J Comp Physiol A (2014) 200:251–264

DOI 10.1007/s00359-014-0891-5

Review

Neuroethology of male courtship in Drosophila: from the gene

to behavior
Daisuke Yamamoto · Kosei Sato ·
Masayuki Koganezawa 

Received: 19 July 2013 / Revised: 29 August 2013 / Accepted: 11 February 2014 / Published online: 25 February 2014
© Springer-Verlag Berlin Heidelberg 2014

Abstract  Neurogenetic analyses in the fruit fly Dros- dTrpA1 Drosophila transient receptor potential
ophila melanogaster revealed that gendered behaviors, cation channel subfamily A1
including courtship, are underpinned by sexually dimor- EcR Ecdysone receptor
phic neural circuitries, whose development is directed in EPSPs Excitatory postsynaptic potentials
a sex-specific manner by transcription factor genes, fruit- fru  fruitless
less (fru) and doublesex (dsx), two core members compos- Gr Gustatory receptor
ing the sex-determination cascade. Via chromatin modifi- HDAC1 Histone deacetylase 1
cation the Fru proteins translated specifically in the male HP1 Heterochromatin protein 1
nervous system lead the fru-expressing neurons to take on IR84a Ionotropic receptor 84a
the male fate, as manifested by their male-specific sur- mAL Medially located just above the antennal
vival or male-specific neurite formations. One such male- lobe
specific neuron group, P1, was shown to be activated Or67d Olfactory receptor 67d
when the male taps the female abdomen. Moreover, when P2X2 ATP-activated P2X purinoceptor 2
artificially activated, P1 neurons are sufficient to induce ppk  pickpocket
the entire repertoire of the male courtship ritual. These SOG Suboesophageal ganglion
studies provide a conceptual framework for understanding TIF1 Transcription intermediary factor 1
how the genetic code for innate behavior can be embodied TNT Tetanus toxin light chain
in the neuronal substrate. tra  transformer

Keywords  fruitless · Sexual dimorphism ·

Identified neurons · Pheromones · Chromatin Introduction

Abbreviations The courtship ritual of Drosophila melanogaster is histori-

7-T 7-Tricosene cally considered as a fixed action pattern that is composed of
7,11-HD 7,11-Heptacosadiene several discrete behavioral elements (Bastock and Manning
bon  bonus 1955; Hall 1982; Greenspan and Ferveur 2000) (Fig. 1a).
cVA  cis-Vaccenyl acetate These sequential actions are indeed genetically programmed
DEG/ENaC Degenerin/epithelial sodium channels and have been dissected by the genetic method. In the past
dsx  doublesex few years, spectacular advances have been made in unrave-
ling the causal links among the genes, circuitry and behaviors
involved in the courtship as fuelled by many newly developed
D. Yamamoto (*) · K. Sato · M. Koganezawa  methodologies such as optogenetic and thermogenetic tools
Division of Neurogenetics, Tohoku University Graduate School
even at single-cell resolution (Luo et al. 2008; Rideout et al.
of Life Sciences, 2‑1‑1 Katahira, Aoba‑ku, Sendai 980‑8577,
Japan 2010; Venken et al. 2011; Ruta et al. 2010; Kohatsu et al.
e-mail: daichan@m.tohoku.ac.jp 2011; Pan et al. 2011; von Philipsborn et al. 2011).

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252
Fig. 1  Courtship behavior and neural sex determination in Drosoph-
ila. a Steps (1–6) of courtship behavior and the time of FruM action
in development. When a male finds a moving female, he orients his
body axis toward the female and starts to chase her (orientation and
following). Upon approaching the female, the male fly often touches
her abdomen with his foreleg, on which pheromone-sensitive gus-
tatory organs are distributed (tapping). At the same time, the male
fly vibrates, for seconds to minutes, a wing of the side closer to the
female while circling around her (wing extension and vibration).
These unilateral wing vibrations generate so-called courtship songs,
which calm the female (Bennet-Clark and Ewing 1968; von Schil-
cher 1976a, b). When a female becomes sufficiently receptive upon
the exposure to courtship songs, the male licks her genitalia with his
proboscis (licking) and attempts to mount her (attempted copulation).
The receptive female responds to the male by her raising wings and
opening the vaginal plate, assisting the male in his mounting and,
when his attempt is rewarding, allowing copulation to start (copula-
Among dozens of mutant flies isolated, fruitless (fru)
mutant flies have opened up a new avenue for the study of
the molecular and cellular bases of courtship behavior. In
this review, we discuss the molecular mechanism whereby
the master regulator Fru instructs the formation of sexually
dimorphic neural circuitries and how these circuitries oper-
ate to guide sex-specific courtship behavior. Finally, we
attempt to present a general framework which will assist in
the exploration of the mechanistic basis for sexually dimor-
phic behavior in other organisms.
Molecular basis of fruitless function in neural
sex determination
Fru sexually dimorphic expression
The first mutant allele of fru (now called fru1) was iso-
lated in 1963 by Gill (1963) as a byproduct in screens for
male sterile mutants with impaired spermatogenesis. Gill
13
J Comp Physiol A (2014) 200:251–264
tion). Copulation usually persists for 15–20 min. At the end of copu-
lation, the pair releases their genital coupling and the male dismounts
from the female’s back (disengagement). The inseminated female
becomes unreceptive for a few days to a week and engages in ovipo-
sition. Fru deficiency in males during the prepupal and pupal stages
leads to altered courtship behavior after adult emergence (fru mutant).
b The sex-determination cascade. The sex chromosome composition
for female (XX) and male (XY) individuals is translated into the dif-
ferential transcriptional states, active (ON) and silence (OFF), of the
primary female-determinant gene Sxl, respectively. The female-spe-
cific Sxl regulates splicing of its target, tra, ensuring the female-spe-
cific production of Tra. The presence of Tra represses the production
of FruM in females, whereas its absence in males allows the produc-
tion of FruM, which masculinizes a population of neurons. Tra also
acts on dsx, yielding male-type DsxM and female-type DsxF, each of
which supports the sex-specific development of neural and non-neural
cells
noted the complete lack of copulation accompanied by a
strong tendency for homosexual courtship in fru1 males
(Gill 1963). Phenotypic analyses placed fru downstream of
transformer (tra) (Hall 1977; Gailey and Hall 1989; Gailey
et al. 1991; Taylor 1992; Taylor et al. 1994), a female-deter-
minant gene in the sex determination cascade (Salz 2011)
(Fig.  1b). Subsequent molecular cloning of the fru gene
verified this proposal (Ito et al. 1996; Ryner et al. 1996).
Specifically, the second exon of fru was found to contain
three repeats of a 13-nucleotide stretch that matched a Tra-
binding consensus sequence that had been found in the
doublesex (dsx) gene, the only Tra target known at that time
(Nagoshi et al. 1988). Tra is a female-determinant protein
that is translated only in females and stimulates splicing
near its binding site on the primary RNA transcript (also
known as precursor mRNA or pre-mRNA) (Nagoshi et al.
1988). Due to the presence or absence of Tra, the fru pri-
mary transcript derived from the most distal promoter is
subjected to sexually dimorphic splicing, yielding female-
type transcripts with premature stop codons and male-type
J Comp Physiol A (2014) 200:251–264 253
transcripts with long open reading frames (Ryner et al.
1996; Heinrichs et al. 1998; Lam et al. 2003). Because of
this differential splicing, functional Fru proteins are trans-
lated in the male but not the female nervous system, where
the most distal promoter is active (Usui-Aoki et al. 2000;
Lee et al. 2000). The fru gene has at least three additional
promoters that are active in both sexes outside the nervous
system (Ito et al. 1996; Ryner et al. 1996). These non-sex-
specific Fru proteins are poorly characterized (Anand et al.
2001) and will not be further considered here. In addition to
the second exon, the terminal exon is also subject to alter-
native splicing, resulting in five different types of C-termini
of Fru (Ito et al. 1996; Ryner et al. 1996; Usui-Aoki et al.
2000; Goodwin et al. 2000).
The Fru proteins have a BTB domain near the N-termi-
nus and 2 zinc-finger motifs in the C-terminus, with the
exception of two isoforms lacking the Zinc-finger portion
(Ito et al. 1996; Ryner et al. 1996; Usui-Aoki et al. 2000).
Because the BTB domain is involved in protein dimeriza-
tion and zinc-fingers are well-known DNA-binding motifs,
Fru proteins likely contribute to transcriptional regulation
that functions only in the male nervous system. The func-
tional differences between the five Fru isoforms have not
been firmly established (Song et al. 2002; Billeter et al.
2006).
Switching the neural sex by manipulating fru
Evidence that fru acts as a neural sexual switch was first
provided by the observation that artificial expression of
Fru in a female nervous system leads to the formation of a
male-specific trait in such females. For example, a pair of
male-specific muscles (the muscle of Lawrence) (Lawrence
and Johnson 1984, 1986; Gailey et al. 1991; Currie and
Bate 1995; Taylor and Knittel 1995; Nojima et al. 2010) is
induced when a motoneuron innervating the muscle is mas-
culinized by the forcible expression of a normal type of fru
(fru+) from a transgene integrated into an unspecified site
of the genome (Usui-Aoki et al. 2000). However, expres-
sion of fru+ from this transgene in females was unable to
switch the behavioral gender (D. Yamamoto, unpublished
observation), presumably because CNS effects probably
require a precise balance in the expression of different Fru
isoforms, unlike the simple induction of the male fate in a
muscle by a single motoneuron.
One study described how the endogenous fru locus was
engineered with the aim to generate an entire set of Fru-
encoding transcripts that can be expressed regardless of
the sex of the animal (Demir and Dickson 2005). Females
homozygous for this constitutive fru allele (fruM) displayed
male-typical courtship behavior toward other females,
although this courtship activity was not as strong as that
seen in wild-type males (Demir and Dickson 2005). This
observation prompted the authors to propose that Fru acts
like a master regulator gene in organizing the CNS circuitry
for male courtship behavior (Demir and Dickson 2005). A
similar result has been reported (Manoli et al. 2005) based
on the analysis of other fru knock-in alleles.
Sexual dimorphism in single neurons
A subsequent study (Kimura et al. 2005) documented a con-
spicuous sexual dimorphism of a cluster of fru-expressing
interneurons. The cluster was identified using a technique
known as Mosaic Analysis with a Repressible Cell Marker
(MARCM; Lee and Luo 1999), which can be used to label
a group of cells that are derived from a single neuroblast (a
neuroblast clone) or even a single cell (a single-cell clone).
This cluster, which is “medially located just above the
antennal lobe (mAL)” (Lee et al. 2000), exhibits sex differ-
ences in three distinct features (Fig. 2a): the cluster com-
prises 5 neurons in females and 30 in males; in females,
the neurons have a single contralateral neurite descending
to the suboesophageal ganglion (SOG), whereas in males,
some of the neurons have ipsilateral as well as contralat-
eral neurites; in the SOG, the neurite bifurcates to form
a Y-shape in females, whereas it terminates in a simple
horsetail-like structure in males. In males with strong loss-
of-function fru mutations, the mAL cluster is feminized
with respect to all three features described above. Wild-
type females have a smaller number of mAL neurons due
to active elimination of a proportion of cells, as evidenced
by the fact that deleting three cell death genes—hid, grim
and reaper—with the deficiency chromosome Df[H99]
dramatically increases the number of mAL neurons in the
female but not the male brain (Kimura et al. 2005; Kimura
2011). It is possible that mAL neurons are protected from
cell death by Fru in the male brain. The supernumerary
mAL neurons that escaped cell death in the female brain by
the action of Df[H99] have the Y-shaped dendritic arbors,
retaining the female-typical structure (Kimura et al. 2005),
but extend an ipsilateral neurite toward the SOG, a male-
specific structure. These observations indicate, first, that
the sexual dimorphism in the three features—cell number,
the presence or absence of the ipsilateral neurite and the
tip shape of the contralateral neurite—is produced by dis-
tinct molecular mechanisms and, second, that Fru controls
all three of these characteristics (Kimura et al. 2005; Goto
et al. 2011).
Exhaustive identification of other fru-expressing neurons
by subsequent studies using the MARCM or intersectional
technique revealed structural sex differences in approxi-
mately one-third of ~2,000 fru-expressing neurons (Cash-
ero et al. 2010; Yu et al. 2010; Chiang et al. 2011). The
development of methods to scale individual neurons on a
standardized brain (Jefferis et al. 2007; Chiang et al. 2011)
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254 J Comp Physiol A (2014) 200:251–264

Fig. 2  Mechanism of action of
Fru in the production of sex dif-
ferences in single neurons of the
mAL cluster. a The mAL cluster
exhibits sex-related differences
in cell number, the presence or
absence of ipsilateral neurites,
and the shape of contralateral
neurites. b The mAL cluster in
males carrying a hypomorphic
fru mutation has an intersexual
appearance. Single-cell clones
that carry the fru hypomorphic
mutation showed either a male-
typical or female-typical struc-
ture (left-hand side the result
was consistent with hypothesis
1) and did not show an inter-
sexual structure (right-hand side
the result was consistent with
hypothesis 2), indicating an all-
or-none mode of the sex deter-
mination at the single-cell level.
c Single mAL neurons adopt
either a female fate or a male
fate depending on the amount
of Fru they express. d Changes
in chromatin factor expression
phenocopy fru mutants in the
mAL neuron development. e
Proposed mechanism by which
Fru and interacting factors alter
the expression of target genes
that promote or inhibit neural
masculinization

has enabled researchers to deduce the presence of connec-
tions among neighboring arbors of different neurons, and
this has resulted in a putative circuit diagram in which fru-
expressing neurons are found to connect with each other
(Yu et al. 2010), although it needs to be verified whether
these putative connections are actually functional.
Molecular mechanism of Fru action
To obtain insights into the molecular function of Fru,
genetic screens were conducted for mutations that modify
the action of Fru (Goto et al. 2011). The results showed
that overexpression of a normal type of fru (fru+) in the
developing eye disc distorts the regular alignment of the
ommatidia (unit eyes in the compound eye), leading to
the so-called rough-eye phenotype. The severity of this
phenotype is regulated by genetic modifiers, as shown in
experiments in which heterozygous loss-of-function alleles
of 435 different genes with a known function in neural
development were introduced. These studies showed that in
the case of 14 genes, a loss-of-function allele enhanced fru-
induced roughness, and in the case of 4 genes, a loss-of-
function alleles of 4 genes suppressed it (Goto et al. 2011;
Ito et al. 2012). One of the enhancers thus obtained was an
Ecdysone receptor mutation (Goto et al. 2011), which had
previously been reported to synergistically interact with fru
mutant alleles to enhance male–male courtship and abro-
gate neural sexual dimorphism in the antennal lobe (Dalton
et al. 2009). A potent suppressor of the fru-induced rough-
eye phenotype was a loss-of-function allele in bonus (bon)
(Ito et al. 2012). Bon is a fly homolog of the mammalian
transcription intermediary factor 1 family of transcriptional

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J Comp Physiol A (2014) 200:251–264 255
cofactors, which are known to participate in gene silenc-
ing by associating with histone deacetylase 1 (HDAC1)
and heterochromatin protein 1 (HP1) (Nielsen et al. 1999;
Schreiber and Bernstein 2002). The involvement of Bon
and these chromatin factors in fru-dependent development
of the sex-specific circuitry was suggested by genetic inter-
actions between the respective mutations; the reduced male
courtship in hypomorphic fru mutants was dominantly
enhanced by a loss-of-function allele in bon or HDAC1 (on
the genetic locus Rpd3) and alleviated by a loss-of-function
allele in HP1a (the genetic locus Su(var)205) (Ito et al.
2012). Thus, all these factors likely interact in fru-depend-
ent sexual development of the nervous system. However,
rather unexpectedly, HDAC1 and HP1a appear to counter-
act each other, i.e., HDAC1 normally supports Fru-depend-
ent masculinisation of the nervous system, whereas HP1a
supports de-masculinization (Ito et al. 2012) (see also Liu
et al. 2005; Shirangi et al. 2006).
The hypothesis stemming from these behavioral obser-
vations was supported by the examination of single-neu-
ron anatomy (Fig. 2b). In males carrying hypomorphic
fru mutations, the mAL cluster (visualized en masse as a
neuroblast clone) exhibits intersexual characteristics: it
has ~10 cells, ipsilateral neurites and Y-shaped branches
in the contralateral terminal. However, single-cell clones
of mAL cluster neurons show no intersexual characteris-
tics, i.e., each mAL neuron is either male-type or female-
type. Interestingly, in such fru hypomorphic males, knock
down of HDAC1 increases the proportion of male-type
cells, whereas knock down of HP1a decreases it. Therefore,
HDAC1 drives masculinization of single neurons and HP1a
impedes it. This indicates that, in these neurons, Fru func-
tions as an all-or-none sexual switch (Fig. 2c), in which
HDAC1 and HP1a have opposing actions to each other (Ito
et al. 2012) (Fig. 2e).
Immunostaining of polytene chromosomes has revealed
that Fru binds approximately 130 locations. Bon binds to 90
Fru-target sites and an additional ~100 locations. HDAC1
binds to 86 out of 90 Fru-Bon labeled sites and HP1a to 20
of these sites, including several sites shared by HDAC1 and
HP1a. The binding of HDAC1 and HP1a to the Fru target
sites is abrogated in the absence of Bon (Fru itself remains
to bind to these sites), indicating that Bon is required for
binding of HDAC1 and HP1a to the Fru target sites (Ito
et al. 2012). Immunoprecipitation assays demonstrated that
Bon, HDAC1 and HP1a are contained in the Fru-protein
complex and HDAC1 and HP1a compete with each other
in the recruitment to this complex (Ito et al. 2012). As
HDAC1 mediates histone deacetylation that silences tran-
scription (Schreiber and Bernstein 2002; Kharchenko et al.
2010; The modENCODE Consortium 2010) and HP1 has
been shown to play a role in both silencing and activation
of euchromatic gene transcription (Piacentini et al. 2003), it
is tempting to hypothesize that, in masculinizing a neuron,
Fru recruits HDAC1 to the target sites for inducing chro-
matin condensation and gene silencing and that this process
is fine-tuned by HP1a, which counteracts it (Fig. 2e).
Cellular basis of Fruitless function in courtship circuit
development
The male‑specific P1 neuron cluster
Sex differences are prevalent among fru-expressing neu-
rons. Some are sexually dimorphic as is the mAL cluster
described above. Others are sex-specific, including the
male-specific P1 cluster (Fig. 3a, b). This cluster consists
of 20 interneurons that are located in the dorsal posterior
brain and that have a trans-midline neurite connecting lat-
eral protocerebra (Fig. 3c). The P1 cluster plays a central
role in initiating male courtship. In a behavioral applica-
tion of the MARCM technique (Kimura et al. 2008), tra−
mutant clones were generated in the female brain to exam-
ine whether tra−-induced masculinization of a particular
subset of fru-expressing neurons is sufficient for a female
to exhibit male-like courtship behavior. Out of ~200 tra−
mosaic females, 16 females courted a female, using a male-
like behavioral pattern. In 13 of these 16 females (81 %)
the P1 neuron cluster had been masculinized unilaterally or
bilaterally (Fig. 3d3). For another ~40 fru-expressing clus-
ters assessed in this study, there was no correlation between
masculinization and the occurrence of male-like behavior
in the tra− mosaic female flies. Thus, the P1 cluster is a
potent activator of male-type courtship and may function
as a decision-making center that initiates this behavior
(Kimura et al. 2008).
The P1 cluster is not present in females unless the femi-
nizing factor tra is inactivated, as in the MARCM flies used
in the experiment described above (Kimura et al. 2008). As
in the case of the mAL cluster (Kimura et al. 2005), the P1
cluster is absent in females as a result of the female-specific
death of neurons (Kimura et al. 2008). However, unlike in
the case of mAL, Fru proteins have no role in regulating
the male-specific presence of the P1 cluster. Instead of fru,
another Tra target, dsx, is the key factor here. The female-
specific product of this gene, DsxF, eliminates the P1 clus-
ter from the female brain (Kimura et al. 2008; Kimura
2011). In fact, approximately 65 neurons (Rideout et al.
2007, 2010) (out of ~150 dsx-positive neurons) (Lee et al.
2002; Rideout et al. 2007, 2010; Sanders and Arbeitman
2008; Robinett et al. 2010) in the brain express both fru and
dsx and the P1 cluster is composed of this type of neuron
(Kimura et al. 2008). Consistent with this, females carry-
ing a knock-in fru allele, fruM, lack the P1 cluster because
they have the DsxF protein, yet these females display
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256 J Comp Physiol A (2014) 200:251–264

a d1

male

d2

female

d3

c female carrying P1 neurons

artificially induced

d4

male whose P1 neurons are

activated by TrpA1

Fig. 3  P1 neurons with the ability to initiate male courtship. Neu-
ronal staining with a GAL4 enhancer trap that drives expression in
the P1 cluster highlights the presence and absence of the P1 cluster
in the male a and female b brain, respectively. The somata of P1 neu-
rons in the male brain are circled in (a) and the corresponding region
of the female brain is circled in (b). c A neuroblast clone of the P1
cluster labeled by MARCM. The somata of P1 neurons are circled.
The midline of the brain is indicated with a broken line and the con-
tralateral arbors associated with the trans-midline neurites are shown
by an arrow. d The correlation of the P1 neurons with the male court-
ship initiation. d1 Wild-type males carrying a pair of P1 cluster in the
brain courted a female. d2 Wild-type females lacking the P1 cluster
in the brain did not court a female. d3 Females in which a P1 cluster
was artificially produced by MARCM courted a female in a pattern
similar to that of males. d4 Solitary males that carried the dTrpA1
transgene in the P1 cluster initiated courtship when dTrpA1 was acti-
vated by a temperature increase

male-type courtship behavior toward females (Kimura
et al. 2008). This means that, even though the P1 cluster
strongly promotes the initiation of male-type courtship,
it is not absolutely necessary for this behavior to occur.
Another implication of results of the behavioral MARCM
approach is that the female neural network is able to pro-
duce motor outputs for male-type courtship when driven to
do so by, for example, ectopically producing the P1 cluster
in the female brain as has been shown in females carrying
tra− neurons.
P1 neurons initiate male courtship
Forced activation or inactivation of a subset of neurons is a
powerful technique to determine their functions in behav-
ior. Transgenic expression of transient receptor poten-
tial cation channel subfamily A member 1 (dTrpA1), a
warmth-activated channel, in a subset of neurons—using
the MARCM technique or the intersectional GAL4-UAS
strategy (Luo et al. 2008)—induces firing in these neu-
rons when the ambient temperature was increased (Venken
et al. 2011). This approach has revealed two sets of neurons
that can induce courtship behavior in a male placed alone,
i.e., without any courtship target (Kohatsu et al. 2011; Pan
et al. 2011; von Philipsborn et al. 2011). One of the two
sets of neurons was the P1 cluster (Fig. 3d4). The other set
of neurons, which includes P2b (Kohatsu et al. 2011) and
pIP10 (von Philipsborn et al. 2011) (Fig. 4a), comprised
fru-expressing descending interneurons with terminals in
the thoracic ganglia, where the presumptive motor pattern
generator for courtship exists. pIP10 is a single neuron,
whereas P2b consists of ~10 neurons (Kohatsu et al. 2011;
von Philipsborn et al. 2011). It is tempting to postulate
that the P2b–pIP10 set functions as a command center that

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J Comp Physiol A (2014) 200:251–264 257

Fig. 4  The core neural com-

a
ponents that drive a male to
sing. a A diagram showing the
presumptive circuitry for male
courtship. b The male court-
P1: Decision making center
ship song. c A tethered male
fly placed on a treadmill (upper
panel) was subjected to neural
activity recordings with a Ca2+
indicator expressed in fru-
positive neurons (lower panel).
P2b: Command fiber
d The fly shown in c was
stimulated by a female contact,
which resulted in a Ca2+ rise
in the lateral protocerebrum in
the brain dPR1: Song generator c

vPR6: IPI regulator

vMS11: Output gate

d
Sine song Pulse song

drives the thoracic motor pattern generator when activated
by the P1 cluster neurons, whose arborizations are closely
apposed to P2b and pIP10 dendrites in the lateral protocer-
ebrum (Fig. 4a).
Physiological correlates of the courtship-initiating capa-
bility of P1 neurons were recorded from a behaving fly by
means of optical imaging with a Ca2+ sensor protein, Yel-
low cameleon, expressed specifically in all fru-expressing
neurons (Fig. 4c, d) or P1 MARCM clones (Kohatsu et al.
2011). For this purpose, the authors invented the ‘teth-
ered male’ preparation in which a male fly is anchored to
a metal wire on his dorsal thorax and is forced to grasp a
Styrofoam ball on which he “walks” while remaining in
a stationary position. This allows neural activity record-
ings of the brain through a window opened on the head
capsule. The tethered male will readily start to chase a
moving female presented in front of him, provided that he
has touched the female with his foreleg (tapping). While
chasing the female, the tethered male typically extends and
vibrates a wing (Kohatsu et al. 2011), a behavior regarded
as a hallmark of male courtship. Touching the female (but
not seeing a moving female) induced a large Ca2+ transient
in the P1 neurons in these males. Ca2+ transients were
induced in P1 neurons when the tethered male contacted,
by a foreleg, a glass rod coated with an extract of body
surface hydrocarbons, which are major contact-chemical
pheromones, whereas touching an uncoated glass rod did
not induce any response (Kohatsu et al. 2011). Indeed,
receptors for contact chemicals (gustatory receptors) are
distributed in leg sensory hairs, some of which contain
pheromone-responsive cells (see below). These observa-
tions support the notion that the activation of the P1 neu-
rons by chemosensory cues initiates courtship behavior in
males.
Ethological significance of input–output neural
regulation
Motor control of courtship song
The decision to court made by P1 neurons is conveyed,
presumably by P2b/pIP10 neurons, to the song pattern gen-
erator, which is demonstrated to be located in the thoracic
ganglia (Clyne and Miesenböck 2008). Clyne and Miesen-
böck (2008) showed that decapitated male flies generate

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258
quasi-normal courtship songs composed of pulse and sine
songs when fru-expressing neurons are forcibly excited via
ATP-activated P2X purinoceptor 2 channels that have been
transgenically introduced. These flies show a strong ten-
dency to use the same wing in successive courtship bouts,
in contrast to wild-type males, which typically use two
wings alternately (Clyne and Miesenböck 2008). In addi-
tion, compared to songs of normal male flies (Fig. 4b), the
songs generated by decapitated flies have longer intervals
between pulses in pulse songs and lower frequency oscil-
lations in sine songs (Pan et al. 2011). The inter-pulse
interval is unaffected by thorax-restricted fru knockdown
in intact (non-decapitated) males (Rubinstein et al. 2010).
Female flies (which do not express functional Fru proteins)
also generate courtship song-like sounds, but they are dis-
torted in pattern (Clyne and Miesenböck 2008; Kohatsu
et al. 2011; Pan et al. 2011; von Philipsborn et al. 2011).
Together, these findings indicate, first, that the motor pat-
tern generator for male courtship songs resides in the tho-
racic ganglia of both males and females; and second, that
the brain is dispensable for the motor pattern generation per
se, but signals descending from the brain normally modu-
late the thoracic pattern generator. The signals descending
from the brain also seem to be necessary for alternate use
of the two wings and boosting up of the song pitch. Note
that although the male-specific P1 cluster is a strong driver
of male courtship initiation, the thoracic motor center can
generate song motor outputs if it is activated by some other
means regardless of the presence or absence of P1 and P1
activity.
Then which neurons constitute the song pattern genera-
tor? The mesothoracic ganglion contains a sexually dimor-
phic cluster of interneurons expressing both fru and dsx
(Rideout et al. 2007, 2010; von Philipsborn et al. 2011).
Some of these neurons extend arbors in the “wing neuro-
pile”, which contains dendrites of motoneurons innervat-
ing wing muscles. dTrpA1-induced stimulation of a subset
of these neurons known as vPR6 (Fig. 4a) drives the male
to sing, and stronger activation with higher temperature
increases the pulse rate (i.e., it shortens the inter-pulse
interval). Conversely, blocking synaptic transmission in
vPR6 through targeted expression of tetanus toxin light
chain (TNT) prevented the male from singing. The other
fru and dsx double-positive interneuron cluster is located
in the prothoracic ganglion (Rideout et al. 2007, 2010;
von Philipsborn et al. 2011). One of these neurons, named
dPR1 (Fig. 4a), produces pulse songs when activated with
dTrpA1 and inhibits male singing when inactivated through
TNT expression. Activation of dPR1 had no effect on the
inter-pulse interval. dPR1 is likely male-specific and it
bilaterally innervates the wing neuropile in the mesotho-
racic ganglion. Another fru-expressing neuron, vMS11
(Fig.  4a), has been identified as a component of the song
13
J Comp Physiol A (2014) 200:251–264
pattern generator. The vMS11 neurons are located in the
mesothoracic ganglion and innervate the ipsilateral wing
neuropile. vMS11 activation via dTrpA1 results in wing
extension without vibration and its inactivation via TNT
abolishes or disturbs song generation. Unilateral wing
extension is correlated with ipsilateral vMS11 activation.
Based on these observations, it has been proposed (von
Philipsborn et al. 2011) that vMS11 determines the side on
which patterned motor outputs are released into a periph-
eral nerve to move a wing and that dPR1 and vPR6 could
directly receive a descending command from the brain and
be involved in patterning of motor outputs by, for example,
determining the inter-pulse interval of pulse songs (Fig. 4).
Gustatory inputs in male courtship
The courtship motor pattern generator is controlled by the
central decision center, in particular, P1 neurons. P1 neu-
rons are activated by pheromone stimulation given to the
male’s foreleg. What are the pheromones and how are
they perceived by the flies? The composition of cuticular
hydrocarbons in D. melanogaster is sexually dimorphic;
7-tricosene (7-T) is much more abundant in males than in
females and acts as a male-derived aphrodisiac for females
(and an anti-aphrodisiac for males), whereas 7,11-heptaco-
sadiene (7,11-HD) is predominantly produced in females
and acts as a female-derived aphrodisiac for males (Jal-
lon 1984; Billeter et al. 2009; Yew et al. 2009; Everaerts
et al. 2010). Both compounds are non-volatile and per-
ceived by gustatory receptors in the legs and the mouth
part. Exposure to 7-T at physiological concentrations of
10−12–10−8  M evokes impulses in L2 gustatory neurons in
the mouth part of male flies (Lacaille et al. 2007), and stim-
ulation with 7,11-HD induced impulses in the “Tm4c” sen-
sillum on the leg of a male fly (Toda et al. 2012). Further-
more, a study using G-CaMP-aided Ca2+ imaging revealed
that one of two cells paired in a single sensory hair on the
leg of a male fly responds to 7-T and the other responds
to 7,11-HD (Thistle et al. 2012). These pheromone-respon-
sive sensory cells are defined by the presence of pickpocket
(ppk)-23 and, possibly, ppk-29 expression (Toda et al.
2012; Thistle et al. 2012). ppk genes encode a family of
ion channels known as Degenerin/epithelial sodium chan-
nels and are expressed in receptor cells for various sensory
modalities. Mutations in ppk23, ppk25 and ppk29 and/
or knock down of any of these genes reduce male court-
ship activities toward a female (Lin et al. 2005; Toda et al.
2012; Thistle et al. 2012; Liu et al. 2012; Lu et al. 2012;
Starostina et al. 2012). Remarkably, sensory neurons that
express ppk23, ppk25 and/or ppk29 in the leg sensilla
also express fru (Lin et al. 2005; Lacaille et al. 2007; Yew
et al. 2009; Toda et al. 2012; Thistle et al. 2012; Liu et al.
2012; Lu et al. 2012; Starostina et al. 2012). In females,
J Comp Physiol A (2014) 200:251–264 259
the axons of these fru-positive gustatory neurons terminate
exclusively in the ipsilateral thoracic neuromere, whereas
in males the axons cross the midline and also form termi-
nals on the contralateral side (Mellert et al. 2010; Thistle
et al. 2012; Liu et al. 2012; Lu et al. 2012; Starostina et al.
2012). The functional importance of this midline crossing
in males is unknown, despite the fact that this conspicu-
ous sexual dimorphism was reported nearly 2 decades ago
(Possidente and Murphey 1989). It is also unknown how
these pheromone inputs from the leg sensilla are relayed to
the higher centers to initiate male courtship.
Gustatory receptors implicated in pheromone perception
It remains to be determined whether Ppk family proteins in
the leg sensory cells function as receptors that bind phero-
mone ligands. Instead, some of the members of the gusta-
tory receptor (Gr) family, which is distinctly different from
the Ppk family, have been suggested to function as recep-
tors for contact-chemical pheromones in Drosophila. Grs
are seven-pass transmembrane proteins with no homology
to vertebrate chemosensory receptors. Grs are expressed
in gustatory neurons locating primarily in the mouth part,
leg and wing margin (Bray and Amrein 2003; Lacaille
et al. 2007; Ejima and Griffith 2008; Moon et al. 2009;
Koganezawa et al. 2010; Watanabe et al. 2011). Although
Gr68a was initially reported to be a candidate receptor
for female-derived pheromones (Bray and Amrein 2003),
subsequent studies supported the alternative possibility
that Gr68a predominantly functions in mechanosensory
neurons (Ejima and Griffith 2008). Gr39a, a close rela-
tive of Gr68a, however, might be a receptor for a female-
derived pheromone, as its inactivation curtails male court-
ship toward a female (Watanabe et al. 2011). Several Gr
proteins have been shown to play roles in inhibiting male
courtship. Gr33a is likely a co-receptor that presumably
forms complexes with several Grs to constitute functional
receptors in bitter-sensing cells (Moon et al. 2009). Gr33a
mutant males engage in male–male courtship at a higher
rate than wild-type males do. In addition, after remov-
ing Gr66a-expressing bitter cells, 7-T loses its inhibitory
effect on male courtship, resulting in an increased male–
male courtship (Lacaille et al. 2007). Similarly, knockout
of the bitter-taste receptor Gr32a leads to an elevated level
of male–male courtship (Miyamoto and Amrein 2008).
These observations are compatible with the idea that some
of the bitter-taste receptors are activated by binding of
contact-chemical pheromones. Although these receptors
were shown to prevent males from courting other males in
D. melanogaster, they might also function to inhibit male
courtship toward females of other species that have dif-
ferent pheromone compositions (Billeter et al. 2009; Fen
et al. 2013).
Gustatory pheromone signal integration
Blocking synaptic outputs of Gr32a-expressing gustatory
neurons disrupts the male courtship posture (Koganezawa
et al. 2010). Specifically, males with inactivated Gr32a-
expressing neurons are not able to retain a wing in a resting
position while they are vibrating the other wing, resulting in
bilateral wing extension (Koganezawa et al. 2010), in con-
trast to the unilateral wing extension observed in courting
wild-type males. This anomaly is most evident under dark
(red light) conditions (Koganezawa et al. 2010), probably
because visual cues can normally compensate for the lack
of pheromone cues in these male flies. The Grs on the left
and right forelegs may sense an asymmetry in the concen-
tration of pheromones on the ground spilled by a female.
Notably, in males, the axon terminals of Gr32a sensory
neurons are juxtaposed to the mAL contralateral neurites in
the SOG, whereas in females these two neuronal endings
are spatially separated. This finding leads to the postula-
tion that the sex difference in mAL dendritic arborization
patterns is correlated with sexually distinct ways to inte-
grate pheromone inputs (Koganezawa et al. 2010). mAL
interneurons are GABAergic and mutual inhibitory connec-
tions between the left and right pairs are expected because
arbors of the lateral pairs superimpose and mutual synaptic
connections are not rare in insects. Such lateral inhibition
would enhance the left–right asymmetry in ascending out-
puts of mALs, which terminate in a so-called “lateral trian-
gle” in the lateral protocerebrum and a tract in the superior
medial protocerebrum, the regions where P1 dendrites are
densely distributed (Kimura et al. 2008). It remains to be
seen whether mAL outputs affect the excitability of P1 or
P2b neurons.
Olfactory pathways in male courtship
Although gustatory pheromones play a primary role in
Drosophila courtship, olfactory pheromones are also
important for successful mating. Major olfactory organs
in Drosophila are the antenna and maxillary palp. The
olfactory receptor neurons project to the primary olfac-
tory center antennal lobe, which is composed of approxi-
mately 50 glomeruli, similar to their counterparts in the
mammalian olfactory bulb (Jefferis et al. 2007). The pro-
jection neurons extend their axons toward the lateral horn,
with or without making en passant synapses on the mush-
room body, a multisensory integration center (Jefferis et al.
2007). Three dorsolateral glomeruli, DA1, VA1v and VL2a,
are enlarged in males compared to females (Kondoh et al.
2003; Stockinger et al. 2005). In male moths (e.g., Man-
duca sexta) and cockroaches (e.g., Periplaneta americana),
a few dorsolateral glomeruli form a macroglomerular com-
plex that represents a hotline for integrating sex pheromone
13

260
information (Hildebrand 1996). DA1, VA1v and VL2a glo-
meruli in Drosophila may form a similar pheromone hot-
line, segregated from the general odor pathway (Kondoh
et al. 2003). Intriguingly, fru-positive olfactory receptor
neurons selectively innervate these three sexually dimor-
phic glomeruli (Stockinger et al. 2005).
cis-Vaccenyl acetate (cVA) is a volatile pheromone that
is synthesized in the male accessory gland and transferred
to the female during copulation, resulting in attenuation of
her sexual attractiveness, i.e., acting as an anti-aphrodisiac
for males (Butterworth. 1969; Everaerts et al. 2010). The
sexually dimorphic, fru-positive glomerulus DA1 receives
innervation of olfactory receptor neurons expressing olfac-
tory receptor 67d, which responds to cVA (Kurtovic et al.
2007). The projection neurons from the DA1 glomerulus
innervate a sexually dimorphic terminal region in the lat-
eral horn (Datta et al. 2008), where they form functional
connections. Indeed, DA1 stimulation induces postsynaptic
excitatory responses in at least three groups of fru-express-
ing third-order interneurons, DC1, LC1 and LC2, which are
male-specific or sexually dimorphic (Kohl et al. 2013; Ruta
et al. 2010). DC1 interneurons evoke excitatory postsynap-
tic potentials (EPSPs) in a fourth-order descending neuron,
DN1, which is male-specific (Ruta et al. 2010). The DN1
neuron extends an axon to the thoracic and abdominal gan-
glia, implying that it plays a direct role in controlling motor
outputs (Ruta et al. 2010). If this is indeed the case, it is
surprising that the inhibitory pheromone cVA can modu-
late motor output at such a late step of integration (i.e., by
provoking discrete EPSPs in the fourth-order descending
neuron).
Multimodal regulation of courtship behavior
Male courtship is not only regulated by pheromones, but
also by other sensory stimuli, including food odors, sounds
and visual stimuli. The antennal lobe glomerulus VL2a
is innervated by olfactory receptor neurons that express
Ionotropic Receptor 84a (IR84a), which responds to food-
derived odorous compounds such as phenylacetic acid and
phenylacetaldehyde (Grosjean et al. 2011). In general, the
neural pathway for processing pheromones is segregated
from that for general odors including food odors, each pro-
jecting to a different region in the lateral horn. Although
VL2a responds to the food odors, the VL2a PNs project to
the pheromone integration region in the lateral horn (Gros-
jean et al. 2011). Intriguingly, these food odors accelerate
male courtship in an IR84a-dependent manner (Grosjean
et al. 2011).
Male courtship also relies on audition and vision (Eberl
et al. 1997; Sakai et al. 1997; Ewing 1978; Kamikouchi
et al. 2009; Krstic et al. 2009; Kim et al. 2012; Tootoo-
nian et al. 2012). Under dark conditions, males use noise
13
J Comp Physiol A (2014) 200:251–264
generated by females to locate them for courtship, and inhi-
bition of Gr68a-expressing mechanosensory cells impairs
this ability of males (Ejima and Griffith 2008). By contrast,
under light conditions female tracking by a male is guided
primarily by vision (Sakai et al. 1997), although males do
not court moving females unless they are primed by phe-
romonal stimulation (Kohatsu et al. 2011). However, if the
courtship ‘decision maker’ P1 is artificially activated by
dTrpA1, the male initiates courtship of a moving female, or
even a moving rubber band, in the absence of pheromonal
stimulation (Pan et al. 2012) (only courtship directed to
females is considered here). It is notable that a population
of fru-positive visual interneurons in the lobular plate inter-
digitates with DC1 dendrites (Ruta et al. 2010), implying
that they make synaptic connections. Input–output relations
of fru-positive interneurons in the male-specific superior
medial protocerebrum tract and lateral triangle are thus key
to understanding the multimodal integration underpinning
courtship decisions.
Figure  5 shows the model which integrates the experi-
mental data on the circuitry, pheromones and receptors
involved in male courtship.
Conclusions and perspectives
Studies on a mating behavior mutant, fru, in a model organ-
ism, D. melanogaster, successfully unveiled the logic by
which a dedicated single gene orchestrates the genome-
wide transcriptional states and ultimately organizes the
sexually dimorphic neuronal circuitry underlying gendered
behavior. Several recent papers have reported the success-
ful perturbation of mating behavior by the inhibition of
fru with the aid of RNA interference in cockroaches and
grasshoppers, suggesting that fru also instructs the forma-
tion of sex-specific circuitries for gendered behavior in
these insects. It is an open question whether the same logic
underlies the translation of the genetic code for sex-specific
behavior into the operation principle of the neural circuitry
in a wide variety of animals. In fact, the existence of a fru
ortholog in mammals remains controversial. However,
Sry, the male determinant in many mammals (Sekido and
Lovell-Badge 2009), is expressed in the brain in addition
to the gonad (Bocklandt and Vilain 2007), the established
site of its masculinizing action. As Sry is suggested to act
through chromatin modification, this finding invites the
speculation that this gene plays a role analogous to fru in
the mammalian brain.
Courtship behavior is highly diversified among species,
presumably because it is exposed to strong pressure of sex-
ual selection. How are these species differences in courtship
behavior established? Comparative studies with Drosophila
species on neural substrates for courtship behavior will make
J Comp Physiol A (2014) 200:251–264 261
Or67d
cVA Or47b
IR84a
Gr68a
Antenna
7-T Gr32a
Gr33a Gr39a?
Gr66a
7,11-HD ppk23
ppk25 ppk29
Foreleg
Fig. 5  The input and output channels that regulate male courtship.
Indicated at the left of the figure are pheromones known to regulate
male courtship. Suggested receptors for pheromones are shown in
the boxes at left, which represent the sensory organs that house the
pheromone receptors. The boxes in the middle and at right represent
neural centers, in which neuron groups involved in the regulation of
courtship behavior are indicated by abbreviations. The squares drawn
with broken lines indicate neuropiles, i.e., AL antennal lobe, AMMC
it possible to determine which differences in the identified
neurons are critically responsible for species differences in a
given behavior and which genetic changes in a given gene in
evolution are causally associated with the behavioral differ-
ences. Rapid advances in the techniques for genetic manipu-
lation in nonmodel organisms, such as those using TALEN
or CRISPR (Gaj et al. 2013), will open an avenue for the
evo-devo-neuro approach to the question as to how behaviors
were diversified in these species at the molecular and cellular
levels (Yamamoto and Ishikawa 2013).
Acknowledgments  The authors’ work is funded by Grants-in-
Aid for Scientific Research (24113502, 23220007, 1802012 to D.Y.,
25132702 and 24700309 to K.S. and 24570082 and 23115702 to
M.K.) from MEXT, the Strategic Japanese-French Cooperative Pro-
gram from JST (D.Y.) and a Life Science Grant from the Takeda Sci-
ence Foundation (D.Y. and K.S.). The authors thank M. Suyama and
S. Abe for secretarial assistance.
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DA1 DC1
DA1
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LH
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AL
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PG
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