Drosophila suzukii - Proceedings of the Royal Society B

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Research
Cite this article: Dekker T, Revadi S,
Mansourian S, Ramasamy S, Lebreton S,
Becher PG, Angeli S, Rota-Stabelli O, Anfora G.
2015 Loss of Drosophila pheromone reverses
its role in sexual communication in Drosophila
suzukii. Proc. R. Soc. B 282: 20143018.
http://dx.doi.org/10.1098/rspb.2014.3018
Received: 11 December 2014
Accepted: 22 January 2015
Subject Areas:
evolution, ecology, neuroscience
Keywords:
Drosophila suzukii, pheromone, evolution,
ejaculatory bulb, fruitless, sensory drive
Author for correspondence:
Teun Dekker
e-mail: [email protected]
†
These authors contributed equally to this
work.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2014.3018 or
via http://rspb.royalsocietypublishing.org.
Loss of Drosophila pheromone reverses
its role in sexual communication in
Drosophila suzukii
Teun Dekker1, Santosh Revadi1,2,†, Suzan Mansourian1,†, Sukanya Ramasamy2,
Sebastien Lebreton1, Paul G. Becher1, Sergio Angeli3, Omar Rota-Stabelli2
and Gianfranco Anfora2
1
Unit of Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences,
23053 Alnarp, Sweden
2
FEM, Fondazione Edmund Mach, Research and Innovation Centre, Chemical Ecology Group, Via E. Mach 1,
San Michele all’Adige (TN) 38010, Italy
3
Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Universita` 5, Bolzano 39100, Italy
The Drosophila pheromone cis-11-octadecenyl acetate (cVA) is used as pheromone throughout the melanogaster group and fulfils a primary role in sexual
and social behaviours. Here, we found that Drosophila suzukii, an invasive
pest that oviposits in undamaged ripe fruit, does not produce cVA. In fact,
its production site, the ejaculatory bulb, is atrophied. Despite loss of cVA production, its receptor, Or67d, and cognate sensillum, T1, which are essential
in cVA-mediated behaviours, were fully functional. However, T1 expression
was dramatically reduced in D. suzukii, and the corresponding antennal lobe
glomerulus, DA1, minute. Behavioural responses to cVA depend on the
input balance of Or67d neurons (driving cVA-mediated behaviours) and
Or65a neurons (inhibiting cVA-mediated behaviours). Accordingly, the shifted
input balance in D. suzukii has reversed cVA’s role in sexual behaviour:
perfuming D. suzukii males with Drosophila melanogaster equivalents of cVA
strongly reduced mating rates. cVA has thus evolved from a generic sex pheromone to a heterospecific signal that disrupts mating in D. suzukii, a saltational
shift, mediated through offsetting the input balance that is highly conserved
in congeneric species. This study underlines that dramatic changes in a
species’ sensory preference can result from rather ‘simple’ numerical shifts
in underlying neural circuits.
1. Introduction
Melanogaster-clade species generically use male-produced cis-11-octadecenyl acetate (cVA) as sex pheromone [1]. The underlying cVA circuitry is arguably the
best-studied pheromone communication system, from production to detection,
and from processing to resulting muscle output [2–4]. Different from ‘typical’ sex
pheromones in insects, cVA acts at short range and not only fulfils a role in orientation and sexual attraction, but also most prominently in a variety of sexual and
social behaviours in Drosophila melanogaster: it increases male mate acceptance by
females [5,6], reduces attractiveness of newly mated females, reduces male–male
courtship, while increasing aggression between males [7,8]. cVA is a sex pheromone
found throughout and basal to the melanogaster group species, such as in the obscura
and immigrans group species [1], underlining its primal role in Drosophila.
The fact that all melanogaster group species studied thus far use cVA as
volatile sex pheromone [1] would preclude a role of cVA in species recognition,
a function that is otherwise typical for insect sex pheromones [9]. Moths in particular are well known for their species-specific pheromone blends, which are
used to discriminate between conspecific and heterospecifics [10]. Whereas
males are finely tuned to their conspecific female-produced pheromone, they
often also display acute sensitivity to pheromones of other, often sympatric,
species. In the latter case, such pheromones elicit responses on sensory neurons
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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(a) Flies
A Drosophila suzukii colony was established in 2010 from around
1000 individuals emerging from infested blueberries and raspberries collected in Valsugana, Trentino Province, Italy. The US strain
of D. suzukii was derived from a colony established by D. Walsh
(Washington State University, Prosser, WA, USA). Drosophila
biarmipes (14023-0361.02) and Drosophila subpulchrella (140230401.01) were obtained from the San Diego stock centre.
For D. melanogaster, we used the wild-type Dalby strain [16,17].
Flies were reared in a quarantine facility on a standard cornmeal–
yeast–agar medium at 218C under 12 L : 12 D conditions.
(b) Behavioural assays
Sexes of D. melanogaster and D. suzukii (Trentino strain) were
separated within 1 h post emergence and placed in groups of five
(females) and seven (males). They were placed in standard food
vials and flipped after three days to new food vials. After
four days, seven males were placed into the vial containing five conspecific females. We used groups of individuals to increase the
mating incidence of D. suzukii, which had a lower mating rate compared with D. melanogaster. The higher ratio of males to females was
there to offset any potentially negative effect of the perfuming of the
flies (see §2c, below) on the ‘availability’ of male mates, although no
clear negative effects of the experimental procedures were observed.
No individuals died during the mating experiments. As individuals
could not be reliably recognized and followed during the course of
the experiments, we scored mating rates only. Matings were noted
every 15 min. We observed the mating behaviour for both species
during five consecutive hours. A total of 21 and 30 groups of 12
flies (five females, seven cVA-perfumed males) were perfumed
D. melanogaster and D. suzukii, respectively, against a total of 19
and 26 control groups, respectively. Flies only mated once during
our assay.
(c) Perfuming flies
Three millilitre vials were coated on the inside with a total of
50 mg cVA. The procedures were roughly similar to those
described in Billeter et al. [13]. Fifty microlitres of hexane with
or without 1 mg ml21 of cVA were pipetted into a 3-ml glass
screw cap vial. The hexane was slowly evaporated while the
vial was placed in a horizontal position and slowly rotated.
Seven males were briefly immobilized on ice (1 min) and
placed in a 3-ml treatment or control vial. Vials, coated with
cVA or hexane-treated only and containing seven flies, were
(d) Chemical analysis
Pheromones were extracted from the fly cuticle (n ¼ 5, 6 for male
and female D. suzukii, respectively) by leaving individual flies in
100 ml of hexane for 5 min at room temperature. One hundred nanograms of heptadecenyl acetate were added as an internal standard.
These extracts were analysed on a gas chromatograph coupled with
a mass spectrometer (GC-MS; 6890 GC and 5975 MS, Agilent technologies Inc., Santa Clara, CA, USA). Extracts were concentrated to
ca 10 ml, and 2 ml were injected into a HP-5MS silica capillary
column (30 m 0.25 mm 0.25 mm film thickness; Agilent technologies Inc.) that was temperature-programmed from 508C
(2 min), 88C min21 to 3008C (5 min).
Compounds of interest were identified based on their mass
spectrum, retention time, comparison with previously published
works on D. melanogaster CHs [18] and injection of synthetic corresponding compound (for cVA). They were quantified by peak
integration and comparison with the response of the internal
standard. CH extracts of both the Trentino and US strains of
D. suzukii were analysed (indicated in §3).
(e) Sensory physiology
Using a strong airflow, flies were pushed head-first into a truncated
pipette tip. The pipette tip was cut distally from the head and the
fly was gently pushed forward until the head protruded from
the narrow end. The pipette tip was placed on a wax surface on a
microscope slide, and using a glass micropipette the right antenna
was gently bent backwards and stably positioned on a coverslip.
The fly was placed under a microscope (Olympus BX51W1), with
a magnification less than or equal to 1500. Via a glass tube, a
1 l min21 charcoal-purified and humidified airflow was constantly
blown over the fly head. Tungsten microelectrodes, sharpened in a
KNO2-solution, were used for recording of action potentials of
antennal sensory neurons. A motor-controlled micromanipulator
(Ma¨rzhauser DC-3K, Wetzlar, Germany) equipped with a piezo
unit (Ma¨rzhauser PM-10) was used for fine positioning. A reference
electrode was inserted into the eye with a manually controlled
micromanipulator (Narishige MM33, Tokyo, Japan). Touch stimulation was performed using an additional piezo unit to move the
electrode at micrometre scale towards the sensillum. A glass electrode drawn to a sharp tip was dipped briefly in a 1 mg ml21 cVA
solution, air dried and used for stimulation. After A/D conversion
(Syntech IDAC PCI card), spikes were visualized and stored on a
PC. Analysis was done using AUTOSPIKE v. 3.2 software (Syntech,
Kirchzarten, Germany). For our sensory physiological analysis
of D. suzukii, we used the Trentino strain. For each species, six
individual replicates were used.
(f ) Counts of sensilla trichodea
Antenna of WT Dalby (n ¼ 8) and D. suzukii (n ¼ 9, Trentino
strain) were mounted using spacer rings (Secure-Seal TM imaging
spacers, Sigma-Aldrich) and Vectashield mounting medium Hard
Mount (Vector Laboratories, Burlingame, CA, USA). Highresolution confocal scans (Zeiss LSM 510 confocal microscope,
Carl Zeiss, Jena, Germany), using a 20 objective, were made
through the antennae. The contrast of the relatively thick-walled
2
Proc. R. Soc. B 282: 20143018
2. Material and methods
placed on a rotator and rotated at 4500 rotations min21 for
2 min, in a cycle of 6 s on, 4 s off. In D. melanogaster, cVA deposits
are mostly found on the abdominal segments. However, perfuming the fly with cVA results in more equal distribution of
pheromone across the body. We therefore increased the total
amount of cVA slightly to compensate for lower local concentrations on the fly. After shaking, the group of seven treatment
(þcVA) or seven control (2cVA) male flies, both shaken on the
rotator, were introduced into the food vial containing five
virgin females.
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that mediate behavioural antagonism, and typically disrupt
orientation to conspecifics [11].
Perhaps owing to the complex role that cVA fulfils in a
range of sexual and social behaviours in Drosophila, evolution
of the signal to convey species-specificity may be constrained.
Besides, other, non-volatile Drosophila pheromones fulfil a
function in species recognition instead [12]. These are nonvolatile pheromones, produced by enocytes and embedded
in the cuticular hydrocarbons (CHs), and sensed through
the taste sensilla on the legs and proboscis [13–15].
However, in spite of the evolutionary constraints cVA may
have, here we report how this conserved pheromone has
undergone a radical functional reversal in Drosophila suzukii,
a melanogaster group species. Drosophila suzukii does not produce cVA. Here, we describe the changes that are at the basis
of the inversion of the role of cVA from a broadly used sex
pheromone into a behavioural antagonist in this species.
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trichoid sensilla permitted identification and counting of trichoid
sensilla on the entire antennal surface.
(h) Immunocytochemistry
We verified antennal lobe projection patterns of T1 neurons in
D. suzukii (Trentino strain and US strain, as indicated in §3)
using anterograde-neurobiotin (Molecular Probes, Carlsbad,
CA, USA) backfills. Neurobiotin is readily taken up by neurons
and transported throughout the neuron, including its axonal targets in the antennal lobes. A glass microelectrode with a 0.25 M
KCl þ 2% neurobiotin was placed over the tip of a T1 neuron.
Neurobiotin was allowed to diffuse into the sensillum and
taken up by the neuron for 1 h. Preparations were then fixed in
0.1 M PBS with 0.25% Triton-X þ 4% formaldehyde for 3.5 h at
48C, dissected, washed 3 with PBS containing 0.25% Triton-X
(PBST) and incubated with fluorescein-avidin 488. Ten per cent
mouse a-synapsin antibody (Hybridoma, University of Iowa,
Iowa, IA, USA) was included to identify targeted glomeruli in
the antennal lobes. After 24 h at room temperature on the rotator,
brains were washed 3 with PBST and incubated with antimouse conjugated with Alexafluor 546 (Molecular Probes).
After another 24 h on the rotator at room temperature, brains
were washed 3 with PBST and mounted in Vectashield Hard
Mount (Vector Laboratories), 0.12 mm thick, using spacer rings
(Secure-Seal TM imaging spacers, Sigma-Aldrich). The abovedescribed technique for mouse a-synapsin antibody staining
was also used for overview stainings and reconstructions of
antennal lobes of D. suzukii, D. biarmipes and D. subpulchrella.
(i) Confocal microscopy and reconstructions
Whole-mount brains were viewed in a Zeiss LSM 510 confocal
microscope (Carl Zeiss) equipped with a 40, 1.4 oil-immersion
DIC objective lens. Structures labelled with Fluorescein Avidin
were excited with an Argon laser at 488 nm with detection of
reflected light in the range of 505–515 nm. Alexa 546-labelled structures were excited with a HeNe laser at 543 nm and detected using
a 560 nm long pass filter. Stacks of 50–200 confocal images were
scanned and the images were stored at a size of 1024 1024
pixels. The three-dimensional reconstructions, volumetric measurements of the glomeruli were done using AMIRA v. 3.0 software
(Visage Imaging, Berlin, Germany). In every optical section, the
contours of glomeruli were demarcated by hand (i.e. image segmentation) and interpolated. Volumes were obtained from AMIRA,
based on reconstructed images.
(k) Phylogenetics
For each of the gene families (Desaturases, Elongases, Fruitless and
Odorant receptors), we constructed nucleotide datasets and
checked for the correct reading frame. The translated protein datasets were aligned using MUSCLE v. 3.8.31 [25], and preliminary
tree searches were done to assess the actual orthology of genes.
These preliminary analyses led to recognition of a few wrong
orthologies (false positives owing to gene loss) and characterization of new orthologues in unannotated genomes. After this
manual curation, a second round of MUSCLE alignment using
option—refine was done followed by phylogenetic reconstruction
using PHYML [26] and employing 100 non-parametric bootstrap
replicates and the LG þ G model [27] of replacement.
(l) Statistics
Behavioural data (mating rate) were analysed using a Kaplan–Meier
survival probability estimator and curves were statistically compared
using a log-rank test. Correlation between cVA depletion after
application (measured through GC-MS analysis, see above) and
the relative mating rate in D. suzukii were analysed with a Pearson
product–moment correlation coefficient, with the relative mating
rate of cVA-perfumed D. suzukii being the fraction of the mating
rate of cVA perfumed over control flies. Anatomical data (EB and glomerular volumes) were analysed using independent two-sample
t-tests. Sensory physiological data (spiking rate) were analysed
with a one-way ANOVA, followed by Tukey’s HSD post hoc test.
3. Results and discussion
(a) cVA production is lost in Drosophila suzukii, and its
production site atrophied
Using gas chromatography-coupled mass spectrometry
(GC-MS), we analysed the CH profile of D. suzukii. Unlike
the CH profile of D. melanogaster, D. suzukii’s CH profile was
isomorphic, with just small quantitative differences between
sexes (figure 1a,b). This was also true for its sibling species
D. biarmipes and D. subpulchrella (electronic supplementary
3
Proc. R. Soc. B 282: 20143018
Drosophila suzukii (Trentino strain) and D. melanogaster (WT Dalby)
adult males were collected two days after emergence. For each
species, a total of 10 males were selected. After brief anaesthetization in the freezer, individuals were dissected in phosphatebuffered saline (PBS). The abdomen was clipped and immersed
in 5% KOH for 5 h to remove soft tissues and expose intact hard
cuticular structures, washed in distilled water and partly dehydrated in 70% ethanol. Afterwards, the ejaculatory bulb (EB
[19,20]) was separated from the rest of the male genitalia and
mounted on a glass slide with glycerine. Observations were
made with microscope (Leica LMD7000, Wetzlar, Germany). The
EB dimensions were measured using a Leica LMD7000 microscope
with LEICA APPLICATION SUITE IMAGE ANALYSIS SOFTWARE (n ¼ 10). As
individuals within and among species differed in size and in
order to obtain values comparable from one animal to the others,
the estimates of EB dimensions of D. melanogaster were compensated for by the smaller body length compared to D. suzukii
(multiplication of the dimensions by the ratio (average D. suzukii
length/average D. melanogaster body length)). Volumes were calculated by assuming a sphere (4/3 p fw/2g fh/2g fd/2g).
Measurements from 10 individuals were averaged.
Orthologues searches and assembly. We downloaded various types of
genome data (gene, transcript and protein sequences) for odorant
receptors and other cVA-related genes (desaturases, elongases,
Fruitless, Transformer) of D. melanogaster, Drosophila simulans, Drosophila sechellia, Drosophila erecta and D. ananassae from Flybase [21]
and OrthoDB [22] repositories. For D. biarmipes and D. suzukii,
the protein sequences of D. melanogaster were used for queries to
identify the corresponding orthologues through exhaustive blast
searches. First, TBlastN (BLOSUM 62 matrix with an e-value
threshold of 1025 [23]) was applied for preliminary search of all
the gene families. For the elongases superfamily alone, orthologues were searched using HMMER (http://hmmer.janelia.org/
v3.1b1, using an e-value threshold of 1025) and the following specifications: profile ‘hmmbuild’ was constructed from GNS1/SUR4
(ELO;PF01151) HMM profile of the PFAM database [24], the result
of which was run against D. suzukii and D. biarmipes genomes using
‘hmmsearch’; the output was manually checked for all the positive
hits, specifically looking for the presence of elo-specific domain
HXHH and hydrophobic transmembrane regions. For fruitless, we
underwent a complex manual curation directly from D. suzukii
genome contigs, using the 11 main isoforms of D. melanogaster to
construct putative exons in D. suzukii. This is because the fru gene
spans over a long genome region, contains long introns and codes
for various different isoforms, which are unlikely to be present in
transcriptomic data or being correctly annotated in gene models.
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(g) Ejaculatory bulb
( j) Bioinformatics and phylogenetics
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(a)
(b)
4
2000
males
12
Dm
1600
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IS
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15 17
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16 18 20 22
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eicosane
9-heneicosene
x-heneicosene
heneicosane
unknown
x-docosene
x-docosene
docosane
2-methyldocosane
5,9-tricosadiene
9-tricosene
7-tricosene
5-tricosene
tricosane
3-methyltricosane
9-tetracosene
x-tetracosene
tetracosane
5,9-pentacosadiene
9-pentacosadiene
7-pentacosadiene
pentacosane
heptacosane
2-methyloctacosane
nonacosane
*
*
*
*
0
1
*
3
5
7
9
11
15
13
compounds
17
19
21
23
25
Figure 1. (a) Chromatograms showing female and male D. suzukii CHs. Arrow indicates retention time where cVA would elute. Inset: chromatogram of
D. melanogaster with the arrow indicating cVA (see also electronic supplementary material, figure S1). Note the dominance of tricosenes in both sexes. Tricosenes
are more abundant in male D. melanogaster’s CH profile. IS internal standard (heptadecenyl acetate, 17:OAc). Asterisks (*) indicate significantly higher amount in
females ( p , 0.05). (b) Comparison of the CH profile of male and female D. suzukii (n ¼ 6 and n ¼ 5, respectively). Stars indicate significant differences between
males and females (Mann – Whitney test, a , 0.05). x ¼ unknown double bond position. (Online version in colour.)
material, figure S1c,d). We note that D. suzukii and D. biarmipes
lacked the desaturase desat2 and the elongases eloF, which have
been implied in the biosynthesis of species-specific and sexually dimorphic CH profiles in Drosophila ([28,29]; electronic
supplementary material, figure S2). However, more striking
was the lack of cVA in the chemical profile of D. suzukii
(figure 1a, arrow, verified with D. suzukii from the USA: electronic supplementary material, figure S1d). The lack of cVA
in D. suzukii casts the question of whether this pheromone
might have been replaced by another compound similar to
cVA. However, no other cVA-type compounds (including
the C20 variant present in some species [1]) were found in
D. suzukii cuticular extractions (figure 1a,b and electronic supplementary material, figure S1d). In fact, the EB, the production
site of cVA [30], was dramatically reduced in volume in
D. suzukii compared with that of D. melanogaster (by a factor
of 7.0, p , 0.001, unpaired t-test, n ¼ 10, d.f. ¼ 18, t ¼ 10.1,
d ¼ 3.22, figure 2a). Intact, conserved homologues of corresponding genes for cVA production, elo68 and desat1, were
nevertheless present in the genome of D. suzukii (table 1 and
electronic supplementary material, figure S2), as were
miRNA-124 binding sites upstream of transformer, a factor
involved in cVA production in male D. melanogaster (electronic
supplementary material, figure S3e).
(b) The relative volume of glomeruli tuned to cVA is
reversed in Drosophila suzukii
We then determined how loss of cVA might have influenced its
corresponding olfactory circuitry. In D. melanogaster, the trichoid sensillum T1 and its cognate Or67d-expressing neuron [5]
are key in cVA-mediated behaviours. T1s project to a large glomerulus, DA1 [31,32]. T1s are the most abundant sensillum
type in D. melanogaster [33]. Although T1s were fully functional
(figure 1c), and its receptor, Or67d, highly conserved (table 1
and figure 3), T1 sensilla were rare in D. suzukii antenna: we
identified only around 7–10 T1s per individual, compared to
55–60 found in D. melanogaster [33]. Accordingly, the relative
volume of DA1 was minute in both sexes of D. suzukii when
compared with D. melanogaster ( p , 0.0001, two-tailed independent t-test, t ¼ 10.5, n ¼ 8, d.f. ¼ 6, d ¼ 3.7, figure 4 and
electronic supplementary material, figure S4).
At close range, cVA also induces responses in Or65aexpressing OSNs housed in antennal trichoid T4 [32,34,35].
Or65a neurons counteract behaviour induced through Or67d
OSNs, and reduce cVA-mediated male–male aggression [8],
as well as male attraction to recently mated females [35]. In
D. suzukii, Or65a was highly conserved (table 1), and T4 sensilla were abundantly present and responded to cVA touch
stimulation [34] (figure 2c). The neuron may be slightly more
sensitive to cVA in D. suzukii than in D. melanogaster, responding prior to contact, unlike in D. melanogaster (less than 1 mm
distance). The cognate antennal lobe glomerulus, DL3, which
receives input from Or65a neurons, was enlarged in D. suzukii
(two-tailed independent t-test, t ¼ 12.9, d.f. ¼ 6, p , 0.001, d ¼
4.6, figure 4) and 19% larger in male than female D. suzukii
(two-tailed independent t-test, t ¼ 3.13, d.f. ¼ 6, p ¼ 0.01, d ¼
1.1). Other glomeruli innervated by sensilla trichodea and sensilla intermedia neurons [32,33] were similar in volume
between the two species (except for DA4m) and sexually isomorphic (figure 4). The total number of trichoid sensilla was
similar between species (figure 2b).
Proc. R. Soc. B 282: 20143018
11
amount (ng ± s.d.)
1400
compound
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*
females
1800
peak no.
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(a)
(b)
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120
4
EB volume (×10–3 mm3)
5
3
80
2
40
1
***
Proc. R. Soc. B 282: 20143018
0
0
Ds
100 mm
Dm
Ds
(c)
Dm
(d)
Ds
Dm
T1 response
Ds
120
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Ds
80
DL3
VA1D
40
DA1
VA1V
Dm
0
0
2 6
8 10
12
1
3
log dose (ng)
5
Figure 2. (a) Micrographs of the EB, lateral view, light and autofluorescence microscopy (insets). Overlay: volumetric estimates of the EB of D. suzukii (Ds) and
D. melanogaster (Dm) (n ¼ 10 for each species, independent t-test p , 0.001). ***, p , 0.001. (b) Number of sensilla trichoidea and sensilla intermedia in
D. suzukii and D. melanogaster females. (c) Top: sample trace of T1 sensillum responses to 0.5 s 1 mg cVA stimulation. Bottom: sample traces of T4 sensilla
in D. suzukii and D. melanogaster to cVA, using the ‘touch’ stimulation [24], with the time (s) indicated at the bottom of the traces. Blue: before stimulation,
orange: just prior to contact, red: contact. Side panel: dose response curves of T1 sensilla in D. suzukii and D. melanogaster to 0.5 s cVA stimulation (red bar,
n ¼ 6 per species). (d ) Neurobiotin backfill of T1 neuron (a spurious fill of a neighbouring AB7 neuron toVM5v is also visible). Letters indicate various anterior
trichoid glomeruli. Arrowhead indicates DA1.
Table 1. Olfactory receptor, desaturase and elongase genes are conserved and under purifying selection. Values are Ka/Ks (dN/dS) with the actual value of the
ratio (omega) in brackets. Values have been calculated using http://services.cbu.uib.no/tools/kaks under a maximum-likelihood framework providing input trees
according to trees from figure 3 and electronic supplementary material, figures S2 and S3. In all cases, D. suzukii has an omega substantially lower than 1,
suggesting purifying selection and therefore conserved function. Asterisk (*) denotes values obtained using topology: (((mel1,(ere1,ere2)),(bia,(suz1,suz2))),ana);
similar results were obtained when using an alternative topology: ((((mel1,ere1),(bia,suz1)),ana),(suz2,ere2)); compare with tree A of electronic supplementary
material, figure S2.
DesatF
Desat1
Or65a
Or67d
Elo68a
EloC2*
Elop
D. suzukii
0.013/0.288
0.008/0.124
0.033/0.140
0.024/0.181
0.016/0.114
0.034/0.121
0.045/0.142
D. biarmipes
(0.044)
0.014/0.115
(0.062)
0.005/0.07
(0.234)
0.037/0.166
(0.135)
0.021/0.156
(0.1447)
0.026/0.113
(0.280)
not present
(0.317)
0.043/0.075
(0.225)
(0.136)
(0.233)
(0.124)
(0.074)
(c) Glomerular volume and reversal from pheromone to
antagonist
Or65a and DL3 provide context-dependent suppression of cVA
responses in D. melanogaster, putatively by suppressing output
of DA1 via antennal lobe inhibitory interneuronal connections
[8,17]. We therefore hypothesized that the reversal of glomerular volume ratios in D. suzukii’s cVA circuit (DA1/DL3 from
3.3 in D. melanogaster to 0.41 in D. suzukii, figure 4) would
(0.575)
favour Or65a-mediated behavioural responses to cVA, generating opposite behavioural outputs to those observed in
D. melanogaster. We tested this by applying cVA to male
D. suzukii cuticle with doses equivalent to those found on
male D. melanogaster and assaying the effect on conspecific
courtship behaviour. Because mating rates are low in
D. suzukii, and because applied cVA rapidly decreases over
time on the cuticle (figure 5, red line), we grouped seven
virgin males and five virgin females to ensure sufficiently
Downloaded from http://rspb.royalsocietypublishing.org/ on March 2, 2015
D.biaOR67d
D.suzOR67d
D.anaOR65a3
D.secOR65a
D.anaOR83b
D.ereOR83b
D.melOR83b
D.secOR83b
Or83b / Orco
D.simOR83b
–
0.1
D.biaOR83b
D.suzOR83b
Figure 3. cVA odorant receptors are conserved in D. suzukii. Phylogenetic tree
of Or67d and Or65a in D. suzukii and other Drosophila. These genes are extremely conserved in D. suzukii and the other species sampled. This suggests
that these genes are indeed expressed in neurons of T1 and T4 sensilla
and are structurally constrained to recognize cVA, as indicated by the electrophysiological reponses. The tree is rooted with the Or coreceptor Orco.
Abbreviations and supports are as in electronic supplementary material,
figure S2. (Online version in colour.)
0.30
D. melanogaster
fraction of total ‘trichoid volume’
*
0.25
Dm
Ds
Ds
D. suzukii
D. suzukii
*
0.20
*
0.15
*
0.10
0.05
0
DA1
DL3
VA1d VA1v
DA3 DA4l DA4m DC1
glomerulus
DC3
VL2a
Figure 4. Volume of DA1 and other trichoid glomeruli, relative to the total
volume of all glomeruli receiving input from sensilla trichodea and intermedia.
VL2a, which receives input from coeloconic Ac4a neurons, is included as it is part
of the fru circuitry. Red-outlined bars: DA1 of D. melanogaster and D. suzukii F
and C. Insets are reconstructions of the antennal lobes, with DA1 in bright
red, and other glomeruli that receive input from sensilla trichodea and sensilla
intermedia neurons in light red. *, p , 0.05. Scale bar, 20 mm.
high courtship and mating rates in the bioassay. As we predicted, application of cVA on male D. suzukii strongly
suppressed mating (figure 5 insets, Kaplan–Meier estimator,
Z ¼ 4.45, p , 0.001, n ¼ 150). By contrast, perfuming male
D. melanogaster with cVA did not affect the mating rate
cumulative mating
6
+ cVA
– cVA
0.50
600
***
0.25
0
400
0.5
Y = –0.74ln(x) + 4.44
R = 0.927, p < 0.025
200
0
0
1
2
4
3
5
6
time (h)
Figure 5. Effect of cVA perfuming on mating in D. suzukii and D. melanogaster.
The relative mating rate increased (blue line, n values see below) with a decrease
in cVA levels on the male flies over time (n ¼ 5 per data point). Insets: cumulative mating in Dm and Ds in response to the perfuming with cVA (þcVA, red
lines, n ¼ 21, 30 for Dm and Ds, respectively) or hexane (control, 2cVA, grey
lines, n ¼ 19, 26 for Dm and Ds, respectively). ***, p , 0.001.
(figure 5 insets, Kaplan–Meier, Z ¼ 0.33, p ¼ 0.36, n ¼ 110,
[5,6]). Following an initially nearly complete shutdown of
mating in D. suzukii, when cVA doses were high, mating
rates slowly increased over experimental time (figure 5, blue
line, the relative mating rate of perfumed or non-perfumed
flies). This was significantly correlated with a gradual loss
of cVA on the cuticle (figure 5, Pearson’s r ¼ 0.93, t ¼ 4.38,
p , 0.025). The down-regulation of T1 sensilla expression, the
volume decrease of DA1 and the suppression of mating in
cVA-perfumed D. suzukii support a model in which Or65a
and DL3 suppress Or67d-mediated behaviours similar to
observations in D. melanogaster [7,8,17,35]. Apparently, in
D. suzukii, the balance of DA1 and DL3 input and output has
shifted through evolution, resulting in a chronic suppression
of cVA-induced behaviours in this species, when compared
with other melanogaster group species. This effectively has
reversed the role of cVA from a pheromone to a heterospecific
signal that inhibits mating.
The concurrent miniaturization of DA1 volume and the
behavioural shift in response to cVA are reminiscent of observations in Drosophila’s coding of general odour preference
[36,37] and preference coding of pheromones in moths [38].
In these studies, changes in relative glomerular volume
were associated with shifts in olfactory preference. Increased
glomerular volume is associated with an increased preference
for the ligand of these glomeruli. In this study, however, we
observed the opposite: a severe reduction in glomerular
volume converts attraction into inhibition, suppressing the
cascade of behaviours associated with its ligand (figure 6a).
What factors underlie the reduced T1 expression and diminution of DA1 are unknown. An important factor in driving
sexual behaviours in D. melanogaster is the transcription factor
fruitless ( fru). A male-specific splicing variant of fru, FruM,
causes sex-specific neuronal growth, targeting and corresponding behaviour [31,39 –42]. Our gene annotations show
nevertheless that the fru region, spanning 100 kb of genomic
DNA, contains all putative exons to build the various isoforms found in D. melanogaster, including FruM (electronic
supplementary material, figure S3; [31]). Other well-known
genes involved in sexual dimorphism of the olfactory circuitry, such as sexlethal (sxl [43,44]), transformer (tra [43,45]) and
Proc. R. Soc. B 282: 20143018
100
D.anaOR65a2
D.anaOR65a1 Or65a
D.suzOR65a
(conserved in
D. suzukii and
D.biaOR65a
duplicated in
D.ereOR65a
D. ananassae)
D.melOR65a
D.simOR65a
1.0
800
Ds
0.75
amount of cVA (ng fly–1)
100
Or67d
(conserved in D. suzukii)
relative mating rate (treatment/ctrl)
D.melOR67d
Dm
1.5
rspb.royalsocietypublishing.org
D.anaOR67d
100 D.ereOR67d
D.simOR67d
D.secOR67d
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D. suzukii
DL3
OSNs
–
67d
DA1
65a
DL3
OSNs
67d
(b) D. subpulchrella
PNs
–
DA1
PNs
D. biarmipes
Figure 6. (a) Schematic overview of the change in the circuitry of D. suzukii
compared to D. melanogaster. Based on earlier work [6], we conclude that
reduction in volume of DA1 in D. suzukii lead to the observed chronically
depresses DA1 output and the observed inhibition of sexual behaviour.
OSNs, olfactory sensory neurons; PNs, antennal lobe projection neurons.
(b) Reconstructions of the antennal lobes of D. subpulchrella and D. biarmipes.
In bright red DA1, which received input from T1 neurons, and in light red
other glomeruli receiving input from sensilla trichodea neurons. Note: the
small volume of DA1 in D. subpulchrella, which was comparable to D. suzukii
(see figure 4).
doublesex (dsx [33,34]), were also well conserved in D. suzukii
(electronic supplementary material, figure S3). However, we
noted that the volume of DA1 is sexually isomorphic in
D. suzukii (figure 4). This contrasts with D. melanogaster,
where Fru causes a substantial dimorphism in volume and
behaviour between males and females [31,42]. Similarly, the
two other glomeruli receiving input from sensory neurons
in the fru circuitry, VA1v and VL2a, were also sexually isomorphic (figure 4). However, DL3 was 19% enlarged in
male compared with female D. suzukii (see above), whereas
DL3 is isomorphic in D. melanogaster [31]. This may indicate
an altered expression of Fru in the olfactory circuitry of
D. suzukii males and females. This notion of an altered
expression pattern in D. suzukii is substantiated by the observation that Fru is translated in brains of both sexes of
D. suzukii, whereas in D. melanogaster only in males [46].
(d) Loss of cVA is associated with the species’ shift to
undamaged fruit
The loss of cVA as sex pheromone in D. suzukii may be an adaptation associated with its new niche, or reflect this species’
higher reliance on visual cues (males have a conspicuous
wing spot) over olfactory cues in sex discrimination. It may
also be simply the result of drift, although the presence of
cVA throughout the melanogaster group may render this a less
4. Conclusion
Sex pheromones are intraspecific signals involved in influencing behaviours of conspecifics during sexual communication.
cVA is a sex pheromone produced by male Drosophila species
and fulfils a complex role in intraspecific communication. We
show that, in spite of the fact that cVA signalling fulfils a
primal role and is highly conserved in the melanogaster group,
it has been lost in D. suzukii. We furthermore demonstrate
that its underlying circuit can rapidly evolve to serve a radically
opposite behavioural and ecological function, mediated by offsetting the balance of sensory input. Although this study does
7
Proc. R. Soc. B 282: 20143018
D. melanogaster
65a
likely scenario. We therefore compared D. suzukii to the other
two suzukii group species, D. subpulchrella and D. biarmipes,
males of which also have conspicuous wing spots [47]. Whereas
both species were found to have sexually monomorphic
CH profiles (electronic supplementary, material figure S1),
D. biarmipes, a species that oviposits on rotten fruit, produced
large amounts of cVA, whereas D. subpulchrella, a species with
a similar ovipositor and oviposition preference to D. suzukii,
lacked cVA. As expected, the relative volume of DA1 in
D. subpulchrella was much reduced and comparable to DA1
in D. suzukii, whereas that of D. biarmipes was similar to
D. melanogaster (figure 6b). Apparently, loss of cVA is not associated with the use of a conspicuous wing spot. Instead, our data
suggest that the loss co-occurs with the shift to fresh fruit.
Although the above results demonstrate that cVA (i) has
been lost in D. suzukii and (ii) reduces mating rates in D. suzukii,
the selection pressures that have led to these shifts are unknown.
Loss of cVA production to increase species recognition seems
unlikely, as CHs, and not cVA, appear paramount in mate preference and species recognition of drosophilids [12]. The fact
that cVA reduces mating in D. suzukii may thus be a consequence
rather than a cause.
Further, it is uncertain whether reduction of T1 abundance
preceded loss of cVA production or the other way around. The
first scenario would support the sensory drive hypothesis,
whereby the sensory characteristics of the detection system
drive the evolution of intraspecific signalling [48]. For instance,
T1 loss may have been selected for to suppress, e.g. oviposition
in the presence of interspecific competitors (i.e. other Drosophila
spp. that use cVA deposits for oviposition aggregation [1]).
cVA loss would then have followed the adaptation in the
detection circuit.
Alternatively, cVA production may have been lost first.
A variety of unrelated factors may, alone or in combination,
have caused loss of cVA. For instance, cVA production
may have been lost owing to pressure of natural enemies
cueing in to cVA in habitats where the species evolved [49].
Or, the shift in ecology to fresh fruit, along with a change
in oviposition behaviour (single ovipositions in undamaged
fruit), may have obliterated the need for cVA as oviposition
aggregation pheromone.
It should be noted that cVA production constitutes a large
cost for male Drosophila: a male D. melanogaster carries approximately 1 mg cVA, which is roughly 1/200th of the dry weight of
a male Drosophila fly [50]. If uncountered by other fitness
consequences, loss of cVA would be highly selected for. Be it
as it may, further work on the evolutionary forces that have
led to the change in pheromone communication in D. suzukii
is needed.
rspb.royalsocietypublishing.org
schematic overview cVA circuit
(a)
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8
Author contributions. Conceived the idea: T.D. Experimental design and
data acquisition: T.D., S.M., S.R., S.Ra, S.L., P.G.B., S.A., S.L., O.R.-S.,
G.A. Data analysis and interpretation: T.D., S.M., S.R., S.Ra, O.R.-S.,
G.A. Data analysis and interpretation: T.D., S.R., S.Ra, S.L., P.G.B.,
S.A., O.R.-S., G.A.. First draft of manuscript: T.D. Comments and
revision: T.D., S.M., S.R., S.Ra, S.L., P.G.B., S.A., O.R.-S., G.A.
Funding statement. This work was supported by the FORMAS 2007-1491
(IC-E3 to the division of Chemical Ecology), 220-2007-1491 (T.D.) and
2011-390 (P.G.B.), SLU funding on Invasive Species (T.D., S.R., S.M.),
The Nilsson’s Minnesfond (T.D., S.M.), the Autonomous Province of
Trento (Accordo di Programma 2012– 2013) (S.R., O.R.-S., S.R., G.A.)
and the Free University of Bozen-Bolzano internal funds (S.A.).
Conflict of interests. The authors declare no conflicting interests.
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