The Role of Anorexia in Resistance and Tolerance to
Infections in Drosophila
Janelle S. Ayres¤, David S. Schneider*
Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America
Most infections induce anorexia but its function, if any, remains unclear. Because this response is common among animals,
we hypothesized that infection-induced diet restriction might be an adaptive trait that modulates the host’s ability to fight
infection. Two defense strategies protect hosts against infections: resistance, which is the ability to control pathogen levels,
and tolerance, which helps the host endure infection-induced pathology. Here we show that infected fruit flies become
anorexic and that diet restriction alters defenses, increasing the fly’s tolerance to Salmonella typhimurium infections while
decreasing resistance to Listeria monocytogenes. This suggests that attempts to extend lifespan through diet restriction or
the manipulation of pathways mimicking this process will have complicated effects on a host’s ability to fight infections.
Citation: Ayres JS, Schneider DS (2009) The Role of Anorexia in Resistance and Tolerance to Infections in Drosophila. PLoS Biol 7(7): e1000150. doi:10.1371/
Academic Editor: Daniel Promislow, University of Georgia, United States of America
Received February 26, 2009; Accepted June 4, 2009; Published July 14, 2009
Copyright: ? 2009 Ayres, Schneider. 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.
Funding: This work was funded by a National Science Foundation Graduate Research Fellowship, National Institutes of Health grant 1RO1AI069164, and an
Ellison Medical Foundation Senior Scholar’s Award in Aging. 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.
* E-mail: email@example.com
¤ Current address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America
Infectious diseases are predicted to drive the natural selection of
behaviors that increase fitness. Loss of appetite (anorexia) is a
common behavior that sick animals exhibit when faced with an
immune challenge [1–5]. Traditionally, anorexia was thought of as
an adverse secondary response to infection that served no function
to the host; immune responses are energetically expensive and thus
an infection-induced reduction in food intake seems paradoxical
[1–3]. Since this phenomenon occurs in so many animals,
including both vertebrates and invertebrates, an alternative
explanation is that this response is a conserved adaptive strategy
to increase the chance of surviving an infection [1–3]. Exper-
imental evidence from anorexia and acute starvation studies are
consistent with this notion and suggest that this behavioral change
is actively induced by the host during infection and is
advantageous [3,6–9]. For example, mice infected with L.
monocytogenes (a firmicute and facultative intracellular pathogen)
that were fed ad libitum had increased survival when compared to
similarly infected but force fed mice . The mechanism behind
these changes in survival is unknown.
Diet restriction is a common method used for increasing an
animal’s lifespan. One explanation for this is that diet restriction
increases responses required to survive stress . Much progress
has been made in determining the signaling pathways that trigger
this process but the effector mechanisms remain elusive . Most
diet restriction experiments are done in the lab and a side effect of
this is that the tested organisms are not exposed to a normal range
of pathogens; thus, we do not have a deep understanding about
how diet restriction can affect immune defenses. Individual
immunological indicators of a potential immune response often
improve upon diet restriction [1,4,11]. However, in the few cases
where diet restricted animals have been given an infectious
challenge, the diet restricted host often fared poorly, in spite of
molecular indicators that its immune system would prevail [12–
16]. Thus there seems to be a disconnect between the potential
and realized immune response in diet restricted animals,
suggesting that we are not measuring the relevant parts of the
Hosts can evolve two ways of defending themselves against
infections [17–19]. The first, resistance, is the ability of the host to
reduce pathogen levels. The second, tolerance, is the ability to
limit the impact of infections. The theoretical basis for this
distinction is grounded on work in plants, but recent work, from a
number of groups, demonstrated that animals can also vary in
their tolerance. In animals, tolerance traits appear relatively
common and simple to identify genetically [17,20–22]. For
example, when screening for mutant flies with altered sensitivity
to L. monocytogenes, we found that one-third of the mutants we
recovered had no apparent defects in resistance and succumbed to
infection because of defects in tolerance. At least when studying
Drosophila, it seems clear that much has been missed in our studies
of immunity by focusing on resistance mechanisms and ignoring
Our work here was provoked by our identification of a mutation
in a fly gustatory receptor, gr28b, that altered immune defenses
. We found that gr28b mutant flies had reduced appetites.
This led us to the hypothesis that the feeding changes induced by
anorexia might alter the immune response in an adaptive manner.
We found that L. monocytogenes and S. typhimurium (a gamma
proteobacterium and intracellular pathogen), both induce anorex-
ia in infected flies, suggesting that diet restriction can be a normal
part of the fly’s response to infection. Mimicking this diet
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restriction by testing the gr28b mutant or by feeding the flies
diluted food, we found that diet restriction reduced resistance
against L. monocytogenes but increased tolerance against S.
typhimurium. We propose that the degree of anorexia that is
exhibited by an infected fly and the changes this decrease in food
consumption imposes on the immune response will be continu-
ously shaped by the pathogens a fly encounters in the wild.
The innate defenses important for resistance can be divided in
three: the humoral, cellular, and melanization responses [23,24].
The humoral response is the most deeply characterized and
involves the secretion of antimicrobial peptides into the hemo-
lymph (circulating ‘‘blood’’) of the fly. Antimicrobial peptide
transcript levels are regulated by the Toll and Imd pattern
recognition pathways and these peptides are secreted predomi-
nantly by the fat body into the circulation of the fly and kill
invading microbes . Flies with mutations blocking the
activation of these pathways quickly succumb to infections and
have higher bacterial loads than do wild-type flies, suggesting that
the principal defect in these mutant flies is in resistance [24–30].
The humoral response is induced over the course of several hours
following a systemic infection. The cellular response is an
immediate acting response and involves hemocytes (fly ‘‘blood’’
cells), which phagocytose small particles, encapsulate large
particles, and secrete antimicrobial compounds [23,24]. Melani-
zation is a second immediate immune response in the fly occurring
at sites of tissue damage and infection. Melanin deposits are visible
as dark brown patches at these sites and its synthesis requires the
proteolytic activation of the enzyme phenoloxidase. Reactive
oxygen species are produced as a byproduct of this response that
can cause damage to the fly thus affecting tolerance in addition to
We examined the three arms of the fly immune response for
changes caused by anorexia and diet restriction and found that
melanization drops drastically upon diet restriction; the pattern of
antimicrobial peptides induced during infection changes; but there
were no apparent change in phagocytosis. The changes in
melanization alone can explain the loss of resistance to L.
monocytogenes. The explanation for the increase in tolerance to S.
typhimurium is more complicated because the loss of melanization is
expected to decrease resistance. We suggest that the fly
compensates with another resistance mechanism, possibly antimi-
crobial peptides, while at the same time increasing tolerance. This
work suggests that diet restriction will have complicated effects on
immune defenses as it can alter both resistance and tolerance and
its effects are microbe specific. This work supports the idea that the
environment does not just affect a fly’s immune response but
rather is an integral part of immunity.
Infection-Induced Anorexia in Drosophila
We measured infection-induced feeding changes in adult
Drosophila challenged with three different bacterial pathogens of
humans and Drosophila, L. monocytogenes, S. typhimurium, and
Enterococcus faecalis (a firmicute and extracellular fly pathogen)
(Figure 1) [18,23,31–33]. We chose these microbes because they
represent very different types of bacteria and cause well
characterized lethal infections in the fly; lethal microbes let us
measure both increases and decreases in survival rates whereas
nonpathogens only allow us to measure decreases. Feeding rates
were determined by measuring how quickly flies took a meal when
presented with new food and by recording how much food they
consumed during this meal. The feeding rate assays were used
primarily to determine the appropriate time window to perform
the less subjective consumption assays. L. monocytogenes and S.
typhimurium infections reduced food intake in both assays compared
to unmanipulated and media-injected controls. By contrast, we
detected no effect of E. faecalis infection on either feeding assay,
demonstrating that illness-induced anorexia occurs in the fly in a
microbe dependent manner. Dead L. monocytogenes also induced
anorexia, suggesting that a simple immune response and not an
active infection is sufficient to reduce the fly’s appetite (Figure 1;
Tables S1, S2, S3). Together, these results demonstrate that flies
may enter a state of diet restriction when infected.
gr28b Mutants Are Constitutively Anorexic
We sought to determine how immune-induced diet restriction
might alter the resistance and tolerance of the fly to a variety of
pathogens. We previously identified a mutation in a taste receptor
(gr28b) that reduced flies’ resistance to L. monocytogenes infection
while increasing defenses for S. typhimurium  and show here that
these mutant flies eat less than wild-type controls (Figure 2; Table
S4). We measured the feeding rates and ingestion volume of gr28b
mutants and found that they ate at a significantly reduced rate
compared to wild-type flies and that their ingestion volume was
also reduced (Figure 2). These mutant flies also lived longer than
parental controls when left unmanipulated (Figure S1) as would be
expected for diet restricted flies . Thus the gr28b mutant
pointed to a potential functional link between anorexia and an
altered immune response and provided a simple method of
creating a constitutively anorexic fly.
Anorexia and Diet Restriction Alter the Realized Immune
To determine how anorexia affects the realized immune
response of flies, we measured the survivorship of infected gr28b
mutants and compared these rates to those of infected wild-type
flies (Figure 3). Consistent with what we had observed previously
, we found that when infected with L. monocytogenes, gr28b
mutants died faster than wild-type flies; mutant flies died with a
median time to death (MTD) of 4 d compared to 6–7 d for wild-
type flies. In contrast, when infected with S. typhimurium, gr28b
mutants lived longer than wild-type flies with a MTD of 15 d
compared to 8 d. When infected with E. faecalis, gr28b and wild-
type flies died at the same rate. Thus, gr28b flies have altered
Two routes to decreasing susceptibility to infection are
resistance (the ability to clear pathogens) and tolerance
(the ability to limit damage in response to pathogens).
Anorexia induced by sickness puts animals into a diet-
restricted state, a state that is generally believed to extend
lifespan. We asked whether anorexia induced by sickness
would alter the immune response. We measured the
effects of diet restriction on both resistance and tolerance
to two different infections in the fruit fly, Drosophila
melanogaster. In one case we found that infection induced
anorexia and the resulting diet restriction increased
tolerance to this infection, thereby increasing survival of
flies infected with this pathogen; however, this is not a
universal effect. In a second case we found another
pathogen that induced anorexia but here diet restriction
lead to a reduction in resistance that collapsed the
immune response and caused the fly to die faster. The
relationship between diet restriction and immunity is
complicated and must be evaluated on a pathogen-by-
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interactions with microbes but this could be due to the mutant’s
reduced appetite or to pleiotropic effects of the mutation. Anorexia
is a symptom and there are potentially many different ways of
becoming anorexic; thus we wanted to test another method that
would simply restrict food intake.
To test the hypothesis that a reduction in food intake is
responsible for the array of survival phenotypes we observe in
gr28b mutant flies, we measured the effects of diet restriction on
wild-type flies that we raised on standard diet that was diluted with
1% agar so that each stage of their lifecycle was completed on the
diluted food. Typically in diet restriction studies, the introduction
of restricted food occurs at the adult stage. We chose to utilize
adult flies that had been raised their entire life on restricted food
for two reasons: First, we reasoned that because gr28b mutants are
inherently anorexic, they experience reduced food consumption at
all stages of their life and we wanted to better emulate the reduced
food intake of gr28b mutants. Second, for our initial experiments
we used adult flies that had been diet restricted at 24 h prior to
infection and we found that the phenotypes were enhanced as the
amount of time on diet restricted food increased and we chose to
maximize the effect. We infected food-restricted wild-type flies and
compared survivorship to wild-type flies raised on standard food
(Figure 3). Flies fed a 0.56diet had phenotypes similar to the gr28b
mutation in every way tested: diet restricted flies were more
sensitive to L. monocytogenes; less sensitive to S. typhimurium; and
showed no change in sensitivity to E. faecalis. Our results are in
agreement with past observations that diet restriction has no effect
on the survival rate of E. faecalis infected wild-type flies .
Previous studies examining the effects of diet restriction in the fly
have reported neutral or weak positive effects on fly survival for
Pseudomonas aeruginosa (gamma-proteobacterium and extracellular
pathogen). Libert et al. reported that diet restriction has no effect
on survival when challenged with P. aeruginosa. The pathogen load
was not measured in this study and thus it cannot be determined
whether there were compensatory changes in resistance and
tolerance . Diet restriction was reported to have positive effects
on survival of P. aeruginosa-infected flies in an age dependent
manner, where an increase in survival was seen in flies 30 d old or
older but not 20 d or younger. This result demonstrated that the
life history of a fly is another important factor to consider when
measuring the interactions between diet restriction and immunity
. Pathogen load was not determined in this study and thus it
cannot be determined whether the changes in old flies were due to
changes in resistance or tolerance.
The lifespan of unmanipulated flies raised on a 0.56 diet was
extended, which is in agreement with what has been previously
observed in diet restricted flies and similar to what is seen in gr28b
mutants. As diet restriction produced a complete phenocopy of the
mutant phenotypes, we concluded that gr28b influences fly
immunity by regulating food intake.
Resistance and Tolerance Are Affected by Anorexia
In Drosophila, we can determine whether a fly succumbs to an
infection because of defects in resistance or tolerance mechanisms
by monitoring both fly survival and pathogen growth over the
course of the infection [12,35]. We found that both resistance and
tolerance mechanisms are affected by anorexia and the effect
depended on the type of infection. Both gr28b mutant flies and
diet-restricted wild-type flies exhibited increased growth of L.
monocytogenes during infection (Figure 4). This growth, combined
Figure 1. Effect of infection on appetite. Flies were infected with live or heat killed L. monocytogenes, live S. typhimurium, live E. faecalis, medium
as a control, or left unmanipulated. Feeding was monitored by measuring the rate that flies took a meal (A–C) and the volume that they consumed
during this meal (D–F). Feeding rate measurements: (A) L. monocytogenes 24 h postinfection; (B) S. typhimurium 24 h postinfection; (C) E. faecalis 24 h
postinfection. To measure the volume of food consumed, fed flies were homogenized and the absorbance of an added blue dye was measured 24 h
postinfection. (D) L. monocytogenes, (E) S. typhimurium, (F) E. faecalis. Error bars indicate standard error of the mean. Significance for (A–C) was
assessed using a Fisher’s exact test. Green asterisks represent live bacteria significantly different compared to both unmanipulated and media-
injected flies. Blue asterisks represent dead bacteria (L. monocytogenes only) significantly different than both unmanipulated and media-injected flies.
Black cross represents live bacteria significantly different from unmanipulated flies only. Pink cross represents live bacteria significantly different from
media-injected flies only. Actual p-values are listed in Tables S1, S2, S3. Statistical analysis for (D–F) was done using ANOVA and a Tukey post-test;
black asterisk indicates p,0.01 with respect to both unmanipulated and media-injected flies, black cross indicates p,0.05 with respect to
unmanipulated flies only, and pink cross indicates p,0.05 with respect to media-injected flies only.
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with the increased death rate we observed in both models,
suggested that these flies died because of defects in resistance to L.
monocytogenes; that is, reduced food intake blocked the ability of a fly
to limit L. monocytogenes growth and thus the flies died faster.
Because of the way we measure resistance and tolerance in the fly
we cannot always measure changes in tolerance as microbe levels
are changing; therefore, it is possible tolerance also changes under
food restricted conditions in L. monocytogenes–infected flies. By
contrast, during S. typhimurium infections, food restricted and gr28b
mutants exhibited similar levels of bacteria to what we observed in
wild-type flies (Figure 4) yet they lived longer. This suggested that
resistance was unchanged but tolerance was increased.
A drawback of our diet restriction protocol is that it raises the
caveat that lifelong food limitation has effectson immunitybecause of
developmental changes. To determine whether short-term diet
restriction could produce symptoms similar to those seen in gr28b
flies or flies diet restricted since hatching, we placed flies on diet
L. monocytogenes–infected flies showed significantly decreased survival,
whereas S. typhimurium-infected flies showed increased survival
comparable to that seen in gr28b flies. These experiments support
the idea that diet restriction in adults affects defenses by altering the
fly’s physiology without causing developmental changes.
In summary, diet restriction has varied effects on tolerance and
resistance in the fly: diet restriction causes no change during E.
faecalis infections, reduces resistance to L. monocytogenes, and
increases tolerance to S. typhimurium.
Anorexia Affects Multiple Arms of the Drosophila Innate
To determine the mechanism behind the changes in resistance
and tolerance we observe under diet restriction, we examined the
three resistance mechanisms of the Drosophila innate immune
response that are important for limiting microbial growth:
phagocytosis, antimicrobial peptide production, and melanization
[23,24]. We saw no change in phagocytosis rates in anorexic flies
(Figure S2) but found significant differences in the other two
We measured the levels of antimicrobial peptide transcript levels
in L. monocytogenes–infected gr28b mutants and food-restricted flies
and found that they elicit similar effects (Figure 6; unpublished
data). In gr28b mutants we found that postchallenge transcript
levels for drosomycin and drosocin were significantly reduced
Figure 2. Effect of mutation of the gustatory receptor gr28b on appetite. Isogenic wild-type and gr28b mutants were assayed for feeding
rates (A) and meal volumes (B). Error bars indicate standard error of the mean. Statistical analysis for (A) was done using a Fisher’s exact test. Green
asterisk indicates Listeria-infected gr28b mutants are significantly different than unmanipulated wild-type flies. Blue asterisks indicate that
unmanipulated gr28b mutants are significantly different than unmanipulated wild-type flies. Green crosses indicate that Listeria-infected mutants are
significantly different from unmanipulated wild-type flies only. Actual p-values are listed in Table S4. Statistical analysis for (B) was done using ANOVA
and a Tukey post-test. Black asterisk indicates p,0.001 compared to unmanipulated wild-type flies.
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Figure 3. Effect of mutation of the gustatory receptor gr28b and diet restriction on sensitivity to infections. Isogenic wild-type and
gr28b homozygous mutant flies were challenged with (A) L. monocytogenes (p,0.0001); (B) S. typhimurium (p,0.0001); (C) E. faecalis (p=0.2779); or
(D) medium alone (p,0.0001); and survival rates were measured and compared between flies given the two treatments. Wild-type flies fed on 16and
0.56diets, and were challenged with (E) L. monocytogenes (p,0.0001); (F) S. typhimurium (p,0.0001); (G) E. faecalis (p=0.6053); or (H) medium alone
(p,0.0001) and survival rates were measured. Significance was determined by log-rank test. Effects of gr28b mutations and diet restriction in
unmanipulated flies on lifespan are shown in Figure S1.
Figure 4. Effect of gr28b mutation and diet restriction on the growth of L. monocytogenes and S. typhimurium. Isogenic wild-type and
gr28b homozygous mutant flies or wild-type flies fed on 16and 0.56diets were challenged with pathogens. Because L. monocytogenes infections
showed a change in pathogen levels, half of the infected flies were injected with gentamicin to determine the relative abundance of intracellular and
extracellular bacteria. Wild-type versus gr28b mutants: (A) L. monocytogenes; (B) S. typhimurium; and (C) E. faecalis. Regular food versus diet restriction:
(D) L. monocytogenes; (E) S. typhimurium; (F) E. faecalis. Error bars indicate standard deviation. Statistical analysis was done using an unpaired two-
tailed t-test. One asterisk indicates p,0.01 and two asterisks indicates p,0.005.
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Figure 5. Effects of diet restriction on immunity when introduced post-eclosure. Five to 7-d-old adult males flies were collected and placed
on a restricted diet or left at a 16diet 24 h prior to infection. Survival of (A) L. monocytogenes (0.256compared to 16, p=0.0154); (B) S. typhimurium
(0.56compared to 16, p,0.0001); (C) growth of L. monocytogenes. Because L. monocytogenes infections showed a change in pathogen levels, half of
the infected flies were injected with gentamicin to determine the relative abundance of intracellular and extracellular bacteria. Asterisk indicates
p=0.0173 as determined by an unpaired two-tailed t-test.
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compared to wild-type flies (gr28b, 206). However, anorexia did
not affect all antimicrobial peptide transcripts in the same way;
attacin transcripts were found at higher levels in gr28b flies
(Figure 6), whereas diptericin transcripts showed no consistent
change (unpublished data). The antimicrobial peptide response
has been well-characterized for newly infected but otherwise
healthy flies; We found that three antimicrobial peptides typically
described as being coordinately regulated (attacin, diptericin, and
drosocin) are regulated independently during diet restricted
conditions. We show that the rules governing AMP expression
are variable and depend not only upon the specific immune
challenge but also upon environmental conditions.
To determine if diet restriction affected the melanization
response, we infected flies with L. monocytogenes or S. typhimurium,
both of which elicit a robust disseminated melanization response in
the fly, and examined flies for evidence of melanization (Figure 6)
. We found that approximately 90% of our wild-type flies fed a
standard diet exhibited melanization, whereas less than 10% of
gr28b mutants melanized when infected with either S. typhimurium
or L. monocytogenes. We also observed a significant reduction in
melanization in diet restricted wild-type flies. Nutrient deprivation
studies in the mosquito and the darkling beetle have also
demonstrated that melanization is reduced under food restricted
conditions [36,37]. These results demonstrate that diet restriction/
anorexia causes down-regulation of infection-induced melaniza-
In wild-type flies, L. monocytogenes establishes an intracellular
infection; in CG3066 fly mutants defective in melanization, we find
an extracellular population of bacteria in addition to the typical
intracellular population . Because diet restriction causes a
reduction in the melanization response, we hypothesized that flies
will also produce an extracellular population of L. monocytogenes
when diet restricted. To test this idea we performed a gentamicin
chase experiment (Figures 4 and 5) . Infected flies were
injected with the antibiotic gentamicin or with water at 0, 24, and
48 h postinfection and surviving bacteria were counted; gentami-
cin kills extracellular bacteria while the intracellular bacteria are
protected. Indeed, gr28b flies and diet restricted flies had a large
extracellular population of L. monocytogenes, in contrast to wild-type
and normally fed flies, which did not. We also observe this effect in
flies that were diet restricted only 24 h prior to infection but the
phenotype is dramatically enhanced in flies that were raised on a
restricted diet (Figures 4 and 5).
The effects of diet restriction on melanization seem easily
interpretable with respect to L. monocytogenes infections but reveal
an exciting complexity with S. typhimurium. Inhibition of melani-
zation in a CG3066 mutant has the same effect on L. monocytogenes
and S. typhimurium infections— a loss of resistance . Therefore,
the loss of melanization in diet restricted flies can explain the entire
L. monocytogenes infection phenotype because the phenotype is the
same as that seen in CG3066 mutants. This is not the case with S.
typhimurium; loss of melanization was anticipated to reduce
resistance to S. typhimurium; instead, we found an increase in
tolerance and no change in resistance. If a resistance mechanism is
lost when melanization is removed because of diet restriction,
some resistance mechanism must replace it to prevent S.
typhimurium growth. In addition, the increase in tolerance in these
flies needs to be explained. One possible explanation is that, in diet
restricted flies, the loss of melanization increases the tolerance to S.
typhimurium infections and the rebalancing of antimicrobial peptide
levels replaces the resistance that would have been lost through the
loss of melanization. More complex explanations require propos-
ing the induction of unknown resistance and tolerance mecha-
Regardless of the effects diet restriction has on individual
resistance mechanisms, the practical outcome of this work is its
demonstration that sensitivity to infections changes in diet
restricted flies. This can benefit the host, as is seen in S. typhimurium
Figure 6. Effect of anorexia and diet restriction on antimicrobial peptide expression and melanization. gr28b mutants and diet
restricted flies were injected with L. monocytogenes, and antimicrobial peptide transcript levels were monitored at 6 h postinfection by quantitative
real-time reverse-transcription PCR. Transcript levels were recorded as the ratio of the antimicrobial peptide transcript divided by a housekeeping
transcript (ribosomal protein 15a) and normalized to 1 for unmanipulated wild-type flies. (A) drosomycin; (B) drosocin; (C) attacin. Error bars report
standard error of the mean. ANOVA and Tukey tests were performed for statistical analysis and asterisks indicate p,0.05. Melanized spots were
recorded in (D) L. monocytogenes and (E) S. typhimurium infections. ANOVA and Tukey test were done for statistical analysis. Asterisks represent
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infected flies or harm the host, as seen in L. monocytogenes infections.
In the field, the effect of an anorexic response to infection on
evolution should depend upon the pathogens to which a
population was exposed. For example, Salmonella-like organisms
should drive an increase in anorexia responses while Listeria-like
pathogens would have the opposite effect.
By highlighting the contribution of feeding to defense, this work
has practical implications for fly immunity experiments. Changes
in nutrition due to food variation could explain week-to-week
alterations in survival curves or plating experiments within a lab.
Similarly, differences in food recipes could explain lab-to-lab
variability. The finding that diet affects both specific antimicro-
bial peptide transcript levels and melanization means that
experiments using these responses as outputs must be interpreted
carefully; for example, when pathogens are fed to flies, test
subjects fed a high dose of bacteria receive a different diet than
flies fed food lacking these microbes and might be expected to
have a different immune response just because the food differs.
Microbe dose may be difficult to regulate when feeding sick flies
if anorexia is induced by the infection; this could lead to
confounding results where mutant flies that do not become
anorexic take larger doses of the infecting microbes than do wild-
type flies. In cases like this, nonanorexic flies could pay for their
dietary indiscretion with their lives. Recent work in a variety of
animals demonstrates that our native microbiota affect our
immune system [38,39]. Certainly some of this comes from the
direct interaction between the microbes and pattern recognition
pathways but native microbiota often play a role in contributing
to host nutrition and metabolism. Therefore, care should be
taken when comparing immune responses between axenic and
normally raised flies; not all immune changes will be due to the
mere physical exposure of the fly to microbes.
This work adds to a growing literature on regulatory
loops linking the fly’s immune responses to the environment
[40–46]. A fly’s susceptibility to infection is altered by
temperature and several insects have behavioral fevers induced
by infection; such fevers can affect the outcome of infections [47–
52]. Immune challenges alter circadian rhythms in flies and this
can feedback to change immunity in ways that can be either
helpful or destructive [43,45,46]. It perhaps came as no surprise
that nutrition affects fly immunity but what we demonstrated
here was that during an immune response, the fly actively alters
its nutrition and, again, this leads to feedback loops that can aid
or collapse the immune response. Recent work on the African
armyworm, Spodoptera exempta, demonstrates that this insect
may not only change its appetite, but also changes its preference
for protein or carbohydrate rich foods during infection .
This raises the possibility that the anorexia response we
measure in flies could be complicated as it is difficult to
distinguish an ‘‘I am not that hungry’’ response from ‘‘yuck, I do
not want to eat this junk,’’ if the flies are presented with just one
food choice. All of this suggests that the fly’s immune response
isn’t merely sensitive to ambient environmental conditions,
rather the fly uses the environment as an integral part of its
Diet restriction can increase the lifespan of animals allowed to
come to a generic ‘‘natural death’’ in the lab . Though the
ultimate mechanisms regulating aging remain unknown, signal-
ing pathways linking diet restriction and aging are emerging as
potential drug targets. Our model provided an opportunity to
measure the effects of a naturally induced diet restriction on
deaths induced by different pathogens. The work reported here
should raise a cautionary flag as it demonstrates that diet
restriction can have complex effects on the realized immune
response of a diet-restricted animal. We must determine how diet
restriction affects realized immune responses in addition to basic
immune effectors and anticipate that this will differ in a
The wild-type parental strain used in all experiments is white1118
(Bloomington stock center, stock 6326). The gr28bc01884allele was
obtained from Bloomington stock center (stock 10743). The piggy
bac line was generated on the white1118
backcrossed further onto the white1118background for four
generations. Flies were kept in standard fly bottles containing
dextrose medium and raised under a 12-h light-dark cycle at 25uC
prior to experiments.
L. monocytogenes strain 10403s  was stored at 280uC in brain-
heart infusion (BHI) broth containing 15% glycerol. S. typhimurium
strain SL1344 and E. faecalis strain V583 were stored at 280uC in
Luria Bertani (LB) medium containing 15% glycerol.
Pathogen Culture Conditions
E. faecalis and S. typhimurium cultures were grown overnight at
37uC in LB medium. E. faecalis cultures were shaken, while S.
typhimurium cultures were grown standing. S. typhimurium cultures
were diluted to OD600of 0.1 with fresh LB medium prior to
injection. E. faecalis cultures were diluted to an OD600of 0.05 with
medium. L. monocytogenes was grown overnight in BHI medium. L.
monocytogenes was grown standing and injected at an OD600of 0.01.
5- to 7-d-old males were used for injection. Flies were
anesthetized with CO2 and injected with 50 nl of culture or
medium using a picospritzer (Parker Hannifin) and pulled glass
needle. Flies were injected in the anterior abdomen on the
ventrolateral surface. Flies were then placed in vials containing
dextrose medium in groups of 20 (or ten for feeding assays) and
incubated at 29uC 65% CO2under a 12-h light-dark cycle. Flies
were injected with 1,000 CFUs of live or dead L. monocytogenes,
10,000 CFUs of S. typhimurium, or 5,000 CFUs of E. faecalis
For each microbe tested, w1118and gr28b mutants were injected
with the microbe or medium as a control. Flies were placed in
dextrose vials in groups of 20 after injection and a total of 60 flies
were assayed for each condition. The number of dead flies was
counted daily. Using Prism software, Kaplan-Meier survival
curves were generated and statistical analysis was done using
log-rank analysis. Survival was tested for each microbe at least
three times and gave similar results for each trial. All survival
experiments were done at 29uC.
CFU Determination and Gentamicin Chase
Infected flies were homogenized in media supplemented with 1%
Triton X-100 and serially diluted. Dilutions were plated on LB agar
plates and incubated over night. The data were plotted as box and
whiskers plots using Graphpad Prism software for three independent
experiments. Using an unpaired two-tailed t-test, the p-value was
with50 nlof1 mg/mlgentamicinorwater3 hpriortohomogenizing
and plating. Flies were incubated at 29uC post infection
Diet Restriction, Immunity, and Lifespan
PLoS Biology | www.plosbiology.org8July 2009 | Volume 7 | Issue 7 | e1000150
Quantitation of Antimicrobial Peptide Transcripts
Total RNA was extracted from infected or control flies that
were incubated at 29uC in groups of five flies using the Qiagen
RNeasy kit (Qiagen) at 0 and 6 h postinjection. The samples were
treated with DNase (Promega). Quantitative real-time RT-PCR
was performed with rTth polymerase (Applied Biosystems) using a
Bio-Rad icycler (Bio-Rad) and the following primer sets:
drosomycin 59 59-gacttgttcgccctcttcg-39, drosomycin 39 59-cttgca-
cacacgacgacag-39, drosomycin Taqman probe 59-tccggaagata-
caagggtccctgtg-39, diptericin 59 59-accgcagtacccactcaatc-39, dipter-
caatctgg-39, attacin 39 59-attcctgggaagttgctgtg-39, attacin Taqman
catcgttttcctgct-39, drosocin 39 59-agcttgagccaggtgatcct-39, drosocin
Taqman probe 59-gtttttgccatggctgtggccact-39. Concentrations of
AMP transcripts were normalized to the expression of the
Drosophila ribosomal protein 15a transcript for each sample .
All experiments were performed with three biological replicates
and each experiment was performed at least three times.
Flies were infected as described above with L. monocytogenes or S.
typhimurium and incubated at 29uC for 4 d. Flies were then
visualized by light microscopy and examined for a disseminated
melanization response. Flies that exhibited melanization beyond
what is observed at the injection site are scored as positive for a
melanization response. Flies that observe no melanization or
melanization only at the site of injection are scored as negative for
a melanization response.
Diet Restriction Assays
All experiments were performed as described above using flies
that were raised on restricted diets. Restricted food was generated
by diluting the standard 16 diet 1:2 or 1:4 in 1% agar water to
generate the 0.56 or 0.256 diet. Vials were placed on a rocker
while food solidified to prevent settling of the food.
Feeding rate and ingestion amount were done using standard fly
dextrose diet supplemented with 0.1% bromophenol blue and
0.5% xylene cyanol [56,57]. Our standard fly food recipe contains
the following chemicals in 1 l of cooked food: 129.4 g dextrose,
7.4 g agar, 61.2 g corn meal, 32.4 g yeast, 2.7 g tegosept. Flies
were injected as described above or were left unmanipulated and
were incubated at 29uC under 12-h light-dark cycle for at least
24 h to allow flies adequate time to recover from CO2treatment
on their standard diet without tracking dye. To measure feeding
rate, flies were transferred to vials containing food with tracking
dye and incubated at room temperature for time point collections.
We chose to keep the flies at room temperature because we found
that the opening and closing of the incubator door at each time
point disturbed feeding activity. Experiments were performed at
the same time of day (2 pm, ZT5). At each time point flies are then
transferred to empty vials that contain no food. At the end of the
time course all flies are examined for the presence of blue dye
inside their bodies and the percentage of flies that ingested a meal
was recorded. For each experimental condition three groups of at
least ten flies were tested for each time point. The average
percentage of flies that ingested a meal was plotted and a Fisher’s
exact test was done for statistical analysis.
To measure ingestion amount at the desired time points at least
three groups of ten flies that have been feeding on the tracking
food were collected and homogenized in 100 ml of 16TE buffer
with 0.1% Triton X-100. 1 ml of 16TE was added and then
homogenates were centrifuged at 14,000 rpm for 3 min. Super-
natants were collected and the absorbance at 614 nm was
measured. The average absorbance for each experimental
condition was recorded and ANOVA and a Tukey test was done
for statistical analysis.
extended lifespans. Five to 7-d-old male flies were placed on
standard dextrose diet or on 0.56concentrated dextrose diet and
incubated at 29uC. The number of dead flies was counted daily
until all flies were dead. Survival curves and median time to death
are presented. Survival rates were analyzed by log-rank analysis.
Found at: doi:10.1371/journal.pbio.1000150.s001 (0.53 MB EPS)
gr28b mutants and diet restricted flies have
unchanged. Wild-type and gr28b mutants were injected with
FITC-labeled Escherichia coli and S. aureus and incubated at room
temperature for 1 h to allow phagocytosis to occur. Flies were then
injected with trypan blue, which quenches any extracellular
fluorescence but intracellular bacteria are protected from the
quenching agent. Flies were visualized by fluorescent microscopy
to look for differences in fluorescence levels.
Found at: doi:10.1371/journal.pbio.1000150.s002 (4.24 MB EPS)
The cellular response in gr28b mutants is
genes infected flies. Flies were infected with live or heat killed
L. monocytogenes, medium as a control or left unmanipulated. Flies
were placed on food supplemented with a blue dye and the rates of
feeding were monitored. A Fisher’s exact test to determine the
significance at each time point was done and the p-values are
Found at: doi:10.1371/journal.pbio.1000150.s003 (0.01 MB XLS)
p-Values for rate of feeding in L. monocyto-
infected flies. Flies were infected with live E. faecalis, medium as
a control, or left unmanipulated. Feeding was monitored by
measuring the rate that flies took a meal. A Fisher’s exact test to
determine the significance at each time point was done and the p-
values are reported here.
Found at: doi:10.1371/journal.pbio.1000150.s004 (0.01 MB XLS)
p-Values for the rate of feeding of E. faecalis
infected flies. Flies were infected with live S. typhimurium,
medium as a control, or left unmanipulated. Feeding was
monitored by measuring the rate that flies took a meal. A Fisher’s
exact test to determine the significance at each time point was
done and the p-values are reported here.
Found at: doi:10.1371/journal.pbio.1000150.s005 (0.01 MB XLS)
p-Values for rate of feeding in S. typhimurium
ulated and L. monocytogenes infected gr28b mutants.
Flies were infected with live L. monocytogenes, or left unmanipulated.
Feeding was monitored by measuring the rate that flies took a
meal. A Fisher’s exact test to determine the significance at each
time point was done and the p-values are reported here.
Found at: doi:10.1371/journal.pbio.1000150.s006 (0.01 MB XLS)
p-Values for the rate of feeding of unmanip-
We thank Stanley Falkow, Michael Simon, Julie Theriot, and all members
of the Schneider lab for helpful discussions. We extend special thanks to
Adam, Baxter and Buster Ayres for their support.
Diet Restriction, Immunity, and Lifespan
PLoS Biology | www.plosbiology.org9July 2009 | Volume 7 | Issue 7 | e1000150
Author Contributions Download full-text
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: JSA DSS.
Performed the experiments: JSA. Analyzed the data: JSA DSS.
Contributed reagents/materials/analysis tools: JSA DSS. Wrote the paper:
1. Hart BL (1988) Biological basis of behavior of sick animals. Neurosci Behav Rev
2. Kyriazakis I, Tolkamp BJ, Hutchings MR (1998) Towards a functional
explanation for the occurrence of anorexia during parasitic infections. Anim
Behav 52: 265–274.
3. Exton MS (1997) Infection-induced anorexia: active host defense strategy.
Appetite 29: 369–383.
4. Dunn PE, Bohnert TJ, Rusell V (1994) Regulation of antimicrobial protein
synthesis following infection and during metamorphosis of Manduca sexta.
Ann N Y Acad Sci 712: 117–130.
5. Adamo SA (2005) Parasitic suppression of feeding in the tobacco hornworm,-
Manduca sexta: parallels with feeding depression after an immune challenge. Arch
Insect Biochem Physiol 60: 185–197.
6. Murray MJ, Murray AB (1979) Anorexia of infection as a mechanism of host
defense. Amer J Clin Nutr 32: 593–596.
7. Alexander JW, Gonce SJ, Miskell PW, Peck MD, Sax H (1989) A new model for
studying nutrition in peritonitis: The adverse effect of overfeeding. Ann Surg
8. Wing EJ, Young JB (1980) Acute starvation protects mice against Listeria
monocytogenes. Infect Immun 28: 771–776.
9. Brown AE, Baumbach J, Cook PE, Lipoxygakis P (2009) Short-term starvation
of immune deficient Drosophila improves survival to gram-negative bacterial
infections. PLoS One 4: e4490. doi:10.1371/journal.pone.0004490.
10. Mair W, Dillin A (2008) Aging and survival: the genetics of life span extension by
dietary restriction. Annu Rev Biochem 77: 727–754.
11. Bedoyan JK, Patil CS, Kyriakides TR, Spence KD (1992) Effect of excess
dietary glucose on growth and immune response on Manduca sexta. J Insect Phys
12. Kristan DM (2007) Chronic calorie restriction increases susceptibility of
laboratory mice (Mus musculus) to a primary intestinal parasite infection. Aging
Cell 6: 817–825.
13. Ritz BW, Aktan I, Nogusa S, Gardener EM (2008) Energy restriction impairs
natural killer cell function and increases the severity of influenza infection in
young adult male C57BL/6 mice. J Nutr 138: 2269–2275.
14. Libert S, Chao Y, Zwiener J, Pletcher SD (2008) Realized immune response is
enhanced in long-lived puc and chico mutants but unaffected by dietary
restriction. Mol Immunol 45: 810–817.
15. Burger JMS, Hwangbo DS, Corby-Harris V, Promislow DEL (2007) The
functional costs and benefits of dietary restriction in Drosophila. Aging Cell 6:
16. Sun D, Muthukumar AR, Lawrence RA, Fernandes G (2001) Effects of calorie
restriction on polymicrobial peritonitis induced by cecum ligation and puncture
in young C57BL/6 mice. Clin Diagn Lab Immunol 8: 1003–1011.
17. Ra ˚berg L, Sim D, Read AF (2007) Disentangling genetic variation for resistance
and tolerance to infectious diseases in animals. Science 318: 812–814.
18. Schneider DS, Ayres JS (2008) Two ways to survive infection: what resistance
and tolerance can teach us about treating infectious diseases. Nat Rev Immunol
19. Read AF, Graham AL, Raberg L (2008) Animal defenses against infectious
agents: is damage control more important than pathogen control. PLoS Biol 6:
20. Corby-Harris V, Habel KE, Ali FG, Promislow DE (2007) Alternative measures
of response to Pseudomonas aeruginosa infection in Drosophila melanogaster. J Evol Biol
21. Kraaijeveld AR, Godfray HC (2008) Selection for resistance to a fungal
pathogen in Drosophila melanogaster. Heredity 100: 400–406.
22. Ayres JS, Freitag N, Schneider DS (2008) Identification of Drosophila mutants
altering defense and endurance of Listeria monocytogenes infection. Genetics
23. Dionne MS, Schneider DS (2008) Host pathogen interactions in Drosophila. Dis
Model Mech 1: 67–68.
24. Brennan CA, Anderson KV (2004) Drosophila: the genetics of innate immune
recognition and response. Annu Rev Immunol 22: 457–483.
25. Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, et al. (1995)
A recessive mutation, immune deficiency (imd), defines two distinct control
pathways in the Drosophila host defense. Proc Natl Acad Sci U S A 92:
26. Lemaitre B, Nicolas E, Michaut L, Reichart JM, Hoffmann J (1996) The
dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent
antifungal response in Drosophila adults. Cell 86: 973–983.
27. Hedengren M, Asling B, Dushay MS, Ando I, Ekengren S, et al. (1999) Relish, a
central factor in the control of humoral, but not cellular immunity in Drosophila.
Mol Cell 4: 827–837.
28. Naitza S, Rosse C, Kappler C, Georgel P, Belvin M, et al. (2002) The Drosophila
immune defense against gram-negative infection requires the death protein
Dfadd. Immunity 17: 575–581.
29. Gottar M, Gobert V, Matskevich AA, Reichhart JM, Wang C, et al. (2006) Dual
detection of fungal infections in Drosophila via recognition of glucans and sensing
virulence factors. Cell 127: 1425–1437.
30. Lau GW, Goumnerov BC, Walendziewicz CL, Hewitson J, Xiao W, et al. (2003)
The Drosophila melanogaster Toll pathway participates in resistance to infection
by the gram-negative human pathogen Pseudomonas aeruginosa. Infect Immun 71:
31. Mansfield BE, Dionne MS, Schneider DS, Freitag NE (2003) Exploration of
host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster.
Cell Microbiol 5: 901–911.
32. Brandt SM, Dionne MS, Khush RS, Pham LN, Vigdal TJ, et al. (2004) Secreted
bacterial effectors and host-produced eiger/TNF drive death in a Salmonella-
infected fruit fly. PLoS Biol 2: e418. doi:10.1371/journal.pbio.0020418.
33. Schneider DS, Ayres JS, Brandt SM, Costa A, Dionne MS, et al. (2007)
Drosophila eiger mutants are sensitive to extracellular pathogens. PLoS Pathog 3:
34. Partridge L, Piper MD, Mair W (2005) Dietary restriction in Drosophila. Mech
Ageing Dev 126: 938–950.
35. Ayres JS, Schneider DS (2008) A signaling protease required for melanization in
Drosophila affects resistance and tolerance of infections. PLoS Biol 6: e305.
36. Koella JC, Sørense FL (2002) Effect of adult nutrition on the melanization
immune response of the malaria vector Anopheles stephensi. Med Vet Entomol 16:
37. Siva-Jothy MT, Thompson JW (2002) Short-term nutrient deprivation affects
adult immune function in the mealworm beetle, Tenebrio molitor L. Physiol
Entomol 27: 206–212.
38. Muyskens JB, Guillemin K (2008) Bugs inside bugs: what the fruit fly teaches us
about immune and microbial balance in the gut. Cell Host Microbe 3: 117–118.
39. Cheesman SE, Guillemin K (2007) We know you are in there: conversing with
the indigenous gut microbiota. Res Microbiology 158: 2–9.
40. Lazzaro BP, Flores HA, Lorigan JG, Yourth CP (2008) Genotype-by-
environment interactions and adaptation to local temperature affect immunity
and fecundity in Drosophila melanogaster. Plos Pathog 4: e1000025. doi:10.1371/
41. McKean KA, Nunney L (2005) Bateman’s principle and immunity: phenotyp-
ically plastic reproductive strategies predict changes in immunological sex
differences. Evolution 59: 1510–1517.
42. Lee KP, Cory JS, Wilson K, Raubenheimer D, Simpson SJ (2006) Flexible diet
choice offsets protein costs of pathogen resistance in a caterpillar. Proc Soc B
43. Shirasu-Hiza MM, Dionne MS, Pham LN, Ayres JS, Schneider DS (2007)
Interactions between circadian rhythm and immunity in Drosophila melanogaster.
Curr Biol 15: R353–R355.
44. Feigin RD, San Joaquin VH, Haymond MW, Wyatt RG (1969) Daily
periodicity of susceptibility of mice to pneumococcal infection. Nature 224:
45. Lee JE, Edery I (2008) Circadian regulation in the ability of Drosophila to combat
pathogenic infections. Curr Biol 18: 195–199.
46. Williams JA, Sathyanarayanan S, Hendricks JC, Sehgal A (2007) Interaction
between sleep and the immune response in Drosophila: a role for the NFkappaB
relish. Sleep 30: 389–400.
47. Linder JE, Owers KA, Promislow DEL (2008) The effects on host-pathogen
interactions in D. melanogaster: Who benefits? J Insect Phys 54: 297–308.
48. Inglis GD, Johnson DL, Goettel MS (1996) Effects of temperature and
thermoregulation on mycosis by Beauveria bassiana in grasshoppers. Biological
Control 7: 131–139.
49. Watson DW, Mullens BA, Petersen JJ (1993) Behavioral fever response of Musca-
domestica (Diptera, Muscidae) to infection by Entomophthora-Muscae (Zygomycetes,
Entomophthorales). J Invert Path 61: 10–16.
50. Blanford S, Thomas MB, Langewald J (1998) Behavioural fever in the
Senegalese grasshopper, Pedaleus senegalensis, and its implications for biological
control using pathogens. Ecol Entom 23: 9–14.
51. Elliot SL, Blanford S, Thomas MB (2002) Host-pathogen interactions in a
varying environment: temperature, behavioural fever and fitness. Proc R Soc
London Series B- B Sci 269: 1599–1607.
52. Thomas MB, Blanford S (2003) Thermal biology in insect-parasite interactions.
Trends Ecol Evol 18: 344–350.
53. Povey S, Cotter SC, Simpson SJ, Pum Lee K, Wilson K (2009) Can the protein
costs of bacterial resistance be offset by altered feeding behaviour? J Animal
Ecology 78: 437–446.
54. Portnoy DA, Jacks PS, Hinrichs DJ (1988) Role of haemolysin for the
intracellular growth of Listeria monocytogenes. J Exp Med 167: 1459–1471.
55. Schneider DS, Shahabuddin M (2000) Malaria parasite development in a
Drosophila model. Science 288: 2376–2379.
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PLoS Biology | www.plosbiology.org10 July 2009 | Volume 7 | Issue 7 | e1000150