
molecular basis underlying specific phagocyte activation and
other adaptive aspects of innate immunity.
Materials and Methods
Fly stocks. Fliesweremaintainedonstandarddextrosemediumat258C
R background to limit background effects. In particular, white mutant flies
are very sensitive to S. pneumoniae and do not elicit a primed response
(unpublished data). Mutant lines used in this study include PGRP-SAseml
imd10191is included below.
Molecular information on imd10191. The imd10191line has a 26-
nucleotide deletion that frameshifts the protein at amino acid 179,
which is the beginning of the death domain.
Microbial strains and culture. Microbial strains used in this study
include S. pneumoniae strain SP1, E. coli DH5a, M. luteus, B. bassiana,
L. monocytogenes strain 10403S, S. typhimurium strain SL1344, and M.
marinum strain M. S. pneumoniae cultures were grown standing at 37 8C
5% CO2in brain heart infusion broth (BHI) (BD Bioscience, http://
www.bdbioscience.com) to an OD600of 0.15, and aliquots were frozen
at?808C in 10% glycerol.Forinfection,an aliquotofS. pneumoniaewas
thawed, diluted 1:3 in fresh BHI, and allowed to adjust for 2 h at 37 8C
5% CO2. E. coli,S. typhimurium, and L. monocytogenes cultures weregrown
standing overnight in BHI at 37 8C. M. luteus was cultured standing at
29 8C in BHI for 1 wk or until a sufficient density was reached.
M. marinum was cultured standing at 29 8C in Middlebrook 7H9 broth
(BD Bioscience) supplemented with Middlebrook OADC (BD Bio-
science) and 0.2% Tween. B. bassiana spores were grown on malt agar
(BD Bioscience) at 29 8C for 2 wk or until a sufficient density was
Fly injections. For injection, flies were anesthetized with CO2and
injected with a total volume of 50 nl using individually calibrated
pulled glass needles attached to a Picospritzer III injector (Parker
Hannifin, http://www.parker.com). Flies were always injected in the
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Priming in the Drosophila Immune Response
abdomen, close to the junction with the thorax and just ventral to the
junction between the ventral and dorsal cuticles. Flies were never
anesthetized for longer than 10 min. After each injection, all flies were
transferred to a new vial and maintained at 29 8C and 65% humidity.
Priming doses of microbes. To prepare priming doses of microbes,
concentrated cultures were boiled for 30 min, centrifuged 5 min at
2,000g, and washed three times in PBS. Bacterial cultures were diluted
in PBS to an OD600of 0.1 and stored at?80 8C. Heat-killed B. bassiana
spores were counted on a hemocytometer, adjusted to a concen-
tration of 1 3 107/ml in PBS, and stored at ?80 8C. Aliquots were
plated on the appropriate media to verify that the microbes had been
heat-killed. To prime flies, 50 nl of these solutions was injected into
the fly. Flies were incubated at 29 8C and 65% humidity until they
received their second challenge.
Lethal challenges of microbes. Fresh cultures were washed three
times in PBS and diluted to the appropriate OD600in PBS. For the
different concentrations of S. pneumoniae, appropriate bacterial load
corresponding to different optical densities was experimentally
determined. For reference, an OD600of 0.1 corresponds to 3,000
CFU. Lethal doses of other bacterial species are as follows:
S. typhimurium, OD 0.1 (10,000 CFU); L. monocytogenes. OD 0.01 (6,500
CFU); and M. marinum, OD 0.05 (500 CFU). Bacterial load after
injection was verified for all strains except M. marinum by plating on
the appropriate media (blood agar for S. pneumoniae, BHI agar for all
other strains). For lethal B. bassiana challenges, flies were anesthetized
in groups of 20 and shaken on a plate of spores for exactly 30 s. All
infections were carried out at 29 8C.
Determination of CFU in flies. Individual flies were homogenized
in 100 ll of PBS, diluted serially, and spotted onto appropriate plates.
S. pneumoniae were grown on blood agar supplemented with 500 lg/ml
streptomycin (Sigma, http://www.sigmaaldrich.com), 10 lg/ml colistin
(Sigma), and 5 lg/ml oxolinic acid (Sigma) to eliminate the growth of
bacterial contaminants from the fly. Plates were incubated overnight
at 37 8C 5% CO2. E. coli colonies were grown on LB and incubated
overnight at 37 8C.
29 8C for the indicated time points. At the given times, triplicates of
three flies were anesthetized, placed in 1.5-ml tubes, and homogenized
extracted using the standard Trizol-LS protocol, and remaining
genomic DNA was degraded with DNase I treatment. RT-PCR was
carried out with a Bio-Rad iCycler (http://www.bio-rad.com) using
TaqMan probes and rTth polymerase (Perkin-Elmer, http://www.
perkinelmer.com) as directed by the manufacturer. The following
primers below were used. Relative RNA quantities were determined
with respect to Drosophila ribosomal protein 15a, and all levels were
normalized with respect to the zero time point for media injection:
defensin TTCTCGTGGCTATCGCTTTT (left primer), GGAGAGTA-
GGTCGCATGTGG (right primer), AGGATCATGTCCTGGTGCAT-
primer), ATTCCTGGGAAGTTGCTGTG (right primer), AATGGTTT-
CGAGTTCCAGCGGAATG (Taqman probe); diptericin ACCGCAG-
TACCCACTCAATC (left primer), CCCAAGTGCTGTCCATATCC
(right primer), CAGTCCAGGGTCACCAGAAGGTGTG (Taqman
probe); and ribosomal protein 15a TGGACCACGAGGAGGCTAGG
(left primer), GTTGGTTGCAT-GGTCGGTGA (right primer),
TGGGAGGCAAAATTCTCGGCTTC (Taqman probe).
Bead injections. Carboxylate-modified blue fluorescent 0.2-lm-
diameter polystyrene beads (Molecular Probes, http://www.invitrogen.
com) were injected to block phagocytosis essentially as previously
described . Briefly, beads were washed twice in sterile water and
resuspended in one fourth of the original volume. Flies were injected
with 50 nl of bead solution or water as an injection control. To
confirm that phagocytosis was inhibited, the in vivo phagocytosis
assay was performed as described previously with FITC-conjugated
E. coli or FITC-conjugated Staphylococcus aureus. Phagocytic inhibition
was confirmed each time bead-injected flies were manipulated.
Data analysis. All experiments were performed at least three times.
For survival analysis, a minimum of 45 flies were injected for each
condition. Dead flies were counted daily, and survival data were
graphed and analyzed using GraphPad Prism (GraphPad Software,
http://www.graphpad.com). Mean survival time with standard error
was calculated using R (http://www.r-project.org).
Figure S1. S. pneumoniae–Induced Protection Persists for the Life of
Flies were primed on day 0 with PBS (open circles) or dead
S. pneumoniae (filled squares). Flies were challenged with 5,000 CFU
on the indicated days, and log-rank analysis was performed. A
representative experiment (of three replicates) is shown. (A) Day 1, p
, 0.0001; (B) day 3, p ¼ 0.0038; (C) day 7, p , 0.0001; (D) day 10, p ¼
0.0037; (E) day 14, p ¼ 0.0003.
Found at doi:10.1371/journal.ppat.0030026.sg001 (89 KB PDF).
Figure S2. Specific Protection Elicited by Priming Doses of
S. pneumoniae and B. bassiana
Flies were injected with a priming dose of PBS (open circles), the
same bacteria used for the lethal challenge (triangles) or S. pneumoniae
(boxes). One week later, flies were injected with the lethal challenge
listed, and log-rank analysis was performed (statistics shown below
the graph; ns¼not significant). A representative experiment (of three
replicates) is shown. (A) S. typhimurium; (B) L. monocytogenes; (C)
M. marinum; (D) B. bassiana; (E) S. pneumoniae.
Found at doi:10.1371/journal.ppat.0030026.sg002 (154 KB PDF).
Figure S3. The Toll and imd Pathways Contribute to S. pneumoniae
Wild-type (squares, 5,000 CFU, n ¼ 184) and partial loss-of-function
alleles of imd (triangles, 100 CFU, n ¼ 180) and PGRP-SA (circles, 20
CFU, n ¼ 177) were injected with lethal doses of S. pneumoniae. The
curves corresponding to imd and PGRP-SA differ significantly from
wild-type at p , 0.0001 (log-rank analysis).
Found at doi:10.1371/journal.ppat.0030026.sg003 (20 KB PDF).
Figure S4. AMP Transcription Is Not Strongly Induced by
S. pneumoniae and Does Not Persist for 1 wk
Flies were injected with media (open bars), 250 CFU of S. pneumoniae
(gray bars), or 250 CFU of E. coli (black bars). RNA was extracted from
whole flies. Attacin (A) and diptericin (B) transcript levels were
quantified using qRT-PCR and normalized to 0-h media injection.
Bars represent mean values with standard deviation.
Found at doi:10.1371/journal.ppat.0030026.sg004 (185 KB PDF).
Figure S5. AMPs Are Not Differentially Induced in S. pneumoniae–
Flies were injected with a priming dose of media (white bars) or dead
S. pneumoniae (black bars) and challenged 1 wk later with media, 3,500
CFU of S. pneumoniae, or 3,500 CFU of E. coli (indicated above the
graph). RNA was extracted from whole flies. Attacin (A) and
diptericin (B) transcript levels were quantified using qRT-PCR and
normalized to 0-h media injection. Bars represent mean values with
Found at doi:10.1371/journal.ppat.0030026.sg005 (223 KB PDF).
We thank E. A. Joyce for bacterial strains, P. Ligoxygakis for fly
strains, and J. D. Dunn, J. P. Boyle, J. A. Hammond, and members of
the Schneider lab for comments.
Author contributions. MSD conducted initial infection experi-
ments with S. pneumoniae. LNP conceived the priming experiments
and performed all experiments in the figures. LNP, MSD, MSH, and
DSS wrote the paper. All authors discussed the results and
commented on the manuscript.
Funding. This work was supported by grants R01AI053080 and
NIH A1055651 from the National Institutes of Health (DSS and MSH)
and grants from the Irvington Institute (MSD), and the National
Science Foundation (LNP).
Competing interests. The authors have declared that no competing
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Priming in the Drosophila Immune Response