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Gut microbiota affects development and olfactory behavior in Drosophila melanogaster
Huili Qiao1,2, Ian W. Keesey1, Bill S. Hansson1,3, Markus Knaden1,3*
1 Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena,
Germany
2 Henan Provincial Key laboratory of Funiu Mountain Insect Biology, Nanyang Normal University,
Nanyang, China
3 Authors share last authorship
* Correspondence to: mknaden@ice.mpg.de
KEY WORDS: Drosophila, microbiota, gut bacteria, yeast, behavior, development
SUMMARY STATEMENT
Vinegar flies raised on food enriched with one of their gut microbes, changed their preference for these
microbes and their developmental rate depending on the microbes they were raised upon.
ABSTRACT
It has been shown that gut microbes are very important for the behavior and development of Drosophila,
as the beneficial microbes are involved in the identification of suitable feeding and oviposition places.
However, in what way these associated gut microbes influence the fitness-related behaviors of
Drosophila melanogaster remains unclear. Here we show that D. melanogaster exhibits different
behavioral preferences towards gut microbes. Both adults and larvae were attracted by the headspace
of Saccharomyces cerevisiae and Lactobacillus plantarum, but were repelled by Acetobacter malorum
in behavioral assays, indicating an olfactory mechanism involved in these preference behaviors. While
the attraction to yeast was governed by olfactory sensory neurons expressing the odorant co-receptor
Orco, the observed behaviors towards the other microbes still remained in flies lacking this co-receptor.
By experimentally manipulating the microbiota of the flies, we found that flies did not strive for a
diverse microbiome by e.g. increasing their preference towards gut microbes that they had not
experienced previously. Instead, in some cases the flies even increased preference for the microbes they
were reared on. Furthermore, exposing Drosophila larvae to all three microbes promoted Drosophila’s
development while only exposure to S. cerevisiae and A. malorum resulted in the development of larger
ovaries and in increased egg numbers the flies laid in an oviposition assay. Thus our study provides a
better understanding of how gut microbes affect insect behavior and development, and offers an
ecological rationale for preferences of flies for different microbes in their natural environment.
Journal of Experimental Biology • Accepted manuscript
http://jeb.biologists.org/lookup/doi/10.1242/jeb.192500Access the most recent version at
First posted online on 24 January 2019 as 10.1242/jeb.192500
INTRODUCTION
Gut microbiomes play important roles in different physiological processes of their hosts, such as
nutrition (Wong et al., 2014; Newell and Douglas, 2014; Tefit and Leulier, 2017; Leitão-Gonçalves et
al., 2017), development (Ridley et al., 2012; Shin et al., 2011; Storelli et al., 2011; Tefit and Leulier,
2017), longevity (Guo et al., 2014; Clark et al., 2015), immunity (Sansone et al., 2015) and disease
avoidance (Van Nood et al., 2013; Zhang et al., 2015). The vinegar fly, Drosophila melanogaster, has
been largely used to study host-microbe interactions related to innate immunity and pathogenic
association (Lemaitre et al., 2007; Keesey et al., 2017). Recently several independent studies analyzing
the diversity of gut microbes in D. melanogaster showed that the Drosophila microbiome mainly
consists of yeasts, and two genera of bacteria, Acetobacter and Lactobacillus (Chandler et al., 2011;
Broderick and Lemaitre, 2012; Staubach et al., 2013; Wong et al., 2011, 2017).
The environmental microbes that flies have been exposed to as larvae and adults not only drive the
composition of the flies’ gut microbiome (Chandler et al., 2011), but can also affect the flies’ behaviors,
such as oviposition (Tefit and Leulier, 2017) or foraging (Wong et al., 2017; Leitão-Gonçalves et al.,
2017; Keesey et al., 2017). Furthermore, Drosophila larvae and adults can be attracted by odors
emanating from food patches that have been previously used by larvae (Durisko and Dukas, 2013;
Durisko et al., 2014) and a study performed with axenic Drosophila revealed that at least some of these
attractants are produced by the larval gut bacteria (Venu et al., 2014). These results suggest that
Drosophila adults may rely on microbe derived volatiles for long-distance attraction to suitable feeding
and egg-laying sites. Recent studies have demonstrated that Drosophila prefers a microbe co-culture,
due to the metabolite exchange of the different microbes when grown together (Fisher et al., 2017) and
that gut microbe composition can modify microbial and nutritional preferences of D. melanogaster,
suggesting that microbiota can affect host chemosensory responses, preferences and behavior (Wong et
al., 2017). However, we have limited understanding of how gut microbes, such as yeast and bacteria,
affect Drosophila behaviors.
Several innate dedicated olfactory circuits in Drosophila have been described for detecting pleasant
yeast volatiles for oviposition (Dweck et al., 2015a), as well as circuits for aversive volatiles for
detecting danger by fungal mold or parasitoids (Stensmyr et al., 2012; Ebrahim et al., 2015). However,
it remains unclear whether similar circuits exist that help Drosophila to identify food containing healthy
or preferred gut microbes. Here we investigate whether Drosophila’s health is affected by a diet
containing primarily one microbe species, and whether flies raised on such a diet change their food
preferences (i.e. prefer food with microbes they could not access before, and were missing from their
dietary intake). To do so, instead of treating flies with antibiotics and/or sterilizing eggs by
dechorionation to produce axenic flies (Sabat et al., 2015; Koyle et al., 2016), we raised flies on diet
enriched with either Saccharomyces cerevisiae, Lactobacillus plantarum, or Acetobacter malorum. We
found that Drosophila raised on different microbes later differed regarding their olfactory behavioral
preference, developmental time, and fecundity.
Journal of Experimental Biology • Accepted manuscript
MATERIALS AND METHODS
Drosophila stocks
All experiments were carried out with wild type (WT) or Orco-/- transgenic Drosophila melanogaster
of the strain Canton-S, which were obtained from the Bloomington Drosophila Stock Center
(www.flystocks.bio.indiana.edu). D. melanogaster were raised on standard diet at 25°C with 70%
humidity, and a 12h light:12h dark cycle. For behavioral experiments, 3-5 days old flies of both sexes
or 3rd instar larvae were used.
Microbes strains
All microbes were purchased from Leibniz Institute DSMZ-German collection of microorganisms and
cell cultures, Saccharomyces cerevisiae, DSM1333; Lactobacillus plantarum, DSM-20174;
Acetobacter malorum, DSM-14337. They were kept at -80°C in 50% glycerol for long term storage.
Fresh cultures were generated daily and grown at 30°C 250 rpm in YM medium (S. cerevisiae) or MRS
medium (L. plantarum and A. malorum). To expose flies to specific microbes, 1 ml of stationary phase
microbe culture pellet (OD=1) was washed and re-suspended in 100 μl PBS, then was inoculated on the
surface of antibiotic free fly food contained in a 1.5 cm diameter fly vial as previous described (Tefit
and Leulier, 2017). 1 or 2 day old flies were distributed on fly food associated with the corresponding
microbe, and transferred to a new vial twice per day. In order to test for short- and long-term effects of
exposure to specific microbes, flies were either exposed two days to the corresponding microbes, or
were continuously bred for several generations under these conditions. For the latter, flies were allowed
to lay eggs on medium with one of the microbes for one day and were discarded afterwards. The medium
was not changed until the next generation of flies eclosed. Newly hatched flies were transferred to fresh
medium equipped with the same microbes and their offspring was raised as before. The flies of the fifth
generation raised under these conditions were transferred to fresh medium twice per day and were tested
at an age of 3-5 days.
Trap assays
Trap assays were performed as previously described (Keesey et al., 2017). Briefly, 35 flies (30 female
and 5 male flies, 3-5 days old, starved for 24 h) were introduced into a test chamber, which contains a
transparent plastic cup (length, 10 x 8 x 10 cm) with holes in the lid, and two smaller containers (height,
4.5 cm; diameter, 3 cm) with a cut pipette tip (tip opening, 2 mm). Experiments were always started at
the same time of day and carried out in a climate chamber with the same conditions of fly breeding.
Containers were equipped with a disc of filter paper (diameter, 5 mm) that was loaded either with 50 μl
Journal of Experimental Biology • Accepted manuscript
of growth medium containing the equivalent of the microbe pellet or with 50 μl of growth medium only.
By the use of a hemocytometer we estimated the numbers of cells per pellet as roughly 106 for S.
cerevisiae and each 108 for L. plantarum and A. malorum. The number of flies inside and outside the
traps was counted after 24 h. The attraction index(AI) was calculated as AI=(O − C)/ T, where O is the
number of flies entered the microbe containing trap, and C is the number of flies that entered the growth
medium containing trap, and T is the sum of all flies tested. The resulting index ranges from -1
(complete avoidance) to 1 (complete attraction). A value of zero characterizes a neutral or non-
responsive treatment. Each experiment was repeated 9-10 times.
Oviposition assays
Oviposition assays were carried out in a small container (10 x 10 x 20 cm) that was equipped with two
petri dishes (diameter, 5 cm) containing 0.5% agarose, of which one was loaded with 50 μl of growth
medium containing the equivalent of the microbe pellet or with 50 μl of growth medium only. 20 female
flies, 4-5 days old, were placed in each container. Experiments were carried out in a climate chamber
at the same conditions as the fly breeding. The number of eggs was counted after 24 h. The oviposition
index was calculated as (O − C)/(O + C), where O is the number of eggs on microbe treatment plate,
and C is the number of eggs on the growth medium plate. Each experiment was repeated 10 times.
Feeding assays
25 flies (20 female and 5 male flies) were collected and tested between 3-5 days old. Flies were starved
beforehand for 24 h with constant access to water. Flies were cooled for 3 min at -20°C and then
transferred to the behavioral arena. The capillary feeder (CAFÉ) assays utilized glass micropipettes
with liquid medium that were filled by capillary action and then inserted through pipette tips into the
container holding the adult flies as previously described (Keesey et al., 2016; 2017). One capillary
contained the control growth medium, while the other contained the microbe culture. The volume
consumed from each side was measured after feeding for 4 hours. Feeding index were calculated as (O
– C)/(O + C), where O is the amount of food consumed from the microbe solution and C is the amount
of food consumed from the control solution. Each experiment was repeated 10 times.
Larval two-choice assays
The larval olfactory choice assays were performed as previously described (Ebrahim et al., 2015). The
50 third instar larvae were placed in the center of a Petri dish which was filled with 0.5% agarose. The
petri dish contained two lids of an eppendorf cap which were placed at opposite positions at the
periphery of the petri dish. 30 μl of growth medium containing the equivalent of the microbe pellet or
with 30 μl of growth medium only were loaded in each cap lid. Larvae were allowed to crawl for 5 min
before their position on the petri dish was determined. Attraction index was calculated as (O − C)/ T,
where O is the number of larvae on the side of the dish loaded with microbe, C is the number of larvae
on the medium control side, and T is the total number of larvae. Each experiment was repeated 9-10
times.
Journal of Experimental Biology • Accepted manuscript
Single pair courtship and mating assays
Newly emerged virgin flies were collected, where males were kept individually in separate vials, and
females were reared in groups of 20-30 flies. All courtship experiments were performed with 4-5 days
old virgin flies, and the behavioral experiments were conducted within a circular courtship arena (height,
0.5 cm; diameter, 1 cm). Mating and courtship behaviors were documented for 60 minutes and then
analyzed. Copulation latency was measured as the time that the male and female took until successful
copulation. Copulation success was calculated as the percentage of all pairs that mated within the 60
minutes time span. For each combination of flies, the experiments were repeated 20-24 times.
Adult body weight, body size and ovary size measurement
Male and female adults were collected and exposed to each microbe for 2 days in a similar fashion as
for the oviposition assays. 15 individuals for each treatment were weighed on a Sartorious analytical
balance ME235P (Sartorius Weighing Technology GmbH, Goettingen, Germany). The images of 6 male
and female adults for each treatment were taken under a stereo microscope (Axio Zoom.V16, Zeiss
company, Gernany), where the area of head, thorax, and abdomen were measured with the software
Image J. The ovaries from 10 female flies of each group were dissected in 1 x PBS, where the images
were taken and their area were measured as above.
Fecundity assessment
10 female and 5 male virgin wild type or Orco-/- flies were collected directly after emergence and raised
continuously on control diet or on diet enriched with one of the three microbes. The diet was changed
every 24 h and egg numbers were recorded every day for one week. Each experiment was repeated 10
times.
Larvae developmental timing
Fifty 1st instar larvae were transferred to control diet or diet enriched with one of the three microbes.
The number of pupae appearing was counted twice per day until the last larvae of the population reached
the pupae state. Each experiment was repeated 10 times.
Chemical analysis
To analyze the volatiles emitted by the different microbes a 500 mL laboratory glass bottle was filled
with 400 mL fresh culture of the microbes and closed with a custom-made polyether ether ketone
(PEEK) stopper. The headspace was collected for 24 hrs on a Super-Q filter (50 mg, Analytical
Research Systems; www.ars-fla.com) according to standard procedures. Airflow at 0.5 L/min was
drawn through the bottle by a pressure pump. The filter was eluted with 1 mL hexane, and samples were
Journal of Experimental Biology • Accepted manuscript
stored at -20 °C until analysis. The fly bodywash extracts were obtained by washing 1 fly in 30 µl of
methanol for 6 h as previously described (Keesey et al., 2017; Dweck et al., 2015b), 8-10 individual fly
for each treatment were extracted. GC-MS (HP5 and HP-innowax) analyses were performed on all
volatiles and insect body wash collections. Microbe volatiles as well as fly odors were analyzed via
GC-MS. The GC was equipped with a HP5 (for fly bodywash) or HP-innowax (for microbe headspace)
MS column (30 m long, 0.25 mm id, 25 μm film thickness; Agilent Technologies) with helium used as
carrier gas (1.1 ml min-1 constant flow). The inlet temperature was set to 250°C. The temperature of
the GC- oven was held at 50°C for 2 min and then increased by 15°C min-1 to 280°C. The final
temperature was held for 15 min. The MS transfer-line was held at 280°C, the MS source at 230°C, and
the MS quad at 150°C. Mass spectra were taken in EI mode (at 70 eV) in the range from 33 m z-1 to
350 m z-1 with a scanning rate of 4.42 scan s-1. GC-MS data were processed with the MDS-
Chemstation software (Agilent Technologies). Compounds were identified with the NIST 2.0 mass
spectra database using the NIST algorithm. Identification was confirmed by comparison of Kovats
retention indices with published data. Several compounds were also confirmed by comparison with
synthetic standards (spectrum and retention time), obtained Sigma-Aldrich (http://www.sigma-
aldrich.com) in the of highest available purity. The internal standard bromo-decane was used for
quantification and statistical comparisons between analyzed samples.
Statistics analysis
Statistical analysis were performed using Prism 5, figures were prepared using Prism 5, Microsoft Excel,
and Adobe Illustrator CS5. Data were tested for a normal distribution and afterwards analyzed using
two-tailed, paired t-tests or one way ANOVA with Tukey’s multiple comparison tests.
RESULTS
Drosophila preference for gut microbes
We first performed trap-, oviposition- and feeding assays (Fig. 1A) to analyze innate preferences of
the flies for each of the different gut microbes. We next compared these preferences with those of flies
that were either shortly exposed to one of the three species of gut microbes (S. cerevisiae, L. plantarum,
or A. malorum) or that were reared on one of these microbes for several generations.
Flies raised on control diet without microbes were attracted by the headspaces of S. cerevisiae and L.
plantarum, but were repelled by A. malorum (Fig. 1B). When we repeated the experiments with Orco-
/- flies lacking functional odorant receptors (ORs), the preference to S. cerevisiae was abolished, while
the preference for L. plantarum and the avoidance of A. malorum were not affected (Fig. 1C). We
Journal of Experimental Biology • Accepted manuscript
conclude that flies can detect the headspace of all tested microbes, and that the preference for yeast is
governed by Orco dependent odorant receptors (ORs). We next gave the flies the opportunity to choose
between oviposition sites with or without microbes (Fig. 1A). In contrast to the pure attraction assay,
both wild type flies (Fig. 1D) as well as Orco-/- flies (Fig. 1E) preferred to lay eggs on the plate with
microbes including all three species, S. cerevisiae, L. plantarum, or A. malorum. Hence, flies consider
the presence of microbes during oviposition, and as this preference is conserved in Orco-/- flies,
oviposition preference seems to be governed by ionotropic receptors (IRs) or gustatory receptors (GRs)
that do not depend on the coreceptor Orco for the detection of environmental chemical cues.
In addition, we tested the flies feeding preference for the same set of microbes by performing a CAFÉ
assay. In this assay flies can choose between a solution with or without the microbe (Fig. 1A). As the
liquids are presented in tiny glass capillaries, any preference should mainly be based on cues detected
by the labellum and palps of the flies (although we cannot fully exclude the evaporation of volatile
compounds from the capillaries and thereby the involvement of antenna in any kind of choice). However,
we did not observe any preference for microbes versus the control liquid, and we assert that the labellum
and palps do not seem to be involved in the flies’ preference or avoidance of S. cerevisiae, L. plantarum,
or A. malorum (Fig. 1F-G).
We next asked, whether the adult preferences are conserved in larvae. Therefore, we performed a
larval attraction assay (Fig. 1A). Larvae showed the same preference trend as the adult flies displayed
in the trap assay, i.e. larvae were attracted to S. cerevisiae and L. plantarum, while repelled by A.
malorum, indicating that both larvae and adults might share the same olfactory mechanisms involved
in these consistent preference behaviors (Fig. 1H).
Flies detect cues of high ecological relevance, like harmful microbes or parasitoids via highly
specialized neuronal circuits that are dedicated to detect signature odors (Stensmyr et al., 2012, Ebrahim
et al., 2015). In order to investigate, whether the behavior towards any of the gut microbes of this study
was governed by such an labeled line, we analyzed the headspaces of the different microbe cultures via
gas chromatography-coupled mass spectrometry (GC-MS) (Fig. S1). From our samples we found
compounds like 3-Methyl-1-butanol and 2-Pheylethanol that are described attractants (Knaden et al.
2012, Becher et al. 2012) and Benzaldehyde which has been shown to be aversive (Knaden et al. 2012).
However, all of these compounds become detected by rather widely tuned receptors at the
concentrations that have been tested (Hallem and Carlson, 2006). Although we cannot exclude that we
overlooked novel ligands that might be detected by a dedicated pathway, the presence of a wide range
of general odors that are well known to attract flies, makes the involvement of a labeled line unlikely
in Drosophila melanogaster response towards these microbes.
Journal of Experimental Biology • Accepted manuscript
The effect of gut microbiome on flies’ behavioral preference
Having shown that the flies become attracted to two of the microbe types and that all microbes
positively affect the flies’ oviposition choices, we next asked whether the flies’ behavior would change
after they have been exposed to one of the microbes for a prolonged time. We hypothesized that flies
after exposure to only one microbe should switch their preference to the other microbes in order to keep
a diverse and healthy gut microbiome. As an alternative hypothesis, flies could instead prefer those
microbes they are familiar with. Drosophila were manipulated by raising them on fly food enriched
with one of the microbe species for either 2 days or for several generations. With these manipulated
flies we performed the same behavioral assays as before. Independent of their pre-experimental
exposure to one of the microbes, the flies still became attracted by the headspaces of S. cerevisiae and
L. plantarum and repelled by the one of A. malorum (Fig. 2A-C, Fig. S2). However, pre-exposure to S.
cerevisiae significantly increased the preference to this microbe and the avoidance of A. malorum,
suggesting that exposure to these microbes may generate a learned response that accentuates the
behavioral decisions towards these microbes. We therefore conclude that flies do not increase their
preference towards gut microbes that they had not been in contact with previously. We furthermore did
not find effects of pre-exposure on the oviposition preference of these flies (Fig. 2D-F). Interestingly,
exposing flies to S. cerevisiae and A. malorum significantly increased the total egg numbers that the
flies laid during the oviposition assay (Fig. 2G-I). It remains unclear, why flies are repelled by the
headspace of A. malorum which has a positive effect on the flies’ fecundity.
We next tested whether the oviposition behaviors were correlated with the flies’ courtship and
mating behaviors. The behavioral performance of individual pairs of flies was analyzed in courtship
and mating assays. To do so, we paired flies reared on the different diets in all possible combinations.
The copulation success rates of L. plantarum treated flies were lower than those of all other flies (Fig.
3A) and their copulation latencies were significantly higher (Fig. 3B). Interestingly, a strong increase
in latency was observed only when both sexes were reared on L. plantarum (Fig. 3B). We hypothesized
that the decline in courtship and mating performance of L. plantarum treated flies could be one of the
reasons for their subsequently lower number of eggs in the oviposition assays. To answer what affected
the courtship behavior, we prepared the bodywash of both male and female flies of all treatments and
analyzed the resulting compounds by GC-MS. When testing for effects of the microbes on the amount
of sex- and aggregation pheromones like methyl laurate (ML), methyl myristate, methyl palmitate (MP)
(Dweck et al. 2015b), cis-vaccenyl acetate (cVA) (Bartelt et al., 1985), and the male specific (Z)-7-
tricosene (Lacaille et al. 2007) we found only minor (in most cases non-significant) differences
depending on the treatment (Fig. S3A-D). Furthermore, two female specific cuticular hydrocarbons,
7(Z),11(Z)-heptacosadiene (7,11-HD) and 7(Z),11(Z)-nonacosadiene (7,11-ND) that play important
roles in Drosophila courtship (Ferveur, 1997; Toda et al., 2012) were significantly increased in female
flies after treatment with S. cerevisiae and A. malorum (Fig. 4). However, as L. plantarum-treated flies
Journal of Experimental Biology • Accepted manuscript
did not differ from flies reared on standard diet regarding these compounds, the increased courtship
latency found in L. plantarum treated flies (and specifically associated with females), remains unclear.
Gut microbes affected fly ovary development
To find out how the microbes affect the fly’s egg laying behavior, we tested the effect of the microbes
on single fly body weight, body size, and ovary size. Consistent with the observed increase in egg
numbers for flies treated with S. cerevisiae and A. malorum, females of these treatments were heavier
than control flies and heavier than L. plantarum treated flies. For male flies, only S. cerevisiae treated
flies were slightly (but significantly) heavier than the others (Fig. 5A). We next took images of the
different treated flies and measured the size of the abdomen. Interestingly, there was no difference in
abdomen size in any of the male flies, while females treated with S. cerevisiae exhibit a bigger abdomen
than A. malorum treated flies, and both of them were bigger than those from L. plantarum treated and
from control flies (Fig. 5B, Fig. S4). As the difference of egg number fitted well with the female
abdomen size, we next dissected and measured female ovaries for each microbial treatment. Again,
ovaries of S. cerevisiae treated females were bigger than those of A. malorum treated flies, and the
ovaries of both of these two groups were significantly bigger than those of control flies and flies kept
on L. plantarum (Fig. 5C-D).
The effect of gut microbes on the flies’ fecundity
Having shown that the treatment with S. cerevisiae or A. malorum resulted in more eggs in the
oviposition assay and increased abdomen and ovary sizes in female flies, we asked whether that would
result also in an overall increased fecundity of these flies. When we kept the flies for several days on
the different diets and counted the number of eggs on a daily basis, all flies started to lay eggs after 2
days of reproductive maturation with increasing egg numbers per day until the fourth day. Afterwards,
the egg number kept steady per day until the end of the experiment. As expected from the previous
results of our study, flies treated with S. cerevisiae laid the highest number of eggs (higher during each
day and also in total during the full week), while A. malorum treated flies still laid more eggs than L.
plantarum treated flies and the control flies. We next asked whether a temporary exposure to S.
cerevisiae would be sufficient to increase a female’s fecundity over its entire lifetime. However, when
we transferred the flies back to a control diet after four days on a diet with S. cerevisiae, the daily egg
number decreased dramatically and reached that of flies on control diet or diet with L. plantarum after
2 days (Fig. 6A-B). This indicates that the increased fecundity needs a constant supply of S. cerevisiae,
and that the change in fecundity is temporary. Interestingly, although we could show that Orco-/- flies
become less attracted to the S. cerevisiae (Fig. 1C), the lack of functional OSNs expressing the
coreceptor Orco did not diminish the positive effect of the microbes on the flies’ fecundity (Fig. 6C-D).
Obviously, although Orco-/- flies become less attracted by the headspace of S. cerevisiae, they still
consume this microbes when being reared on them.
Journal of Experimental Biology • Accepted manuscript
Gut microbe mediated larval growth acceleration
Finally, we also tested whether the exposure of flies to the microbes affected the larval development.
It became apparent, that when testing 50 larvae per treatment type (e.g. S. cerevisiae), the number of
larvae that succeeded in pupation was similar for all diets (Fig. 7A). However, the treatment affected
the average timespan until larvae reached the pupal stage, thus the developmental rate was affected by
microbe exposure. While most larvae that were reared on S. cerevisiae reached pupation stage after 3.5
days, all other treatments resulted in durations of 4-4.5 days (Fig. 7B), giving flies reared on S.
cerevisiae a 30% faster development time.
DISCUSSION
In this study, we found that D. melanogaster showed different behavioral preferences to gut microbes.
It is known that Drosophila are highly attracted to volatiles associated with yeast (Becher et al., 2012)
and fermenting fruit (Becher et al., 2010; Keesey et al., 2015). The same attractive compounds, like 3-
Methyl-1-butanol and 2-Phenylethanol, were also identified in S. cerevisiae culture in our experiments.
Previous studies have shown that D. melanogaster flies display positional avoidance towards carboxylic
acids (i.e. one major compound produced by lactic and acetic acid bacteria), while the flies show a
preference to lay eggs on sites containing these acids (Joseph et al., 2009; Chen et al., 2017). When,
however, not presenting the acids alone, but the full headspace of the bacteria, we found that both larvae
and adults were attracted by the lactic acid bacteria L. plantarum and repelled by the acetic acid bacteria
A. malorum in choice assays. Obviously attraction towards lactic acid bacteria is not driven by the acids
but by accompanying compounds in the blend. As at least the preference for S. cerevisiae was absent in
Orco-/- flies and as the flies did not prefer any microbes in a CAFÉ assays (that tests for gustatory
preference while basically excluding the impact of olfactory stimuli), olfaction at least partly is involved
in the flies’ attraction to gut microbes for feeding. However, as Orco-/- flies still targeted L. plantarum
in the trap assay and preferred all microbes in the oviposition assay, our data suggests that ionotropic
receptors (IRs) (Benton et al. 2009) and/or gustatory receptors (GRs) are also involved in this behavioral
preference. Sensory neurons expressing IRs have been reported to mainly detect acids (Ai et al., 2010;
Grosjean et al., 2011; Min et al., 2013; Hussain et al., 2016) and have been shown to mediate Drosophila
oviposition preference on acid containing medium (Chen et al., 2017). Hence, the attraction towards
and the oviposition preference for the different microbes might be governed by several different receptor
types, or sensory modalities.
Compared with Drosophila raised on standard diet, raising the flies on diets enriched with one of the
microbes in some cases resulted in different preferences in the trap assays (Fig. 2A and B) which is in
agreement with Wong et al. (2017) who found different foraging preferences in flies that were either
axenic or those flies that were mono-associated with L. plantarum or A. pomorum. The before
mentioned study used axenic flies, whose guts are basically free of microorganism or due a specific
treatment contain only a single microbe species. This comes with the benefit, that the microbiome of
Journal of Experimental Biology • Accepted manuscript
the studied flies is under full control as compared to our treatment where the flies’ gut microbiome was
manipulated just by exposing them to single microbe types over a specific time. Despite this potential
drawback we assume that our flies had a more conventional gut microbiome like the one would expect
in natural conditions. However, our data suggest that – even in flies with a “normal” gut microbiome –
an exposure to one microbe species can alter the flies’ behavioral preferences.
We also found that the exposure to the different microbes not only affected the flies’ behaviors, but
also promoted their growth and development. It’s known that yeasts are vital for larval development
and survival (Anognostou et al., 2010; Becher et al., 2012) as they seem to provide proteins as well as
most other non-caloric nutritional requirements in Drosophila (Piper et al., 2013). A. pomorum, which
is a close relative of the microbe A. malorum that was used in our study, is able to influence the systemic
larval development of Drosophila by affecting both growth rate and body size via the insulin signaling
pathway (Shin et al., 2011). Furthermore, L. plantarum can promote larval growth and increase the
growth rate of the flies on yeast-poor medium, without affecting the size of flies, by regulating hormonal
signals through TOR-dependent nutrient sensing (Storelli et al., 2011). Finally it is shown that
deprivation of essential amino acids (eAAs) can induce increased yeast intake and decreased
reproduction of Drosophila, but both changes can be rescued by the introduction of healthy gut bacteria,
A. pomorum and Lactobacilli (Leitão-Gonçalves et al., 2017).
In agreement with the aforementioned studies of the microbial impact on the flies’ growth and
development, we found that the gut microbes have different impact on female Drosophila ovary
development and fecundity. More specifically the total egg number of control flies and flies reared on
L. plantarum in oviposition assays was similar and significantly lower when compared to flies from the
other two microbes (Fig. 2G-I). In addition, there was only a slight impact on total fecundity (Fig. 6A
and B) and on larval development (Fig. 7) when flies were treated with L. plantarum. However, we
found clear positive effects when flies were reared on S. cerevisiae and A. malorum, like a faster larval
development time and larger ovaries that came along with increased fecundity. Thus, these results
demonstrate that S. cerevisiae, L. plantarum, and A. malorum can be beneficial partners for D.
melanogaster, and our results help explain the common and natural association of these microbes with
this fly. It remains, however, open why flies become attracted by the former two microbes but avoid the
headspace of A. malorum. In conclusion, our study demonstrates the importance of preference among
microbial associations for the ecological advantage of Drosophila in their natural environment, where
some microbes promote either fecundity or developmental speed, the latter of which aids these insects
in avoiding predation and parasitism during their most vulnerable larval stages.
Journal of Experimental Biology • Accepted manuscript
Acknowledgements
The authors would like to thank Chinese Scholarship Council for providing funding for H Qiao to study
at Max Planck Institute for Chemical Ecology. We also express our gratitude to S. Trautheim, K.
Weniger, and D. Veit for their technical support, guidance and expertise at MPI-CE. We are especially
thankful to M. Wang for his help and advice regarding microbe culture. Stocks obtained from the
Bloomington Drosophila Stock Center were used in this study (NIH P40od018537).
Competing interests
The authors declare no competing or financial interests.
Author contributions
HQ, IWK, BSH, and MK designed the study. HQ performed experiments. HQ and IWK analyzed data.
HQ, IWK, BSH, and MK wrote and/or revised the manuscript.
Funding
This research was supported through funding by the Max Plank Society.
Journal of Experimental Biology • Accepted manuscript
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Figures
Fig. 1. Attraction assays of Drosophila toward different microbes. A: Experimetal designs used for
attraction, oviposition and feeding assays. B-C: Attraction index of naïve wild type (B) or Orco-/- (C)
flies towards the olfactory cues from each microbe culture and medium control. D-E: Oviposition
preference index of wild type (D) or Orco-/- (E) flies towards the olfactory cues from each microbe
culture and medium control. F-G: Feeding index of wild type flies towards each microbe culture and
medium control (F) and the feeding volume (G) after 4 h. H: Attraction index of the 3rd instar larvae
towards the olfactory cues from each microbe culture and medium control. Error bars represent SE.
Significance from zero are denoted by filled boxes in B-F, H (p<0.05, Two tailed, paired t test), no
significant differences are denoted by ns above bars in G (One way ANOVA, Tukey’s multiple
comparison test).
Journal of Experimental Biology • Accepted manuscript
Fig. 2. Attraction and oviposition assays of differently treated Drosophila toward microbes. A-C:
Attraction index of adult Drosophila manipulated with different microbes for 2 days toward the
olfactory cues from S. cerevisiae (A), A. malorum (B) and L. plantarum (C) compared to the cues from
growth medium control. Filled boxplots are different from 0 (Wilcoxon rank sum test, p<0.05). D-F:
Oviposition preference index of adult Drosophila manipulated with different microbes for 2 days
toward the olfactory cues from S. cerevisiae (D), A. malorum (E) and L.plantarum (F) compared to the
cues from growth medium control. Filled boxplots are different from 0 (p<0.05, Two tailed, paired t
test). G-I: Total egg numbers laid in experiments D-F. Error bars represent SE. Significance from zero
are denoted by filled boxes in A-F (p<0.05, Two tailed, paired t test), significant differences are denoted
by letters in G-I (p<0.01 with upper letter, One way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology • Accepted manuscript
Fig. 3. Percentage of copulation success (A) and copulation latency (B) of control and 2 days
manipulated Drosophila. Error bars represent SE. Significant differences are denoted by letters
(p<0.05 with lower letter, One way ANOVA, Tukey’s multiple comparison test). Sample sizes are given
in brackets above bars.
Journal of Experimental Biology • Accepted manuscript
Fig. 4. GC-MS analysis of body wash from different manipulated Drosophila. Significantly
different cuticular hydrocarbons, (Z),11(Z)-heptacosadiene (A) and 7(Z),11(Z)- nonacosadiene (B)
found in females (Bromodecane as internal standard). Error bars represent SE. Significant differences
are denoted by letters (N=8-10 replicates, p<0.05 with lower letter, One way ANOVA, Tukey’s multiple
comparison test).
Journal of Experimental Biology • Accepted manuscript
Fig. 5. Single fly body weight (A), female abdomen size (B) and ovary size (C) measurement and
ovary images (D) of control and 2 days manipulated Drosophila. Error bars represent SE.
Significant differences are denoted by letters (p<0.05 with lower letter, p<0.01 with upper letter, One
way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology • Accepted manuscript
Fig. 6. Fecundity of Drosophila on control diet or diet with different microbes. A and C:
Comparison of 8 days total egg number of Drosophila wild type (A) and Orco-/- (C) laid on different
diets, 10 samples for each. B and D: Comparison of daily egg number of Drosophila wild type (B) and
Orco-/- (D) laid on different diets, 10 samples for each. Error bars represent SE. Significant differences
are denoted by letters (p<0.01 with upper letter, One way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology • Accepted manuscript
Fig. 7. Larval developmental time on control diet or diet with different microbes. A. Accumulation
curve of pupa number appeared on different diets, 10 samples for each; B. Pupa number appeared every
day on different diet, 10 samples for each.
Journal of Experimental Biology • Accepted manuscript
Fig. S1. GC-MS profile of headspace odors from different microbe culture and its medium
control. Numbers from GC-MS refer to peaks in A: (1) isopentyl acetate; (2) 3-methyl-1-butanol; (3)
isovaleric anhydride; (4) 2-methylhexanoic acid; (5) 2-phenethyl acetate; (6) 2-phenylethanol; (7)
benzaldehyde; (8) phenylacetaldehyde; (9) (E)-1-(6,10-Dimethylundeca- 5,9-dien-2-yl)-4-
methylbenzene. Numbers from GC-MS refer to peaks in B and C: (1) 2-heptanone; (2) 3-methyl-1-
butanol; (3) methylpyrazine; (4) acetoin; (5) 2,5-dimethylpyrazine; (6) 2-nonanone; (7) acetic acid; (8)
2,6-dimethyl-4-heptanol; (9) 2-ethenyl-6-methyl pyrazine; (10) benzaldehyde; (11) isobutyric acid; (12)
2-undecanone; (13) butanoic acid; (14) isovaleric acid; (15) 2-Undecanol; (16) 1-methoxynonane; (17)
2-acetylphenol; (18) hexanoic acid; (19) phenylethyl alcohol; (20) (Z)-4-decen-1-ol, methyl ether; (21)
octanoic acid; (22) benzeneacetaldehyde.
6810 12 14 16 18 20 22
6
7
0
1
2
3
4
5
8
9
10
11
12
13
14
15
1
2
Abundance (millions)
1
2
34 5
6
7
89
6810 12 14 16 18 20 22
1
2
3
4
5
6
7
0
1
2
3
4
5
Abundance (millions)
1
2
34
5
6
78
910
11
12
13
14
1516 17
18 19
20 21
3
5
69
10
22
6 8 10 12 14 16 18 20 22
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
Abundance (millions)
3
5
69
10
22
2
3
5
69
19
S. cerevisiae
A. malorum
L. plantarum
MRS medium Control
YM medium control
A
C
B
Retention Time (min)
Retention Time (min) Retention Time (min)
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S2. Attraction assays of Drosophila manipulated for 1 generation (A-C) and 5 generations (D-
F) toward different microbes. Error bars represent SE. Significance from zero are denoted by filled
boxes (p<0.05, Two tailed paired t test), significant differences between each group are denoted by
letters above (p<0.05 with lower letter, One way ANOVA, Tukey’s multiple comparison test). Filled
boxplots are different from 0 (Wilcoxon rank sum test, p<0.05).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S3. GC-MS profiles of body wash from female (A) and male (B), and quantitative analysis of
compounds from female (C) and male (D) Drosophila control and manipulated for 2 days with
different microbes. Br-D, bromodecane (internal standard); ML, methyl laurate; MM, methyl
myristate; MP, methyl palmitate; 7T, (Z)-7-tricosene; cVA, cis-vaccenyl acetate; 7,11-HD, 7(Z),11(Z)-
heptacosadiene; 7,11-ND, 7(Z),11(Z)-nonacosadiene. (N=8-10 replicates, p<0.05 with lower letter, One
way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S4. Male abdomen size of control and 2 days manipulated Drosophila. Error bars represent SE.
Significant differences are denoted by letters (One way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S1. GC-MS profile of headspace odors from different microbe culture and its medium
control. Numbers from GC-MS refer to peaks in A: (1) isopentyl acetate; (2) 3-methyl-1-butanol; (3)
isovaleric anhydride; (4) 2-methylhexanoic acid; (5) 2-phenethyl acetate; (6) 2-phenylethanol; (7)
benzaldehyde; (8) phenylacetaldehyde; (9) (E)-1-(6,10-Dimethylundeca- 5,9-dien-2-yl)-4-
methylbenzene. Numbers from GC-MS refer to peaks in B and C: (1) 2-heptanone; (2) 3-methyl-1-
butanol; (3) methylpyrazine; (4) acetoin; (5) 2,5-dimethylpyrazine; (6) 2-nonanone; (7) acetic acid; (8)
2,6-dimethyl-4-heptanol; (9) 2-ethenyl-6-methyl pyrazine; (10) benzaldehyde; (11) isobutyric acid; (12)
2-undecanone; (13) butanoic acid; (14) isovaleric acid; (15) 2-Undecanol; (16) 1-methoxynonane; (17)
2-acetylphenol; (18) hexanoic acid; (19) phenylethyl alcohol; (20) (Z)-4-decen-1-ol, methyl ether; (21)
octanoic acid; (22) benzeneacetaldehyde.
6810 12 14 16 18 20 22
6
7
0
1
2
3
4
5
8
9
10
11
12
13
14
15
1
2
Abundance (millions)
1
2
34 5
6
7
89
6810 12 14 16 18 20 22
1
2
3
4
5
6
7
0
1
2
3
4
5
Abundance (millions)
1
2
34
5
6
78
910
11
12
13
14
1516 17
18 19
20 21
3
5
69
10
22
6 8 10 12 14 16 18 20 22
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
Abundance (millions)
3
5
69
10
22
2
3
5
69
19
S. cerevisiae
A. malorum
L. plantarum
MRS medium Control
YM medium control
A
C
B
Retention Time (min)
Retention Time (min) Retention Time (min)
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S2. Attraction assays of Drosophila manipulated for 1 generation (A-C) and 5 generations (D-
F) toward different microbes. Error bars represent SE. Significance from zero are denoted by filled
boxes (p<0.05, Two tailed paired t test), significant differences between each group are denoted by
letters above (p<0.05 with lower letter, One way ANOVA, Tukey’s multiple comparison test). Filled
boxplots are different from 0 (Wilcoxon rank sum test, p<0.05).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S3. GC-MS profiles of body wash from female (A) and male (B), and quantitative analysis of
compounds from female (C) and male (D) Drosophila control and manipulated for 2 days with
different microbes. Br-D, bromodecane (internal standard); ML, methyl laurate; MM, methyl
myristate; MP, methyl palmitate; 7T, (Z)-7-tricosene; cVA, cis-vaccenyl acetate; 7,11-HD, 7(Z),11(Z)-
heptacosadiene; 7,11-ND, 7(Z),11(Z)-nonacosadiene. (N=8-10 replicates, p<0.05 with lower letter, One
way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
Fig. S4. Male abdomen size of control and 2 days manipulated Drosophila. Error bars represent SE.
Significant differences are denoted by letters (One way ANOVA, Tukey’s multiple comparison test).
Journal of Experimental Biology: doi:10.1242/jeb.192500: Supplementary information
Journal of Experimental Biology • Supplementary information
