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Behavioral Neuroscience
2015, Vol. 129, No. 5, 549 –563
© 2015 American Psychological Association
0735-7044/15/$12.00 http://dx.doi.org/10.1037/bne0000089
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Aggression in Drosophila
Edward A. Kravitz
Maria de la Paz Fernandez
Harvard Medical School
Instituto de Investigación en Biomedicina de Buenos Aires,
Partner Institute of the Max Planck Society,
Buenos Aires, Argentina
Aggression is used by essentially all species of animals to gain access to desired resources, including
territory, food, and potential mates: Fruit flies are no exception. In Drosophila, both males and females
compete in same sex fights for resources, but only males establish hierarchical relationships. Many
investigators now study aggression using the fruit fly model, mainly because (a) aggression in fruit flies
is a quantifiable well-defined and easily evoked behavior; (b) powerful genetic methods allow investigators to manipulate genes of interest at any place or time during embryonic, larval, pupal or adult life,
and while flies are behaving; (c) the growth of the relatively new field of optogenetics makes physiological studies possible at single neuron levels despite the small sizes of neurons and other types of cells
in fly brains; and (d) the rearing of fly stocks with their short generation times and limited growth space
requirements can easily be performed at relatively low cost in most laboratories. This review begins with
an examination of the behavior, both from a historical perspective and then from the birth of the
“modern” era of studies of aggression in fruit flies including its quantitative analysis. The review
continues with examinations of the roles of genes, neurotransmitters and neurohormones, peptides,
nutritional and metabolic status, and surface cuticular hydrocarbons in the initiation and maintenance of
aggression. It concludes with suggestions for future studies with this important model system.
Keywords: aggression, Drosophila melanogaster, hierarchical relationships, learning and memory,
peptides
Supplemental materials: http://dx.doi.org/10.1037/bne0000089.supp
may sometimes be seen to spread their wings, run at each other, and
apparently butt heads. One of them soon gives up and runs away. If
the other then runs at him again within the next few minutes he
usually makes off without showing fight. (p. 353)
Aggression in Drosophila: The Behavior
In an article dealing both with sex recognition and sexual
selection, Sturtevant (1915) was the first to mention male–male
aggression and to demonstrate a novel female–male rejection
behavior in Drosophila. Using mainly D. ampilophila for experiments, Sturtevant reported,
This brief description suggested that competition likely exists
between males for mating partners, and also that some sort of short
term memory might exist in the loser fly of having recently been
defeated. Females were reported to frighten away a male occasionally by charging at him with spread wings. Sturtevant was
using a different species of fly than the commonly used D. melanogaster of today, and one pattern described by him involving
males sounds more like the “head-butt” that is commonly seen in
female fights (Nilsen, Chan, Huber, & Kravitz, 2004), but the
suggestion of male–male aggression in this early work was clear.
Some 45 years later, Jacobs published a second important article
on aggression (Jacobs, 1960). He referred to the Sturtevant, 1915
article but only in reference to odor perception as an important
factor in courtship behavior. The discussion of this article began
“The charges and tussles of the male Drosophila appear to be
forms of aggression” (p. 186). Jacobs suggested further that this
might be characterized as territorial behavior that was not long
lasting. Their assay arenas were relatively large Petri dishes that
were illuminated from above and the focus of their studies were on
the effects of melanization on mating behavior. A second article by
Jacobs continued the studies of melanization on mating behavior
[i]f two males are courting the same female, they often grow very
excited, especially if she is unwilling to stay quiet. In such cases they
This article was published Online First September 7, 2015.
Edward A. Kravitz, Department of Neurobiology, Harvard Medical
School; Maria de la Paz Fernandez, Instituto de Investigación en Biomedicina de Buenos Aires, Partner Institute of the Max Planck Society,
Buenos Aires, Argentina.
This research was supported by Grants GM099883 and GM074675 from
the National Institutes of General Medical Sciences and a Pew Latin American
Fellowship. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Correspondence concerning this article should be addressed to Edward A.
Kravitz, Department of Neurobiology, Harvard Medical School, 220 Longwood
Avenue, Boston, MA 02115. E-mail: [email protected], or Maria
de la Paz Fernandez, Instituto de Investigación en Biomedicina de Buenos Aires,
Partner Institute of the Max Planck Society, Godoy Cruz 2390, C1425FQD
Buenos Aires, Argentina. E-mail: [email protected]
549
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KRAVITZ AND FERNANDEZ
and aggression using ebony mutants in which ␤-alanine is not
inserted into developing cuticles, and black variants showing reduced synthesis of ␤-alanine (Jacobs, 1978). Fifteen years later a
more complete description of aggression and its relationship to
courtship success in D. melanogaster was published (Dow & von
Schilcher, 1975). This article made no mention of the earlier study
by Jacobs, although the authors commented that there had been
several descriptions of aggressive behavior in other species of flies
over the previous decade. They suggested that their studies were
the first observations on aggression in laboratory stocks of D.
melanogaster. Three patterns of aggressive behavior shown by
males were described: wing threat, charge, and “boxing”. The
results showed wide differences between males in terms of who
occupied the food surface, who attacked first, who won fights and
who had success in copulation, with a tight correlation shown
between the latter two. The data supported the notion that a linear
hierarchy had been established between the males during the time
in the arena before females were introduced, although this was not
specifically discussed by the authors. Instead their focus was on
fitness and the close linkage between success in aggression and
mating.
The most complete descriptions of aggression in fruit flies prior
to those published during the current renaissance were by Hoffmann (1987a, 1987b). Using laboratory-reared males and females
of the sibling species D. melanogaster and D. simulans, Hoffmann
presented a complete ethogram (word description of the patterns
seen) of agonistic interactions between males of the two species.
These included the following: approach, fly on, lunge, hold, tussle,
on back, fall, wings erect, wing vibration, and fly off. Most of
these terms had been coined in earlier studies. The adult flies used
in all experiments were reared under continuous light conditions,
with males collected as virgins and placed in isolation vials prior
to their use in experiments. Six males and three females were
placed in observation cages similar to those described by Dow and
von Schilcher (1975) in the morning and all interactions between
the flies were monitored for the next 8 hours via videotaping. A
food cup containing fly medium coated with live yeast was placed
in the center of the arena. Winners of encounters were defined as
the male who successfully forced another male (the loser) off the
food surface and remained on the cup afterwards. The percentage
usage of the various behavioral patterns by the two species was
described in text tables. The dynamic aspects of the interactions
between both male and female flies over the extended observation
period were described in the text of the article, which focused on
territorial behavior, including acquisition of and defense of the
territory and examination of the linkage between territoriality and
success in mating behavior. The results showed that males occupying territories had greater success in mating than did males not
occupying a territory, with the author reporting in a note that 80%
of the matings took place on the food surface. Size and weight also
influenced the outcome of the fights, a result further described in
a later short article. Although an impressive description of the
behavior, these articles and the interpretation of the usage of
behavioral patterns suffer somewhat from the complexity of the
behavioral scene. For example: in some male–male interactions
hierarchical relationships might already have been established with
animals recognizing familiar opponents and fighting differently
against them compared with nonfamiliar opponents (Yurkovic,
Wang, Basu, & Kravitz, 2006), or social interactions might change
over an extended 8-hr observation period relating to less-active
periods over the course of a day, rather than as a consequence of
social interactions per se, or the use of mated rather than virgin
females might influence courtship object choice by males and
possibly also by females. To avoid such complications, authors of
current studies of aggression usually pair only two males, with or
without the availability of resources like food and potential mates,
depending on the laboratory.
Sturtevant (1915) also was the first to mention that females
sometimes charge at males as a rejection behavior, an observation
that was briefly repeated in Jacobs (1960) as being directed at
other flies during feeding. Other early studies of female aggression
were field studies with different species of fruit flies (Pritchard,
1969) that clearly documented and partially characterized female–
female aggression in relation to oviposition sites. A more recent
publication, also involving field behavior studies, was by Shelly
(1999) and these studies further defined behavioral patterns seen
during female–female aggression (head-butting, pushing, lunging,
and wing threat) and dealt with the ecological value of such
aggression during egg-laying. The first publication describing
female–female aggression in a laboratory setting with D. melanogaster was by Ueda and Kidokoro (2002). Three behavioral patterns were described by these authors, including approach, lunge,
and wings erect. The lunge described here was different from the pattern
described in male aggression as a lunge in that the female does not rise
above the horizontal. Moreover, the authors state several times in
the article that the “behaviors are similar to those of male aggression in this species” (p. 25). The female patterns of aggression in
a laboratory setting were described more fully and renamed recently, in a publication that included a quantitative analysis of
female aggressive behavior (Nilsen et al., 2004). Ueda and Kidokoro (2002) reported that levels of female aggression were
dependent on rearing conditions (isolated more aggressive than
group housed flies) and on the food resource, with flies showing
higher levels of aggression when yeast colonies were growing on
the food. The latter findings were suggested as a potential link
between the quality of food resources and the defense of potential
future egg laying sites, a notion that has been revived in recent
studies (see the following paragraghs).
Modern Era of Studies of Aggression in Drosophila
Quantitative analysis of the behavior: Two articles appeared
almost simultaneously in 2002 (Baier, Wittek, & Brembs, 2002;
Chen, Lee, Bowens, Huber, & Kravitz, 2002), reviving interest in
aggression in fruit flies after a gap of some 15 years. One analysis
by Baier et al. (2002) used chambers that were the same as those
used in the earlier studies, but these authors focused on exploring
the roles of amines in the behavior. Pharmacological treatments
were used to alter amine levels for dopamine (DA) and serotonin
(5HT) but without verification of the effectiveness of the treatments. For octopamine (OA), a mutant line without the amine
generated by Monasterioti et al. (Monastirioti, Linn, & White,
1996), was used and a severe reduction in aggression was reported
in flies without OA. However, controls were missing for mutants
that expressed a w⫺ mutation that likely interfered with vision and
that also might lead to altered amine levels (Borycz, Borycz,
Kubow, Lloyd, & Meinertzhagen, 2008; Sitaraman et al., 2008).
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AGGRESSION IN DROSOPHILA
Finally, flies were placed in arenas for 2 hr before video recording
was begun.
In the second article, Chen et al. (2002) designed a different
chamber than those used before, aimed at observing aggression
between a single pair of flies. These arenas had features built in
that were modeled on real world situations, including allowing
room for losers to escape from winners by leaving the resourcecontaining area. A small cup containing food that the flies had
been reared on was placed in the middle of the chamber, and either
a headless mated female or a small dab of freshly ground yeast
paste was placed on the food surface as an additional attractant
(Certel & Kravitz, 2012). Other arenas that allow for higher
throughput analysis and less fly handling have been described
recently (Fernandez et al., 2010), and our laboratory has just
designed a new arena that requires no handling of flies prior to,
during, or after fights (Trannoy, Chowdhury, & Kravitz, 2015).
This new arena improves all behavioral parameters measured and
in particular preserves the learning and memory that accompanies
fight outcomes. Fights between pairs of flies usually are monitored
by videotaping for 30-min periods after the first social interaction
(called an encounter) between flies on the food surface, or after the
first higher intensity encounter (lunge) is seen. Quantitative analyses of aggression between pairs of male and pairs of female flies
were carried out in the Chen et al. studies using standardized
ethological approaches to the study of behavior. These involved
observation of fights to generate an ethogram of the behavior and
then scoring all patterns and transitions between patterns during
each encounter during fights. This was followed by construction of
transition matrices and a graphic visualization of the matrices
using a first-order Markov chain (see Figure 1). Nine offensive and
four defensive patterns were listed in the male ethogram, many of
which had been described in earlier studies (Hoffmann, 1987a,
1987b; see Figure 1). Female fights tend to be at lower intensity
levels and some of the patterns seen during male fights also are
seen during female fights: others are male or female specific.
Although male fights tend to end with the establishment of a
hierarchical relationship, female fights end with flies sharing the
resource. Why females should expend the energy to compete in
such a case is unclear. It may be that in real world situations,
preferred egg-laying sites may be scarcer. Among the malespecific offensive actions, the lunge is particularly important, as its
usage predicts the outcome of a fight: the first animal to lunge, if
the opponent retreats, is 16 times more likely to be the ultimate
winner. Ultimate winners lunge more and more as fights progress,
losers less and less, and in second fights losers are highly unlikely
to lunge against familiar opponents and less likely to lunge against
any opponents (Yurkovic et al., 2006). A quantitative analysis of
courtship behavior in fruit using transition matrices and first-order
Markov chain analyses was carried out by Markow and Hanson
(1981).
Of particular interest regarding the patterns that make up the
behavior, is that though they are recognizable to observers (they
are stereotypical) and meaningful to opponents, every time a fly
utilizes a pattern it is different. A high-speed video of lunging
behavior (see Movies S1 and S2 in the online supplemental materials) reveals the complexity of this key pattern of behavior.
During the 30th of a second it takes to complete a lunge (Movie
S1), a smooth sequential activation of different motor programs is
seen (Movie S2).1 Thus in a published video (Hoyer et al., 2008),
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the lunging fly (a) raises the anterior end of its body and forelegs,
(b) lifts the wings up then down as the fly rises higher and higher
above the horizontal, (c) forms a sliding tripod between the mid
and hind legs and the wings as the fly approaches its maximum
elevation, (d) strikes down with the forelegs so fast that at 500
frames/s the image is blurred, and (e) attempts to grab onto the
opponent. If the same animal lunges again during a fight, however,
several or many of the motor sequences that make up the pattern
may be omitted. The ability of animals to change patterns in this
way makes good sense, as the success or failure of a lunge in
striking an opponent depends on what the other animal is doing. Is
the other animal stationary or moving? Is it moving toward or
away from the opponent? How behavioral patterns of such complexity get wired into nervous systems remains unclear (but see the
work of Michael Bate; Bate, 1999; Crisp, Evers, Fiala, & Bate,
2008; Landgraf, Jeffrey, Fujioka, Jaynes, & Bate, 2003; Suster &
Bate, 2002) on the formation of central motor patterns during
development). Another mystery that remains to be solved asks how
animals “decide” to transition from one behavioral pattern to
another during fights. Finally, flies continually adjust their pattern
usage directed at opponents during fights in a form of socially
mediated learning (see the following section). Where and how this
takes place in the nervous system and other important questions
remain unanswered as investigators continue their attempts to
unravel the complexity of behaviors like aggression.
The Dynamics of a Fight: Learning and Memory and
“Winner” and “Loser Mentalities”
Male flies paired in arenas continue to fight for up to 5 hr, but
fewer encounters are seen and the nature of the interactions differ
at later compared with earlier times during fights (Yurkovic et al.,
2006). Early in fights, both animals appear capable of displaying
lunging and retreat behavior and may occupy the food surface.
After the establishment of hierarchical relationships, losers hardly
ever lunge and instead spend most of their time off the food
surface, whereas winners lunge about 30% of the time and do not
retreat and occupy the food surface most of the time. The numbers
of encounters also drop dramatically from ca. 2/min at the start of
a fight in the Canton-S strain we are using to 0.2/min at the end.
When losing flies are paired in second fights 30 min after first
fights; what is observed depends on the opponent. With familiar
opponents, losers will not lunge and will quickly lose again. With
unfamiliar opponents (naïve flies or winners of other fights), losers
do occasionally lunge but will be defeated again quickly. Thus in
both cases, even though the losing flies fight differently depending
on the opponent, they lose (or draw) fights against all opponents,
developing what we call a loser mentality. Depending on the
training protocol (a single long fight vs. four shorter fights in
which they lose every time against different opponents over the
1
Movies S1 and S2: Movie clips were originally photographed at 500
frames/s and are from the publication by Hoyer et al., 2008, pp. 159 –167).
Figure 1A of that publication presents single frames from the movie and
the actual movie is found in the online supplementary material accompanying the article. This is labeled as “Movie S1. Movie supplement to
Figure 1A”. Movie S2 shows the original movie at 12,000 fps or slowed
down from the original by 24 times to allow visualization of the details. As
shown here the movies are at different fps rates than those shown in the
original publication.
KRAVITZ AND FERNANDEZ
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high posture fencing
with a wing threat
thrust with
a wing
threat
wing
threat
chase
shove
walking
retreat
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wing
threat
low
posture
fencing
approach
FEMALE
high
posture
fencing
head
buttt
running
or flying
retreat
approach
MALE
walking
retreat
hold
lunge
boxing
and
tussling
high
posture
fencing
low
posture
fencing
wing
flicking
Figure 1. Behavioral patterns and transitions seen in fights between pairs of male and pairs of female D.
melanogaster. Females and males share five common behavioral patterns (white boxes), whereas several
gender-selective behavioral patterns also are seen (male: blue boxes; female: red boxes). The large light green
arrows indicate similar transition loops in males and females, and the gray arrows indicate gender-selective
transition loops. The male data were collected from 2,526 transitions from 376 encounters in 19 trials and the
female data from 2,597 transitions. Original data and figure were published in “Gender-selective Patterns of
Aggressive Behavior in Drosophila Melanogaster,” by S. P. Nilsen, Y.-B. Chan, R. Huber, and E. A. Kravitz,
(2004), Proceedings of the National Academy of Sciences of the United States of America, 101, p. 12346.
Copyright 2004 by The National Academy of Sciences of the USA. Adapted with permission. The “Thrust with
a wing threat”, and the “High-posture fencing with a wing threat” seen in the female patterns include brief lifting
of one or both wings and are different than the sustained wing threat often seen in males in which both wings
are lifted to an approximately 45-degree angle and held there for greater than 2 s while the animal is not moving.
same time as the long fight) the duration of the loser mentality can
be changed. With a single loss, the flies no longer retain a loser
mentality 1 day later; with multiple losses they do. In many
species, winner effects also are seen, where winning animal are
more likely to win second fights against naïve opponents. Winner
effects tend to last for shorter times than do loser effects, with the
ethological suggestion for this being that it is risky to believe that
you will always win fights. Recently, short lasting winner effects
also have observed as a consequence of fighting in Drosophila
(Trannoy et al., 2015).
Genes and Aggression
Genetic Studies
Several recent reviews have appeared that describe genetic
studies aimed at extending our understanding of aggression (Anholt & Mackay, 2012; Zwarts, Versteven, & Callaerts, 2012).
Aggression is a highly complex behavior; however, and despite
evidence of important genetic contributions to the behavior, the
linkages between the two remain obscure. By inbreeding winners
of fights for multiple generations, lines of hyper-aggressive Drosophila melanogaster flies can be generated starting with a wildtype Canton-S parent strain (Dierick & Greenspan, 2006; Penn,
Zito, & Kravitz, 2010). Males of these strains (called bullies (Penn
et al., 2010) begin fights and start lunging sooner, retaliate against
lunges from opponents more often, and win fights 94% of the time
against males of the parent stock. The high levels of aggression
seen in bully males is not seen in females of the bully stock.
Surprisingly, when bullies are paired in fights, losers of single
fights lose all competitive advantage against males from the parent
stock, but these males revert to bullies a day later. Nurture (losing
a fight) clearly triumphs against nature (inbreeding for aggression)
in this case, but the persistence of this loser effect and how losing
suppresses genetically induced high level aggression remain unknown at present. Bullies spend less time courting and show lower
copulation success rates than the parent Canton-S flies (but see
Dierick & Greenspan, 2006; Penn et al., 2010).
Genome-Wide Studies
Using a similar rearing strategy to the above over 21 generations, Dierick and Greenspan (2006) selected highly aggressive
and less aggressive male flies from starting mixed populations of
Canton-S flies. Via this route, they generated two lines of aggressive and two lines of less aggressive, “neutral” flies. Aggression
levels were scored by measuring the latency to start fighting and
the total numbers of encounters (called fights in these studies)
during which fighting and higher intensity fighting were seen
during a one hour time period. As described previously, aggressive
fly lines showed fewer matings than did neutral lines. Microarray
expression analyses on the heads of the flies demonstrated that
large numbers of genes (474 –775) showed differential expression
levels when any two of the four lines were compared. Therefore
analyses were carried out searching for gene expression patterns
that changed in the same direction when brains from the aggressive
lines were compared with brains of neutral flies: Some 80 genes
were identified using this maneuver and most of the gene expression changes were small. Only a single gene (cytochrome P450
6a20) of those identified, when examined in a mutant line, showed
effects on aggression in directions predicted by the array results.
No genes directly related to amine neurons or to fruitless (see the
following text) were identified in these array studies.
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AGGRESSION IN DROSOPHILA
A series of studies from the Mackay laboratory examined transcript abundance in high- and low-aggression lines derived from
laboratory stock lines and from recently isolated inbred lines in
their attempts to unravel the genetic underpinnings of aggression
(Edwards, Ayroles, et al., 2009; Edwards & Mackay, 2009; Edwards, Zwarts, Yamamoto, Callaerts, & Mackay, 2009; Rollmann
et al., 2008). A high-throughput assay was developed for these
studies, in which behaviorally heterogeneous group-raised flies
were used. With flies reared in this way, low levels of aggression
are seen likely relating to the prior social experience of individual
flies within the population. The animals used in experiments were
deprived of food for 90 min then placed in groups of 4 to 8 males
(in different experiments) into chambers for 2 min to “acclimate.”
Aggression is scored during the following two minutes by counting the numbers of encounters in which any aggressive behavior is
displayed by any individual fly in any of the fights taking place in
the arena. This provides no information on how many flies in the
arena are fighting. The very short time of scoring (2 min) and the
grouping together of all aggressive behaviors as equivalent “units”
of aggression ignore the dynamics of fights and provide no information on whether or when flies use key patterns of behavior like
lunges that are the predictors of the outcomes of fights. In almost
all fights between male flies, the first direct contacts involve
tapping with the tarsi (fencing) or the labium, where abundances of
gustatory and other sensory receptors are concentrated. Gustatory
and olfactory receptors are important for flies in determining the
sex of other flies and play key roles in both courtship and aggression (see the following text). A delay of at least several minutes
has been observed by most investigators before higher intensity
levels of fighting are seen in fights. With the short time of scoring
used in these studies, it is likely that mainly low-level patterns of
behavior relating to aggression are scored and some of those
patterns may not be specifically related to aggression. In their
quest for a high throughput assay, therefore, these authors developed an assay that is quite different than assays used by others who
score aggression. Because these are the parameters used to relate
aggression levels of males to genetic differences between flies, it
is not surprising that a wide variety of genetic differences have
been reported in these studies (hundreds to thousands of genes)
analyzed either as single or as clusters of genes and that there is
little agreement in the genes identified in these studies and in the
whole genome studies of other investigators who use other methods of quantifying aggression.
Aggression is a highly complex behavior involving multiple
steps including: identifying potential resources; approach to potential opponents for the desired resources; identification of a
second fly as a potential opponent or courtship object; recognition
of an opponent as familiar or novel; deciding whether to compete
for the desired resource and at what intensity levels; testing the
“strength” of an opponent through graded levels of aggression;
making decisions during the testing period of which aggression
patterns to use; and then, finally, deciding whether to give up or
whether to continue to fight for the resource. During this entire
time, flies are learning and using newly learned information from
what has gone before in the fight to decide their next moves. With
all this complexity, and with investigators using different methods
of measuring and scoring aggression, it is no wonder that there is
little agreement between investigators seeking to unravel the genetic basis of aggression, and that a very large number of genes
553
concerned with almost all aspects of the physiology of fruit flies
have been suggested to be involved in aggression thus far.
One gene, the fruitless gene of the sex determination hierarchy,
has been directly related to aggression in Drosophila via a candidate gene approach (Billeter, Rideout, Dornan, & Goodwin, 2006).
Male and female flies fight in same-sex pairings (Nilsen et al.,
2004). This, and the observation that many of the key behavioral
patterns seen during fights are sex specific (patterns of aggression
are sexually dimorphic, see Figure 1) raised the possibility that, as
in courtship behavior, genes of the sex determination hierarchy
might be involved in aggression. This was tested directly in studies
in which fruitless (fru), a gene that codes for proteins of the
BTB-zinc-finger family of transcription factors, was expressed in
female brains, where it ordinarily is not made, and was not expressed in male brains where at least three forms of the protein
coded for by the gene normally are made (Billeter et al., 2006;
Vrontou, Nilsen, Demir, Kravitz, & Dickson, 2006). Initially this
was done via the constitutive expression of alleles that were
spliced from transcripts of the gene derived from the P1 promoter
site into male or female forms, regardless of the sex of the flies
(Demir & Dickson, 2005), thereby changing the sex of brain
neurons. In subsequent publications, the sex of neurons in the brain
(sex is cell-specific in flies) also was changed using the Gal4
system to drive expression of UAS constructs that allow or eliminate expression of transformer, a splicing factor translated into
active protein in female but not male fly brains (Chan & Kravitz,
2007; Mundiyanapurath, Chan, Leung, & Kravitz, 2009). Tra
along with Tra-2 (expressed in both males and females) is required
to splice fru and doublesex (dsx) into male or female forms (for
review, see Billeter et al., 2006; Goodwin, 1999; Siwicki & Kravitz, 2009; Villella & Hall, 2008). For fru, truncated transcripts are
made in females, for dsx male and female forms of the protein are
made. In all cases, changing the sex of brain neurons without
changing the sex of the rest of the fly leads to animals fighting
using patterns of aggression appropriate to the sex of the fruexpressing brain neurons and not to the genetic sex of the animals
(Vrontou et al., 2006). One pattern escapes from this generalization: The extended wing threat, a threat posture ordinarily displayed only in male flies, also is seen in males with feminized
brains. The development of hierarchical relationships, also is altered by these changes, which is not surprising, because flies must
lunge to develop such relationships and males with feminized
brain neurons no longer lunge; instead, they show the female head
butt and shove patterns of aggression. A preliminary start was
made on attempting to identify individual neurons involved in
sex-specific circuitry related to aggression (Chan & Kravitz,
2007), but parts of the fru-related circuitries in fly brains (Cachero,
Ostrovsky, Yu, Dickson, & Jefferis, 2010; Yu, Kanai, Demir,
Jefferis, & Dickson, 2010) responsible for aggression, or for courtship, and how and where they intersect were unknown until recently. During the past few years, however, small sets of frutachykinen- and fru-neuropeptide F-expressing neurons were
identified that appear to be important in male higher-level aggression (see the following Peptide section).
Hormonal Modulation of Aggression
As an innate behavior essential to the survival of animals in
complex and often hostile environments, one anticipates that mul-
KRAVITZ AND FERNANDEZ
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tiple sensory modalities and circuitries and numerous hormonal
and neurohormonal substances will influence both the triggering
and the intensity of expression of aggression. In vertebrates, aggression is modulated by amines, peptides, and steroid hormones
(Montoya, Terburg, Bos, & van Honk, 2012; Morrison & Melloni,
2014). In Drosophila, not surprisingly, many of the same or
closely related substances also influence aggression. Amines, including 5HT, OA, and DA have been the most extensively studied
of these hormonal substances, and the results obtained are described in detail in the following paragraphs. More recently. peptides, including peptide F, Drosophila insulin-like peptides, and
tachykinins also have been suggested to be involved in the modulation of aggression. Although little work has been done on
steroid hormones and aggression thus far in fruit flies, one anticipates that these important hormonal substances also will be found
to influence this behavior once suitable experimental protocols can
be established to evaluate behavioral effects of these more difficult
to work with substances.
Amines
At least five amines, all metabolites of common amino acids,
exist and have been proposed to serve specific functions in the fruit
fly nervous system (for a thorough review of four, see Monastirioti, 1999; Roeder, 2005; for tyramine see Bayliss, Roselli, &
Evans, 2013; Roeder, 2005). Studies of amine function in relation
to behavior have focused mainly on three of these: 5HT derived
from tryptophan by ring hydroxylation and decarboxylation, OA
synthesized by decarboxylation followed by side-chain hydroxylation from tyrosine, and DA made by ring hydroxylation and
decarboxylation from tyrosine. Tyramine (TA), the direct decarboxylation product of tyrosine and the direct precursor of OA also
has been proposed as a neurotransmitter/neuromodulator in Drosophila (Bayliss et al., 2013; Roeder, 2005). Finally, histamine
(HA), synthesized from histidine by decarboxylation, has been
suggested to function as a photoreceptor transmitter compound.
HA and its biosynthetic enzyme have been found in small clusters
of neurons in the brain and the ventral cord as well (Nassel, 1999;
Roeder, 2005). Here we focus on OA, 5HT, and DA and their
diverse roles in aggression in Drosophila as these compounds and
their vertebrate counterparts norepinephrine (for OA), DA, and
5HT have been reported to influence aggression in many species
up to and including humans (Yanowitch & Coccaro, 2011). As in
vertebrates, only small numbers of amine neurons are found in the
nervous systems of fruit flies (approximately 100 to 200 of each
subtype, mostly in small clusters in the brain or ventral cord
whereas some 100,000 to 200,000 total neurons are found). Again,
as in vertebrates, they are involved in essentially all behaviors
shown by flies (Alekseyenko, Lee, & Kravitz, 2010; A. Chen et al.,
2013; Crocker, Shahidullah, Levitan, & Sehgal, 2010; Dierick &
Greenspan, 2007; Gasque, Conway, Huang, Rao, & Vosshall,
2013; Hirsh et al., 2010; Lebestky et al., 2009; Van Swinderen &
Andretic, 2011; Waddell, 2013). A key question remains to be
answered, however, in flies as in other species, which is “How do
amines exert these myriad effects?” Several recent reviews have
dealt with amine function in fruit flies (Chen et al., 2013; Foltenyi,
Andretic, Newport, & Greenspan, 2007; Roeder, 2005; Van Swinderen & Andretic, 2011; Waddell, 2013; Zwarts et al., 2012). Here,
the focus will be on amine effects on aggression, even though these
actions overlap with effects on other behaviors like learning and
memory, courtship, feeding, sleep, and so forth (Chen et al., 2013;
Foltenyi et al., 2007; Roeder, 2005; Van Swinderen & Andretic,
2011; Waddell, 2013; Zwarts et al., 2012). It should be added that
a growing literature demonstrates that amines also serve important
roles during the development of nervous systems (Daubert &
Condron, 2010), and investigators should be aware of possible
developmental effects as causes of any behavioral changes induced
when manipulating these systems.
OA
Throughout this review, we emphasize the difficulty of comparing behavioral results from different laboratories where investigators use different assay chambers that contain or do not contain
resources, compute results using different analytical methods, utilize animals reared under different social conditions, and measure
behavior over different time periods. The most difficult part of
such comparisons is that under the diverse experimental paradigms, the results obtained probably are accurate. If anything, the
differing results demonstrate that fruit flies, like other animals, are
highly adaptable to their surroundings. How closely one comes to
the normal behavior of animals in the wild may be the issue under
consideration here rather than whether results are valid. A confusing story in this vein may be the multiple investigations of the role
of OA in aggression. Several publications (Baier et al., 2002;
Hoyer et al., 2008; Zhou, Rao, & Rao, 2008) drew the conclusion
that without OA: Drosophila males cannot go to higher levels of
aggression. More accurately, the results show that without OA,
reduced or absent lunging behavior is seen. Almost all experiments
examining the roles of OA in aggression have used the same or
similar mutant flies, including null mutants for the biosynthetic
enzyme for OA (tyramine ␤-hydroxylase-[Tbh)]), null mutants for
the enzyme that forms both TA and OA (tyrosine decarboxylase[Tdc2]), or the Gal4 system and acute or chronic interference with
transmitter release or function in neurons expressing Tdc2. The
experimental protocols, however, varied widely in the different
publications. In one publication (Hoyer et al., 2008), the collection
of lunge data in fights was automated, lunges were the only
behavioral pattern counted and data was collected 15 to 30 min
after adult male flies were introduced into the fight chambers. At
that time, hierarchical relationships likely would have been established in many but not all of the pairings. Thus the two flies in the
chamber may no longer be behavioral equivalents because after a
hierarchical relationship is established, losers behave differently
than winners in traditional fight chambers: They remain off the
food resource most of the time, they show few or no lunges when
in contact with familiar winners, and they show dramatically
reduced numbers of meetings (encounters) between themselves
and the winners (Yurkovic et al., 2006). To allow automating of
the counting of lunges in these studies, a chamber was designed
with a relatively large center well for food in the base and in which
tall chamber walls were treated with Fluon (tetrafluoroethylene) to
prevent flies from landing on them. In this chamber with little
possibility of escape of losers from winners, higher numbers of
encounters were forced, as noted by the authors, and higher numbers of lunges were seen. Moreover, in quantifying results, these
authors created a new “unit” of measurement: lunge per distance
traveled. This makes the results obtained in this study difficult to
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AGGRESSION IN DROSOPHILA
compare with others that all measure total numbers of lunges per
unit of time. A second publication also reported dramatic reductions in lunge frequency with lowered or abolished OA (Zhou et
al., 2008). These authors reduced the standard chamber size to
increase the numbers of interactions in fights but did allow space
for losers to escape from winners. They then analyzed data for the
first 10 min after both flies were in the chamber, measuring the
latency to the first fight (unit in which they saw physical interactions) in their chambers. They scored lunge frequency and other
patterns and also showed an important change in the latency to
fight in the OA mutant flies, however, lasting 2 to 6 min. The latency
change, which involved a significant portion of the time (almost half)
over which lunging was scored, could result in the decreased number
of lunges seen. An important lengthening in the latency to engage in
social behavior with OA lowering has been seen before, as such
animals appear to have difficulty in recognizing whether conspecifics are courtship or fighting objects (Certel et al., 2010; Certel,
Savella, Schlegel, & Kravitz, 2007), which in turn could influence
aggression levels if that is the main parameter scored in male–male
pairings. That is precisely the effect seen in a recent report (Andrews et al., 2014) in which an aggression vigor index (AVI ⫽
lunges from the first lunge) was computed instead of counting
lunges for a single set period of time. When the AVI was measured, the total numbers of lunges did not change without OA.
Their distribution, however, was altered with two groups seen: one
showing more and another less than the average numbers of lunges
per fight. This supports the notion that OA-less flies have difficulty
determining the sex of a second fly and hence whether to court or
to fight.
Serotonin
Although an early study in male flies reported no effects of
altered 5HT levels on aggression, two subsequent articles did
report actions of 5HT on aggression in flies (Alekseyenko et al.,
2010; Dierick & Greenspan, 2007). Dierick and Greenspan (2007),
using a combination of pharmacological treatments and genetic
methods that raised or lowered 5HT levels in brains were the first
to report that 5HT was not required for aggression in flies, but did
increase the expression of higher level aggression. These authors
also reported an effect opposite to that of 5HT by manipulation of
neuropeptide F levels (see the following text). In a second set of
studies, use of a Ddc-Gal4 driver line to drive expression of a
temperature sensitive dynamin mutant in DA and 5HT neurons led
to hyperactivity displayed as an increase in the numbers of encounters (meetings during fights) between the flies. Encounter
duration was decreased and fights failed to escalate to higher
intensity levels or to yield dominance relationships. Separate manipulation of serotonergic and dopaminergic neuron function;
however, using Th- and dTrh-Gal4 driver lines, revealed differences between the behavioral effects of the amines (Alekseyenko
et al., 2010). Reduced function of 5HT-neurons alone led to
dramatic reductions in the numbers of lunges seen. A third recent
publication used an intersectional genetics strategy (see Figure 2)
to isolate single pairs of serotonergic neurons to try to learn their
function (Alekseyenko et al., 2014). In these studies, the authors
identified one to two pairs of single serotonergic neurons from the
PLP cluster (5HT-PLP neurons) that had extensive arbors of
processes in the ventrolateral protocerebrum, a sensory integration
555
Figure 2. Intersectional strategy. One population of neurons expresses
Gal4 in, for example, amine-neurons: a 2nd population expresses flprecombinase in a different set of neurons. A small subset expresses both,
removes the ⬎stop⬎ in a UAS⬎ stop⬎protein cassette, and makes the
desired protein.
center, and lesser arbors of processes near the mushroom bodies
and the central complex. Silencing the 5HT-PLP neurons alone
reduced the numbers of lunges seen in fights by about the same
magnitude as silencing all serotonergic neurons, whereas activating the neurons enhanced aggression. Endings of these neurons are
in close proximity to neurons expressing 5HT1A receptors: These
reduce cAMP levels when activated in mammalian and fruit fly
systems (see the following text).
The neuronal targets of serotonergic neurons in fly brains should
display receptors for 5HT on their surfaces and manipulation of
receptor function, therefore, might yield all or part of the phenotype displayed by altering 5HT levels. Four orthologs of mammalian receptors have been identified in Drosophila by sequence
homology: d5HT1A and d5HT1B resemble mammalian 5HT1A
receptors and their activation leads to decreases in cAMP synthesis, d5HT2␣ resemble mammalian 5HT2 receptors and influence
phosphoinositide synthesis (recently a d5HT2B receptor subtype
was reported with the suggestion that the original d5HT2 receptor
should be called d5HT2A (Gasque et al., 2013), and d5HT7 receptors resemble the mammalian similarly named receptors and increase cAMP synthesis when activated (Blenau & Thamm, 2011).
Activation or inhibition of these receptor subtypes using drugs of
high specificity that were originally described in mammalian studies have yielded a variety of behavior phenotypes in flies including
actions on circadian rhythms, locomotion, sleep, specific aspects
of learning, and on reproductive functioning (Becnel, Johnson,
Luo, Nassel, & Nichols, 2011; Johnson, Becnel, & Nichols, 2011;
Nall & Sehgal, 2014). One recent report focused on effects on
aggression using agonists and antagonists of various receptor
subtypes (Johnson, Becnel, & Nichols, 2009). While lowered
levels of 5HT itself lead to dramatic reductions in lunging behavior
(see preceding text), these authors reported a variety of behavioral
effects relating to aggression resulting from altering amine receptor function. The difficulties of comparing these effects with
previous results, however, was that a different strain of D. melanogaster was used, different aggression chambers were used, and
most important, very different definitions and hence scoring of the
behavioral patterns displayed by the drug-treated flies were used.
For example, these authors defined wing threat, which is as common in their experiments as fencing behavior, as when a fly
“orients his body in parallel to the other and lifts one wing and
vibrates it” (p. 1294). Other investigators would have called this
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556
KRAVITZ AND FERNANDEZ
male–male courtship behavior because wing threat was defined
previously as a rare posture with both wings erect and motionless
and usually directed by a dominant animal toward an opponent.
Moreover boxing is reported as very common in the recent publication, as common as fencing in fact, which is the initial contact
between flies in other publications, whereas lunging is barely
reported. In all other studies on aggression in wild-type fruit flies,
boxing is rare and lunging is fairly common. There is little doubt
that definition differences are the cause of great confusion in this
case. In the intersectional genetics studies described in preceding
text, it was mentioned that the arbors of the 5HT-PLP neurons
involved in enhancing the usage of higher level aggression during
fights overlapped with regions of high density of 5HT1A receptor
expression. When the neuron pool expressing 5HT1A receptors is
in turn activated, aggression is reduced. This raises the possibility
that 5HT released by the 5HT-PLP neurons inhibits the firing of
5HT1A receptor-bearing neurons via a reduction of cAMP synthesis (see Figure 3) and that members of the pool of receptor
expressing neurons form part of part of an inhibitory descending
pathway holding aggression in check. In this case inhibition by
released 5HT of a descending inhibitory pathway may be the route
through which 5HT-PLP neurons enhance aggression (Alekseyenko et al., 2014).
DA
DA too is involved in multiple behaviors in flies. For example,
it is reportedly involved in learning and memory, sleep, locomotor
activity and stereotypical actions, and so forth (Riemensperger et
al., 2011; Van Swinderen & Andretic, 2011; Waddell, 2013). In
learning studies, a relatively simple hypothesis of how amines
function was proposed initially by several authors. This hypothesis
suggested that DA is involved in aversive and OA in appetitive
learning behavior. Recently, however, this simple hypothesis was
shown to be incorrect with experiments showing that DA is involved both in appetitive and aversive learning, at least in the
mushroom bodies. Moreover, in this system, the OA involvement
appears to be at the level of modulating DA neuron function
(Burke et al., 2012). There is perhaps a lesson to be learned in
these recent results: One should be cautious in proposing simple
linear models for the function of amine neurons. A growing
literature suggests that amine neuron function on systems require
the right amount of amine to be released at the right time and in the
right place in the nervous system to alter neuronal function in
behavior correctly and that addition of amines to levels above or
below this particular set point generates an unstable situation
whereby enhanced or reduced behavioral function may result. In
various test systems, U-shaped rather than simple plus or minus
alterations in function have been noted on the effectiveness of
amine actions on behavior (Van Swinderen & Andretic, 2011). In
aggression, with an acute reduction in DA-neuron function using a
Th-Gal4 driver to express the dynamints mutation (this particular
Gal4 driver has been shown not to influence all DA neurons), flies
fight normally at the permissive temperature but became hyperactive at the restrictive temperature, failing to even land on the food
cup. In a courtship assay such hyperactive flies showed a dramatic
reduction in courtship behavior as well, seeming to show no
interest in normal females (Alekseyenko et al., 2010). More clearcut effects of DA-neurons on aggression are seen by using the
intersectional genetics approach that allows isolation and manipulation of single pairs of DA neurons (Alekseyenko, Chan, Li, &
Kravitz, 2013). These neurons (one pair from the T1 and one pair
from the PPM3 clusters of DA neurons) when altered by expression of the tetanus toxin light chain in the neurons (prevents
transmitter release), display increases in the numbers of lunges
during fights. Hence it appears that these neurons are involved in
holding higher levels of aggression in check during fights, an
effect opposite of that seen in the absence of 5HT. The two pairs
of neurons identified appear to be specialist generating mainly
effects on aggression and no or small effects on other behaviors.
The two distinct pairs of neurons display arbors of axonal endings
in different neuropil regions within the central complex where
each overlap with a different subtype of DA receptor.
Peptides
Neuropeptide F (NPF). NPF is an orthologue of neuropeptide
Y of vertebrate systems, sharing sequence similarity but ending in
phenylalanine (F) rather that tyrosine (Y). Neuropeptide Y has
been suggested to serve a modulatory role in aggression in mammalian systems (Karl et al., 2004). There are 26 NPF neurons per
adult hemibrain in male fly brains found distributed in at least
eight different clusters, but in females only 20 NPF-expressing
neurons are found per hemibrain (Lee, Bahn, & Park, 2006). No
NPF neurons are reportedly found in the ventral nerve cord. This
sexually dimorphic expression pattern depends on FruM expression
in the male specific NPF clusters of neurons (Lee et al., 2006). One
receptor has been identified for NPF, (CG10342) NPFR1, which is
Figure 3. The 5HT-PLP neurons may function to enhance aggression by releasing 5HT onto neurons bearing
5HT1A receptors on their surfaces. 5HT1A receptors act by decreasing cAMP synthesis and inhibiting neurons
expressing these receptors. Activation of neurons with 5HT1A receptors on their surfaces inhibits higher-level
aggression Therefore 5HT released by activation of 5HT-PLP neurons may release higher-level aggression by
inhibiting a tonically active descending inhibitory pathway.
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AGGRESSION IN DROSOPHILA
widely distributed in the brain. NPF has been implicated in multiple behaviors in Drosophila including foraging and feeding,
alcohol sensitivity, evening locomotor activity patterns, and a
longer mating duration of male flies housed with other male flies
in mating arenas (Hermann, Yoshii, Dusik, & Helfrich-Forster,
2012; Kim, Jan, & Jan, 2013; Lee et al., 2006; Nässel & Winther,
2010). As noted above, NPF also has been suggested to hold
aggression in check in male flies (Dierick & Greenspan, 2007).
This was demonstrated both by eliminating peptide release in NPF
neurons with tetanus toxin and by feminizing the male specific
NPF neurons using transformer expression. In both cases large
increases in aggression were reported. The authors also point out
that females, who ordinarily display low levels of aggression,
express no NPF in the male specific clusters of PDF neurons yet
still show low levels of aggression. This suggests that the role of
NPF may be to inhibit a special aggression-promoting pathway
found only in male brains.
Tachykinins. More recently tachykinin-expressing neurons
also were reported to influence aggression and in particular to be
concerned with the differences in the intensities of fighting displayed by male and female flies (Asahina et al., 2014). All tachykinins found in vertebrates share a C-terminal pentapeptide
(FXGLM), whereas the tachykinin-related peptides (TKRPs)
found in invertebrates have a different C-terminal pentapeptide
(FX1GX2R). In Drosophila the TKRPs are derived from a single
gene and six separate peptide isoforms have been found (Tk1– 6).
Two G-protein coupled receptors (Neurokinin receptor from Drosophila [NKD] and Drosophila tachykinin receptor [DTKR]) for
tachykinins have been identified in Drosophila and the genes have
been identified and characterized (Birse, Johnson, Taghert, &
Nassel, 2006; Poels et al., 2009). There are about 150 neurons in
the adult brain that express TKRPs, and these peptides also are
expressed in endocrine cells of the mid-gut (Nässel & Winther,
2010). TKRPs have been shown to serve behavioral roles in fruit
flies in odor detection and in control of locomotion (Winther,
Acebes, & Ferrus, 2006). In a screen for peptides that might
influence aggression, Asahina et al. (2014) found that TKRPs also
might be involved in aggression. The behavioral screen used a
variety of Gal4 driver lines for 16 different peptides (there are 75
predicted varieties of neuropeptides in the Drosophila genome
[Nässel & Winther, 2010]) to express a TrpA1 temperature sensitive potassium channel that allows activation of neurons at elevated temperatures. In same genotype pairings of group-reared
male progeny (group rearing lowers levels of aggression in controls) two of four of the different Tk-Gal4 drivers used (called Tk1and Tk2-Gal4) yielded substantial increases in aggression during
fights after temperature shifts. Only two other peptide-Gal4 drivers
led to increases in aggression in the same screen: One of these was
Npf-Gal4 that in these experiments led to increases rather than
decreases in aggression levels (as reported in preceding paragraphs) after activation of the neurons. It is difficult to compare
these results with the aforementioned studies, however, because a
different Npf-Gal4 driver was used, different proteins were expressed, group-reared flies were used that could only show enhanced levels of aggression, and different behavioral assays and
methods of quantifying of results were used. As with NPF, TKRPs
are expressed in FRUM positive and FRUM negative populations
of neurons and the FRUM positive populations appear to be the
ones important in the regulation of aggression. Deletion mutations
557
were generated within the Tk gene locus (⌬Tk1 and ⌬Tk2), and
when these were examined as either homozygotes or transheterozygotes in isolated male flies (aggression levels are normally
elevated in these male flies) significant decreases but not elimination of lunging behavior were found. In addition when the transheterozygote males, which supposedly have no Tk peptides in the
populations of neurons concerned with the aggression effects, were
used in conjunction with the Tk1-Gal4 line driving TrpA1 expression, a reduction but not elimination was seen in the increased
lunging behavior induced by temperature increases. These results
led the authors to suggest that the residual enhanced lunging
behavior in deletion mutants might result from classical transmitters still present in the involved neurons after elimination of the
peptides. Experiments examining the effects of elimination of
tachykinin receptors yielded confusing results. A homozygous
deletion mutant of the NKD receptor gene reduced the aggression
promoting effects of Tk-Gal4/UAS-TrpA1 driven enhanced aggression, supporting the suggestion that neurons releasing the
DTKR6 peptide that selectively activates NKD receptors (Birse et
al., 2006; Poels et al., 2009) might be involved in the modulation
of aggression. However, rescue experiments for the NKD receptor
only partially restored the Tk1-Gal4/UAS-TrpA1 enhanced aggression levels. Moreover, the same receptor mutant line had no effect
on baseline aggression. An insertional mutant line for the DTKR
receptor had no effect on Tk-Gal4 driven activation of the neurons
or on endogenous aggression levels. The authors suggested that
differential receptor distribution of the two receptor types might
account for these differences. Because the receptor distribution of
NKD and DTKR receptors reportedly are mutually exclusive, this
is a possibility. Clearly further experiments will be required to
clear up some of these findings. No studies were reported aimed at
identification of the DTKR-peptide or peptides involved in the
aggression phenotype or on using antagonists of TK receptors like
Spantides I, II, or III to see if these confirm the results of the
genetic experiments.
Nutrition, peptides, and aggression. A rapidly growing recent literature has implicated the insulin-like peptides (ILPs) and
the nutritional state of animals in the modulation of aggression.
One of these articles has suggested that the quality and quantity of
food available to male flies can influence aggression levels (Lim,
Eyjolfsdottir, Shin, Perona, & Anderson, 2014). The food effects
might be mediated by the insulin-like peptide producing neurons in
flies that are important contributors to the regulation of food intake
and metabolism in flies, and that recently have been reported to
contribute to aggression levels in male Drosophila as well (Luo,
Lushchak, Goergen, Williams, & Nassel, 2014; Nassel, Liu, &
Luo, 2015; Nassel & Williams, 2014). Of particular interest in this
regard are a group of 14 neurons that are included within the
median neurosecretory cell (MNC) group and that release Drosophila insulin-like peptides 2, 3 and 5 into the general circulation.
A subgroup of these MNC neurons also secrete the Drosophila
cholecystokinin-like satiation hormone Drosulfakinin that that has
been suggested to have direct effects on hyperactivity and aggression (Nassel et al., 2015; Nassel & Williams, 2014; Williams,
Goergen, Phad, Fredriksson, & Schioth, 2014). The regulation of
peptide release from the MCNs is complex and includes additional
peptides from the fat bodies and other neurosecretory structures,
GABA, 5HT, and OA (for review, see (Nassel et al., 2015). Less
complete reports have suggested that other peptides influence
KRAVITZ AND FERNANDEZ
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558
levels of aggression in Drosophila, including unidentified peptides
whose synthesis or release from neurosecretory neurons in the
brain is governed by tailless and Atrophin (Davis, Thomas, Nomie,
Huang, & Dierick, 2014). Finally, certain endosymbiotic gut microbes of the Wolbachia family also have been demonstrated to
contribute to various social behaviors in Drosophila, including
aggression, by influencing the biosynthesis of OA and its biosynthetic enzymes (Rohrscheib et al., 2015).
As these studies continue, it will not be surprising to find other
peptides and peptide families that influence levels of aggression,
and that peptide and amine systems likely interact in complex
ways to exert these influences. In this context, it is instructive to
survey the literature on the modulation of the stomatogastric
ganglion of decapod crustaceans (Hamood & Marder, 2014;
Marder, O’Leary, & Shruti, 2014). This 30-neuron network governs the behavior of the stomach as it processes different kinds of
foodstuffs by using large numbers of extrinsic (hormones, neurohormones, and synaptic contacts) and intrinsic (regulation and
distribution of ion channels and receptors) elements interacting
smoothly to ensure proper readout of a wide variety of different
motor outputs from the ganglion.
Pheromones
Drosophila males normally only attack other males; therefore,
displays of aggression depend on sex-recognition. In flies as in
other species, this is mainly via chemosensory communication,
mediated by the olfactory and gustatory systems. Volatile and
semivolatile pheromones, perceived via olfactory signaling, are
believed to be detected first since they act at a distance, whereas
nonvolatile contact pheromones, perceived via gustatory signaling,
are likely second as they require animals to touch or taste each
other (Amrein & Thorne, 2005; Touhara & Vosshall, 2009). Visual
and mechanosensory signals also are likely to contribute to the
recognition of the presence of a conspecific, but it is less certain
whether they can be used to identify the sex of an intruder. A
comprehensive review of the roles of cuticular hydrocarbons in
aggression and courtship has been published recently (Fernandez
& Kravitz, 2013), therefore only several highlights of this topic are
presented in this article.
Fights between male fruit flies typically start with low intensity
interactions, like fencing, during which flies extend legs and contact each other. This step likely involves detection of specific
cuticular hydrocarbons (CHs) or related substances that act as
contact pheromones by gustatory sensilla on the legs, wings, or
labium (Amrein & Thorne, 2005; Billeter & Levine, 2012;
Wicker-Thomas, Guenachi, & Keita, 2009). CHs are complex
mixtures of long chain hydrocarbons synthesized by epidermal
cells called oenocytes and transferred to the cuticle by a still poorly
understood mechanism (Billeter, Atallah, Krupp, Millar, & Levine,
2009; Ferveur, 1997, 2005; Jallon, Kunesch, Bricard, &
Pennanec’h, 1997; Krupp et al., 2008; Wicker-Thomas et al.,
2009). Subsets of CHs act as pheromones carrying information
between flies: for example, dienes within the CH profile are
mainly produced by females and act as aphrodisiacs, activating
male courtship, and monoenes are produced by males and mainly
inhibit their courtship by other males but also may trigger aggression (Amrein, 2004; Antony, Davis, Carlson, Pechine, & Jallon,
1985; Antony & Jallon, 1982; Arienti, Antony, Wicker-Thomas,
Delbecque, & Jallon, 2010; Ebbs & Amrein, 2007; Foley, Chenoweth, Nuzhdin, & Blows, 2007; Jallon, 1984; Tram & Wolfner,
1998; Wicker-Thomas, 2007).
The pheromonal profiles of male and female flies can be
changed to those normally found on surfaces of the opposite sex by
genetic means or by a combination of genetic and chemical manipulations. For example, males can be generated that display
female CHs; such flies are courted by other males (cf. Ferveur et
al., 1997). To determine whether male CH profiles can trigger
aggression, oenocytes were masculinized in an otherwise female
fly via expression of an RNAi transgene for the sex determination
gene transformer (Fernandez et al., 2010). These females displayed
pheromonal profiles similar to wild type males showing high
levels of monoenes and low levels of dienes. In other respects
(surface markings, behavior) they were female. When such mutant
females were paired with wild type males in fight arenas, males
lunged at the females, suggesting that the presence of male CHs
was sufficient to trigger male aggression. It is surprising to note
that, however, males also attacked females with female pheromonal profiles but with a masculinized central nervous system,
indicating that behavioral cues displayed by the females can override the chemical cues provided by the same females (Fernandez et
al., 2010).
Flies without surface CHs also can be generated by destroying
oenocytes using the proapoptotic gene (Billeter et al., 2009). These
“blank”, oenocyte-less females, are courted by males of their own
and sibling species (Billeter et al., 2009; Chertemps, Duportets,
Labeur, Ueyama, & Wicker-Thomas, 2006), indicating that CHs
act as a “tag” that allows males to identify conspecific females.
“Blank” males are vigorously courted by wild type D. melanogaster males but also are attacked by them, although at reduced
intensity (Fernandez et al., 2010). When oenocyte-less males are
perfumed with male CH extracts, the normal aggression levels
elicited from wild type males are restored (Wang et al., 2011).
Blank males perfumed with the male pheromone 7-tricosene (7-T)
have reduced courtship and increased aggression directed toward
them from wild type males. This effect is suggested to be mediated
by sensory neurons expressing the gustatory receptor Gr32a
(Wang et al., 2011), a receptor subtype that had been shown
previously to be involved in sensing 7-T (Lacaille et al., 2007).
The same receptor has been reported to play a role in suppression
of male-male courtship in experiments in which mutant males
were courting immobilized wild-type males (Miyamoto & Amrein,
2008).
Much of the focus on olfactory pheromonal cues used by flies in
social behavior (in courtship and in aggression) has been on the
molecule cis-Vaccenyl acetate (cVA). Originally isolated over 40
years ago this substance has been reported to function as an
antiaphrodisisac, an aggregation factor and most recently as an
enhancer of aggression in groups of males (Bartelt, Schaner, &
Jackson, 1985; J. M. Jallon, Antony, & Benamar, 1981; Wang &
Anderson, 2010). In contrast to the cuticular hydrocarbons, cVA,
and more recently CH503 (a second longer lasting antiaphrodisiac
(Yew et al., 2009) are found in the male ejaculatory bulb and not
in the oenocytes and are transferred to females during copulation
resulting in a reduction in mated female attractiveness to other
males (Brieger & Butterworth, 1970; Butterworth, 1969; Datta et
al., 2008; Ejima et al., 2007; Guiraudie-Capraz, Pho, & Jallon,
2007; Jallon, 1984; Kurtovic, Widmer, & Dickson, 2007; Mane,
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AGGRESSION IN DROSOPHILA
Tompkins, & Richmond, 1983; Ronderos & Smith, 2010; Wang &
Anderson, 2010). When synthetic cVA is added to chambers in
aggression assays between pairs of males, levels of aggression are
increased, an effect that appears to be mediated by the olfactory
receptor Or67d (Wang & Anderson, 2010). In these assays, the
male flies used were group reared, which lowers the aggressiveness of the flies compared to socially isolated naïve flies. A
possible caveat is that hierarchical relationships already have been
established among these males (Yurkovic et al., 2006). A more
complicated scenario was proposed recently with the suggestion
that acute application of cVA acting via Or67d might facilitate
aggression while chronic application of cVA acting via Or65a
receptors might decrease aggression (Liu et al., 2011). The experimental protocols and conditions, however, are so different in these
two sets of experiments that it is extremely difficult to compare
them. A hierarchical relationship in the aggressive responsiveness
to olfactory and gustatory signals has been proposed (Wang et al.,
2011) on the basis of the observation that exposure to cVA does
not increase aggression in gustatory receptor Gr32a-/- mutants. It
remains to be determined whether cVA is sufficient to elicit or
suppress male aggression, but the observation that males attack
mutant females who do not synthesize cVA, suggests that this
compound is not necessary for male aggression (Fernandez et al.,
2010).
Summary and Conclusion
Aggression is essential for the survival and propagation of
animals in the wild. Therefore it is not surprising that many
substances and situations influence the behavior. These include
genes and developmental factors, nutritional state of the animal,
recognition of potential opponents, prior and present social experiences, hormonal and neurohormonal influences on involved circuitries, and then, a complex interaction between all of these in
making what appears on the surface to be simple decisions of
whether or not to fight an opponent, when to switch to different
intensity levels during fights, and when and if to give up in a fight.
There are few, if any, single elements that control aggression as
suggested in various articles, but there are myriad elements that
“influence” the behavior. How then should one proceed in further
exploring the roots of aggression by use of this excellent genetically tractable model organism? Following are a few possible
directions in which studies with the Drosophila model might
move.
As first steps, it seems important for investigators to attempt to
standardize the conditions used to quantify the behavior. This
certainly would allow for better comparisons of experimental
results between laboratories. Thus, elements should be considered
like the genotype and behavioral experience of the flies, how flies
are introduced to fight arenas, the size and shapes of arenas,
whether spaces are available for losers to retreat to, and how and
for how long fights are scored. It also seems important to avoid the
use of indices alone to quantify the behavior, to note whether
hierarchical relationships have been formed and to provide information about fight dynamics in analyses of the behavior.
Standard binary systems in Drosophila (Gal4, LexA, the “Q”
system) are little better than pharmacological approaches that are
used to study behavior in all species, as they tend to manipulate
entire populations of, for example, neurohormonal neurons. Hence
559
these approaches often generate complicated phenotypes. A great
advantage of the Drosophila model is that methods like intersectional genetics, split Gal4 or LexA, and MARCM methods can be
used to bring the study of behavior down to single neuron levels.
The first two of those methods allow readily reproducible manipulation of neurons down to single cell levels and allow definition
of the precise function of those neurons in behaving animals.
When combined with methods that allow labeling of synaptic
inputs and outputs to those neurons using the GFP reconstitution
across synaptic partners (GRASP) technique and other methods
like immunocytochemistry and the more recent optogenetic procedures, circuitries can be worked out entirely at identified neuron
levels.
In the future, increased usage of single neuron technology for
molecular analysis should allow exploration, using RNA-seq and
other analytic technologies that have now been brought to single
neuron levels, to search for molecular differences that might explain functional differences between neurons. In the more distant
future, it might be possible to use optogenetic techniques in combination with intersectional genetic studies to introduce molecular
species to monitor neuronal activity in single identified neurons in
awake behaving animals. This should be possible to do now with
the transparent larvae of flies, but an ultimate goal would be to
explore the possibility of doing such studies in adult flies.
The Drosophila model of aggression is an important and powerful one. The behavior is quantifiable and the behavioral patterns
observed fall readily into the low, medium and high aggression
patterns seen in other species. Decision-making results from male
aggression and loser flies show dramatically altered behavioral
patterns while a lesser effect is seen with winners. Perhaps the
most important advantage that Drosophila has over other species
for the study of complex behaviors like aggression is the speed,
ease and low cost of genetic manipulation of the behavior. An
ever-expanding wealth of genetic tools are available that allow
investigators to: bring the analysis of behavior to identified neuron
levels; elaborate the circuitry through which those neurons function; and that ultimately, should allow insight into how those
circuits function to make the myriad decisions that animals must
compute and act upon in an instant for their survival.
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Received February 19, 2015
Revision received July 7, 2015
Accepted July 7, 2015 䡲
Correction to Aydin, Frohmader, and Akil (2015)
In the article “Revealing a Latent Variable: Individual Differences in Affective Response to
Repeated Injections,” by Cigdem Aydin, Karla Frohmader, and Huda Akil (Behavioral Neuroscience, Advance online publication. July 20, 2015. http://dx.doi.org/10.1037/bne0000084), there was
an error in the abstract. The sentence “However, injections significantly increased time spent
immobile in the forced swim test in LRs, while the identical regimen significantly decreased the
same measure in HRs, compared with handled-controls.” should be “However, injections significantly increased time spent immobile in the forced swim test in HRs, while the identical regimen
significantly decreased the same measure in LRs, compared with handled-controls.” All versions of
this article have been corrected.
http://dx.doi.org/10.1037/bne0000099