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Visual attraction in Drosophila larvae
develops during a critical period and is
modulated by crowding conditions
Zoe Slepian 1
Kelsey Sundby 1
Sarah Glier 1
Jennifer McDaniels 1
Taylor Nystrom 1
Suvadip Mukherjee 2
Scott T. Acton 2
Barry Condron 1,*
Email condron@virginia.edu
1 Department of Biology, University of Virginia, Charlottesville, VA, USA
2 Department of Electrical and Computer Engineering, University of
Virginia, Charlottesville, VA, USA
Abstract
The development of social behavior is poorly understood. Many animals
adjust their behavior to environmental conditions based on a social context.
Despite having relatively simple visual systems, Drosophila larvae are
capable of identifying and are attracted to the movements of other larvae.
Here, we show that Drosophila larval visual recognition is encoded by the
movements of nearby larvae, experienced during a specific developmental
critical period. Exposure to moving larvae, only during a specific period, is
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sufficient for later visual recognition of movement. Larvae exposed to wild­
type body movements, during the critical period, are not attracted to the
movements of tubby mutants, which have altered morphology. However,
exposure to tubby, during the critical period, results in tubby recognition at
the expense of wild­type recognition indicating that this is true learning.
Visual recognition is not learned in excessively crowded conditions, and
this is emulated by exposure, during the critical period, to food previously
used by crowded larvae. We propose that Drosophila larvae have a distinct
critical period, during which they assess both social and resource
conditions, and that this irreversibly determines later visually guided social
behavior. This model provides a platform towards understanding the
regulation and development of social behavior.
AQ1
Z. Slepian, K. Sundby, S. Glier and J. McDaniels contributed equally to this
work.
Electronic supplementary material
The online version of this article (doi:10.1007/s00359­015­1034­3) contains
supplementary material, which is available to authorized users.
Introduction
Social behavior in animals shows a complete range of activity from obligate
eusocial interactions in ants, to largely solitary behavior (Leadbeater and
Chittka 2007 ). Some animals, such as locusts, have the dual option of either
behaving as a group or in a solitary manner. How animals choose to be either
social or non­social is a fundamental question in sociobiology and is critical
for understanding both human disorders (Stanley and Adolphs 2013 ) and the
mechanisms by which social processes develop and evolve (Robinson et al.
2008 ; Sokolowski 2010 ). The key to understanding such social interactions
is to determine the rules, especially those involving the sensory systems,
which govern behavior with conspecifics. The most measureable outcome of
conspecific recognition is clustering, the subject of this study. While olfaction
plays a central role in the recognition and clustering of social animals, the
visual system also plays a key role, such as with wasps (Sheehan and Tibbetts
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2008 ). In many cases, recognition of conspecifics is a learned trait
(Avarguès­Weber et al. 2011 ; Leadbeater and Chittka 2007 ). How this
learning occurs is an open question.
Many model system organisms develop social behavior under laboratory
conditions and serve as good experimental arenas to understand the
mechanisms driving social behavior. For example, Drosophila larvae
demonstrate social behavior in the form of clustering on food (Sokolowski
2010 ; Durisko et al. 2014a ). This clustering is based on food quantity, alleles
of the forager gene, and the presence of olfactory cues from other larvae
(Mast et al. 2014 ). Whether learning is involved in these behaviors is
unknown. The effects of social rearing and overcrowding of Drosophila
larvae have also been examined. Social rearing is advantageous and likely
involves an increase of the yeast subtypes beneficial to Drosophila, as well as
a decrease in toxic fungi (Wertheim et al. 2002 ; Stamps et al. 2012 ).
Overcrowding, on the other hand, results in an increase in toxic waste
products, especially ammonia (Borash et al. 2000 ; Mueller et al. 2005 ).
However, overall larval fitness in laboratory conditions is complex because as
larval density increases, there is an increase in resultant adult size and
fecundity, but also decreased larval survival (Chiang and Hodson 1950 ;
Lewontin 1955 ). Taken together, it would be advantageous for Drosophila
larvae to assess crowding before forming social clusters, to maximize the
number of healthy adults that survive.
Larvae likely employ multiple sensory modalities and strategies to determine
clustering (Durisko et al. 2014b ). Species­specific cuticular hydrocarbons
serve an important role in recognition and clustering (Mast et al. 2014 ).
Vision may also play a role (Justice et al. 2012 ). While larvae possess a
rudimentary visual system, consisting of only 12 photoreceptors per side
(Keene and Sprecher 2012 ), they can nevertheless visually distinguish
moving larvae from dead ones (Justice et al. 2012 ). This is based on the
measured attraction of one test larva to moving, tethered larvae on the other
side of clear plastic. The tethered larval visual target can be replaced with a
video of moving larvae, and this was used to determine the rough speed and
resolution­tuning curve of visual movement recognition. This recognition
process may be a means of finding other larvae in a multilayered,
semitransparent food substrate. If so, it remains to be determined how and
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when this behavior is formed in larvae and whether it can integrate with other
food­based cues. In this study, we report on the development of this behavior
during larval growth and that it is a learned trait. In addition, this behavior is
not formed under conditions of excess crowding. This may therefore be a
useful platform for measuring how social behavior forms.
Materials
Drosophila larvae have three broad stages, or instars, of development: L1, L2,
and L3 after which the animal pupates. Throughout these stages, all motor and
sensory systems are functional. L3 larvae have a foraging phase, L3F, and a
wandering stage, L3W, during which animals cease eating and seek a
pupation site. The L3F phase lasts three 24­h periods, referred to as L3F1,
L3F2, and L3F3. L3W develops during the L3F3 stage. The only difference
between L3F3 and L3W is that L3F3 are found in the food, and L3W are
wandering out of the food looking for a pupation site. For the developmental
sequence, wild­type Canton S eggs were collected over a 4­h period and aged
appropriately, at 22 °C and 40–50 % humidity, in open room light conditions.
Larval stages were confirmed morphologically prior to behavioral testing.
Methods
The behavioral assay is essentially the same as described (Justice et al. 2012 ).
For larval targets, 4–5 L3F Canton S larvae are super­glue tethered at their
rear segments to one spot positioned 1 cm in from the edge of the underside
of a 100­mm plastic Petri dish. This was fitted onto another Petri dish lower
section, which contained a layer of water. Thus, the target larvae were kept
moist and elevated. Most of the target larvae pupate after about 12 h. A water­
dampened, fitted Kimwipe was placed in the base of the upper Petri dish,
upon which a test larva was placed.
For the plastic attraction assay, a section of plastic was glued to the lid above
the test larva with a 1–2 mm clearance from the base, such that the larva
cannot crawl onto it. For the 7­mm­wide section, a white plastic pen was
used. For 2 mm, a cut strip of Fisher slide box plastic lid was used and for
1 mm, a single bristle of a toothbrush was used. The crawling larva was
imaged under ambient room lighting with an iSight camera and recorded
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using PhotoBooth for 30 min. The resultant QuickTime movie was imported
into ImageJ, 50­fold reduced in number of frames, first­frame subtracted from
all other frames, and z­stacked to yield a final trace.
For crossing analysis, a 1­cm circle was placed over the target, and entries
into the area were counted. For cumulative heat plots, larval position was
determined every 20 s using Manual Tracker in ImageJ, and registered using
both the central drop­off point and the larval target that was 2 cm left of
center. Experiments in which larvae spent more than 10 min in one spot,
usually due to injury during transfer, were not considered. Crossovers and
time spent per 1 cm2 were determined using Microsoft Excel and plotted
using R (The R Foundation for Statistical Programming). To autotrack larvae,
a Bayesian particle filter method was used (Arulampalam et al. 2002 ).
For 24­h isolation experiments, larvae were kept on agar­molasses plates with
a centrally placed 2­mm­wide ball of yeast paste. Larvae were kept either
isolated or in groups, depending on the experiment. For co­rearing with tubby
(TM6b, Tb1), a 1­cm­wide ball of yeast was used for 100 larvae. Finally, for
the crowding assay, larvae were incubated singly in 35 mm dishes containing
a 2 × 2 mm piece of Kimwipe floating on 30 ml of a 50:50 mixture of yeast
paste and molasses. The 35­mm dishes were floated in a layer of water in a
lidded 15­cm Petri dish. For grouping, larvae were placed in one 35­mm dish,
but with 100 ml of yeast/molasses mixture.
Results
Developing an assay of visually directed movement and
non-movement behavior
Drosophila larvae can be tested for visual attraction to the movements of
other larvae, using a simple Petri dish assay (Justice et al. 2012 ), (Fig. 1 a). A
group of 5–6 larvae is superglue tethered to the underside of a clear plastic
Petri dish, and a test larva is free to crawl on the upper surface. Attraction is
scored as the number of times the test larva enters a circular 1­cm zone
around the target. The target is placed 1 cm in from the side of the Petri dish,
as test larvae often circle around the sides. The test larva is always deposited
in the center of the plate. The crawling surface is a piece of damp Kimwipe
and this, as well as the test larvae, is changed after each 30­min assay (Justice
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et al. 2012 ). Based on replacing the larval target with a video, the visual
stimulus is likely to be larval movements (Justice et al. 2012 ). Based on the
distribution of time spent by larvae over the surface of the Petri dish, as well
as total distance traveled, alterations of motor behavior do not account for
attraction (Justice et al. 2012 ). However, to enhance this original assay
(Justice et al. 2012 ), another visual attraction protocol was developed. It was
found that larvae are attracted to a section of white drinking straw, glued to
the lid of the Petri dish, that the larvae can crawl below but not onto
(Fig. 1 b). To examine this behavior further, attraction to different­sized white
plastic targets was examined (Fig. 1 c). Plastic targets of 2 mm and larger
show attraction, while 1 mm do not. The ‘0 mm’ data point was a pre­chosen
equivalent 1­cm zone and assayed as a blank. We propose that attraction to
plastic acts as measure of non­specific, visually guided behavior and is
different to attraction to moving larvae (see below).
Fig. 1
Visual attraction assay. a Standard larval attraction assay used in previous
studies (Justice et al. 2012 ). Three to five larvae are glue tethered on the raised
underside of a clear plastic Petri dish. The tethered larvae are placed 1 cm out
from the side of the dish so that larvae circling the edges are not scored. The
test larva is always placed at the center and tracked for 30 min. A test larva is
free to crawl on the upper inside Petri surface and scored for the number of
times at least half of its body enters a 1­cm­diameter circle centered on the
target. The test larva and damp Kimwipe crawling surface are changed after
every assay. b Plastic attraction assay. An 8­mm­long section of a 5­mm­wide
plastic straw was glued onto the underside of the lid of a 15­mm­deep Petri
dish, such that the plastic is about 2 mm above the inside crawling surface.
Larval attraction is scored as either number of times crossing or time spent
under the plastic. c Larvae are attracted to plastic targets 1 mm wide and above.
The white plastic section, width against attraction, is plotted. For 0 mm, no
plastic was used, but the target point was pre­chosen. A 1­cm circle around the
target was considered as score zone, in all cases. At least 20 larvae were used
for each point and statistical analysis was used comparing each data point to the
first, ‘0 mm’
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Visual attraction behavior is learned in a critical period
In previous experiments, test larvae were selected as foraging third instars
from groups on food (Justice et al. 2012 ). To test the effects of group size,
larvae were reared in different group sizes and tested in the middle of third
instar (Fig. 2 a). Testing was as described (Justice et al. 2012 ), in which 4–6
L3F larvae were tethered on the lower side of an elevated 10­cm Petri dish.
Visual attraction was measured by either the number of times the test larva
crossed the target (crossovers) or time spent over the target. Only in group
sizes 20 and above was visual attraction behavior detected. Attraction
behavior was also examined over a developmental sequence (see " Materials ")
and is maximal at the second day of the third instar stage (L3F2, Fig. 2 b) but
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declines as the animal approaches and enters the wandering (L3W) stage. L2
larvae did not demonstrate any attraction to tethered larvae, but did to plastic.
This indicates that visual attraction to other larvae develops only in the third
instar stage, while capacity to see other visual stimuli develops earlier. Third
instar larvae tend to aggregate socially, and this may be associated with visual
attraction behavior (Sokolowski 2010 ). To examine whether the behavior is
learned socially, animals were raised in isolation from L1 to L3F2 and tested.
These larvae showed little visual attraction to tethered larvae, indicating that
this is a learned trait (Fig. 2 b). Socially isolated larvae are attracted to a
plastic target, and this indicates that general, visually driven motor behavior is
unaffected. Larvae that were socially reared in the dark, also show no
attraction to moving larvae (0.5+/0.22, N = 10). Collectively, this indicates
that larvae must be able to see other larvae sometime from L2 to L3F2, to
show visual attraction to tethered larvae.
Fig. 2
Visual attraction is learned during a critical period. a Visual attraction is
learned in group sizes of 20 and above. Staged and synchronized first instar
larvae were reared either in isolation or in groups ranging from 5 to over 100.
Larvae were tested at L3F2. Grouping was on an agar­molasses plate with a
centrally placed 2­mm­wide ball of yeast paste. Attraction appears in group
sizes of 20 and above. The number of samples used is indicated in the bars.
Statistical analysis was performed using one­way ANOVA, followed by
Tukey’s multiple­comparison posttest (**p < 0.01; ***p < 0.001). b Visual
attraction was measured at successive stages of larval development. Well­fed
animals were staged by time and morphology, cultured in a crowded molasses
plate, and attraction was measured such that vision was the only stimulus
(Justice et al. 2012 ). Both tethered larvae and 5­mm white plastic were used as
targets. Attraction is seen only in the first 48 h of the third instar (L3F1 and
L3F2) and decreases as the animal approaches and enters the wandering stage
(L3F3 and L3W). To examine the role of social experience, larvae were raised
singly from L1 to L3F2 and assayed. Numbers in the columns are the number of
larvae independently tested. Statistical analysis was performed using one­
way ANOVA, followed by Tukey’s multiple­comparison posttest (*p < 0.05;
**p < 0.01; ***p < 0.001). c To determine the 24­h period of social experience
necessary for visual attraction behavior, animals were reared in isolation and
then grouped with 20 animals for 24 h on an agar plate with yeast, re­isolated,
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and tested at L3F2. Isolation prior to or after L2–L3F1 had no effect. Animals
isolated at L2–L3F1 showed no attraction, but were attracted to plastic.
Statistical analysis was performed using one­way ANOVA, followed by
Tukey’s multiple­comparison posttest (*p < 0.05; ***p < 0.001)
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To further examine the onset of visual larval attraction, a period of 24­h
isolation was set to either before, during, or after the 24­h L2–L3F1
transition. Apart from periods of isolation, animals were kept in groups of 20,
in a 5­cm fly­agar egg plate, which was found to be sufficient to induce the
visual behavior (Fig. 2 a). Isolation, during L2–L3F1 only, is sufficient to
block the behavior (Fig. 2 c), while isolation before or after this period has
little or no effect. Because the transition in behavioral response between L2
and L3F1 is abrupt, L2 animals undergoing ecdysis were identified in a
crowded plate (Park et al. 2002 ), removed, allowed to complete ecdysis in
isolation, and re­tested at L3F2. These animals showed no attraction to larval
targets (0.29 ± 0.10 n = 34). Because exposure to other larvae is required
during L3F1, but is not effective during L3F2, larval visual attraction
behavior develops within a critical period.
To confirm the results in Fig. 2 b, the movies were re­analyzed for the length
of time spent under target, as opposed to crossovers as described (Justice et
al. 2012 ). This confirms the developmental sequence, in that larvae spend
more time under the target in the middle of the third instar (Fig. 3 a), and this
time is reduced to background levels when the raised in isolation. The manual
tracking used was validated by auto­segmentation of larval position (Fig. 3 c).
Fig. 3
Visual attraction as measured by time over target. a To measure the time spent
over the target, at least twenty 30­s videos of each developmental stage were
reduced to about 100 frames. This is the same as described (Justice et al. 2012 ).
Each frame represents about 20 s of the 30­min experiment, or about 3 body
lengths of locomotion. The xy center of mass position of the test larva was
determined in each frame, registered to a common target coordinate system, and
converted to time spent in each 1 cm2 area of the dish. This was averaged over
>20 videos to generate the average time spent per cm2 and also used to generate
a “heat­map.” Statistical analysis was performed using one­way ANOVA,
followed by Tukey’s multiple­comparison posttest (*p < 0.05; ***p < 0.001). b
Representative traces of individual larvae and heat maps are shown for L2,
L3F2, and L3F2 isolated. The target is indicated by an arrow. Larvae were
dropped off at the center (“d”) of the plate and returned to this location often
(Justice et al. 2012 ). To examine the average distribution of time spent over the
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target at higher resolution, the 20 videos used for L3F2 in S1F were each traced
by auto­segmentation over >600 frames for the 30­s span (“auto­tracked”).
Time spent per ½ cm2 was examined (squared region of the L3F2 heat map).
The resolution is fivefold higher in time and four times higher in space. The
single concentrated point over the target, at low resolution, remains at higher
resolution (arrow in “auto­tracked”)
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AQ2
Atypical visual cues can be learned
If visual attraction behavior is a true form of learning, then alternative larval
body movements presented during the critical period should produce visual
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attraction behavior to only that movement. The alternative is that recognition
and attraction to other larval movements is innate, but is socially activated
only in L3F. To test this idea, a screen was conducted for potential non­
cognate body shape/movements that post­critical period, socially reared L3F2
larvae are not attracted to. Drosophila melanogaster larvae are attracted to
larvae of two other species of fly, D. mauritania and Megaselia scalaris
(Fig. 4 a). However, they are not attracted to Drosophila melanogaster pupae
or mobile but tethered adults. In addition, they are not attracted to the larvae
of the Drosophila melanogaster morphological mutant tubby (Fig. 4 a). Tubby
larvae are round and have different body movements. Only one tubby allele
and genetic background was tested in this study. To test if recognition of
tubby can be learned, larvae were raised from L2 to L3F2 in standard agar egg
plates that contained 100 larvae of varying proportions of wild­type and tubby
mutants. Wild­type larvae were then tested for recognition of either wild­type
or tubby targets. Raising wild­type larvae, with increasing proportions of
tubby larvae, results in the acquisition of visual attraction to tubby and the
loss of attraction to wild type (Fig. 4 b). Therefore, recognized visual cues are
not completely innate. Importantly, raising wild­type larvae with tubby, after
the critical period, does not increase tubby recognition (Fig. 4 b). The
attraction analysis in Fig. 4 b was repeated by calculating the time larvae
spent in each square centimeter and corroborates the crossover data (Fig. s1).
Next, we sought to examine larvae trained to recognize multiple body types.
Animals raised in a 50 % mixture of wild type/tubby result in reduced
responses to both body movements. This could be due to reduced learning of
both body types, either from a limit in the amount of information storage, or
that an individual larvae can only learn one body movement at a time. To
examine this question further, single animals were serially tested against
different targets. However, in regular testing, about 1/3 of larvae do not cross
the target (Justice et al. 2012 ). If this is a stochastic event, wild­type­raised
larvae were repeat tested against a wild­type target (Fig. 4 c). Those larvae
that failed to respond to a target in the first test could recognize the target in
the second test. Likewise, larvae that responded to the target in the first test
could fail in the next (Fig. 4 c). Therefore, failure to recognize target is likely
to be a stochastic trait. Larvae were then raised either as 100 % wild type or
in a 50:50 mixture of wild type/tubby and were serially tested (Fig. 4 d, e).
For larvae raised with 100 % wild type, how they succeed in the first test
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against wild type does not predict their response in the second against tubby
(Fig. 4 d). This pattern does not change substantially when larvae are raised
with 50 % tubby (Fig. 4 e). A portion of larvae can respond to both wild type
and tubby, and this indicates that a single animal can learn to recognize both
targets but with reduced efficiency for each.
Fig. 4
Larvae can learn to recognize alternate body shapes/movements. a Different
targets were used to examine the specificity of visual attraction.
“mauritania” = Drosophila mauritania larvae; “phorid” = Megaselia scalaris
larvae; “pupae” = pupae of Drosophila melano + gaster; “adults” = tethered
Drosophila melanogaster adults; “tubby” = Drosophila melanogaster TM6­
tubby larvae. The number of samples is as indicated in the bars. Responses
were compared to L3F1 (**p < 0.01; ***p < 0.001). Statistical analysis was
performed using one­way ANOVA, followed by Tukey’s multiple­comparison
posttest. b Wild­type larvae were raised L1–L3F2 with increasing proportions
of tubby larvae, which have a round body shape and different movements. The
visual attraction responses to either wild­type or tubby targets, compared with
the percentage of tubby larvae experienced during raising, are plotted. For the
“late tubby exposure,” larvae were raised with tubbys only during the last 24 h,
L3F1–L3F2. Responses were compared between the different targets at 0 and at
80 % tubby (*p < 0.05; ***p < 0.001), or to the same type of target, but
compared between 0 and 80 % tubby (***p < 0.001). Wild type­late and tubby­
late at 50 % tubby raising is indistinguishable from being raised at 0 % tubby.
Statistical analysis was performed using one­way ANOVA, followed by
Tukey’s multiple­comparison posttest. c To examine whether a single larvae
can respond to multiple body shapes, larvae were tested against a wild­type
target and immediately re­tested for a response to a wild­type or tubby target.
The circles represent when a larva showed no response to a larval target. The
larvae came from batches raised with 0 % tubby larvae, (c, d), or 50 % tubby,
(e). In each case, larvae were scored as either responding to the larval target, or
not (open circle)
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Learned visual attraction behavior is sensitive to crowding
conditions
The 24­h isolation experiment described, in Fig. 2 , defines the critical period
required for visual learning. Next, we sought to determine the minimum time
and group size required for learning, using a specialized protocol. To ensure a
more uniform experience, larvae were raised in 35 × 10 mm Petri dishes, and
placed in the center was a 5 × 5 mm section of damp Kimwipe containing a
100­ml 50:50 mixture of Red Star active yeast paste and molasses. This
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approach supplies ample food and allows for regular encounters with other
larvae in the localized food drop. To investigate the grouping periods more,
larvae were raised singly in these Petri dishes from the end of L1 until L3F2.
At L3F1, larvae were grouped in a fresh 35 × 10 mm Petri dish with food for
various times, re­isolated for 24 h, and then tested for attraction to either
larvae or plastic the next day at L3F2. Attraction to plastic did not vary with
any condition of group size or timing in this experiment (Fig. 5 ). Larvae
grouped for 6 h show attraction in group sizes of 5 and 10, but interestingly
not in larger groups (Fig. 5 a). In groups of 10, attraction behavior begins at
4 h, peaks at 6 h, and decreases at longer times (Fig. 5 ). Testing larvae
immediately after the 10 × 6 h showed less attraction (larvae: 1.51 ± 0.32
n = 32; plastic: 1.17 ± 0.27 n = 23) indicating that while learning occurred in
the L3F1 period, a full response was not seen until 24 h later. It was noticed
that with groups of 20 larvae at 8 h, larvae were trying to climb out of the
Petri dish, suggesting that something repulsive was building up in the food.
Larval waste produces toxic ammonia to which larvae are repulsed (Mueller
et al. 2005 ). If the buildup of larval waste is responsible, then 10 larvae
grouped for 6 h in food previously used and over­crowded with larvae should
show no learning of visual attraction. This was the case (Fig. 5 d).
Conversely, 20 larvae were grouped for 8 h, and animals were moved to fresh
food every 2 h. This should lessen the effects of animal waste. These animals
demonstrated visual learning (Fig. 5 d). This indicates that it is a larval­
produced factor that builds up in food and eliminates the visual behavior or
overrides it with a repulsion behavior. At least part of this adverse effect is
olfactory as anosmic, Orco larvae (Louis et al. 2008 ) still learn in 20 × 8 h
groups (Fig. 5 d). The negative effects of old food are required within the
L3F1 critical period, because if animals were exposed to 10 × 6 h in L3F1,
followed by 20 × 8 h in L3F2, they still show normal visual attraction
behavior (Fig. 5 d). This result suggests that the effects of a larval­derived
repulsive agent are also functional only during the same L3F1 critical period
as visual learning.
Fig. 5
Effects of time and group size on visual learning. a Staged larvae were reared
alone in small chambers from late L1 until L3F2 and grouped only during
L3F1. Testing for attraction to either plastic or larval targets was at L3F2. With
a 6 h group time, only 10 grouped larvae showed attraction while 20 did not. b
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Same as a except 10 larvae were grouped for 2, 4 and 6 h. c Three­dimensional
plot of visual attraction versus group size and time. The constituting plots are
shown in Fig. S2A and S2B. Ten larvae at 6 h is the optimal grouping regime
for this experiment. d To test for the role of food waste, food that had contained
20 × 8 h grouped larvae was used for 10 × 6 h, or food was changed every 2 h
for 20 × 8 h. In addition, larvae previously grouped in L3F1 10 × 6 h were re­
grouped 20 × 8 h at L3F2 and immediately tested. The anosmic mutant orco1
was tested 20 × 8 h. Old food has the same effect as crowding and new food
overcomes crowding, as does loss of olfaction. Statistical analysis was
performed using one­way ANOVA, followed by Tukey’s multiple­comparison
posttest (*p < 0.05; **p < 0.01; ***p < 0.001)
AQ3
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Discussion
Drosophila larvae are attracted to visual cues of other larvae (Justice et al.
2012 ). While these experiments do not shed light on the visual cues extracted
by a relatively simple visual system, it does further extend the idea that the
role of this behavior is for social recognition. The experiments described here
indicate that this visual attraction is a learned trait with a distinct critical
period within the first 24 h of the third instar stage of development. In
addition, conditions of crowding modulate this learning. While learning has
been described for Drosophila larvae, the process described requires more
time than that described for olfactory, gustatory, and light associative learning
behaviors (Scherer et al. 2003 ; Gerber et al. 2004 ; von Essen et al. 2011 ).
This might be because of a different cellular mechanism, such as structural
changes to the visual circuit, as has been described for larvae (Yuan et al.
2011 ). A structural change and/or a change in gene expression is also
consistent with the minimum 2­h learning time. However, it remains unclear
as to why maximal response occurs 24 h later. Learned recognition in animals
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could serve many purposes, such as kin recognition either to avoid inbreeding
(Gherardi et al. 2012 ) or to facilitate cooperative breeding (Sharp et al. 2005 ;
D’Ettorre and Heinze 2005 ). A limited visual encoding system might
adequately function in kin recognition, simply by assuming nearby larvae are
from the same egg clutch (Mateo 2004 ). It is possible that visual recognition
in larvae serves the purpose of inducing social clustering, which may result in
later clustering of pupae and mating adults. Alternatively, this learning could
be by simple association of food and movements of other larvae. Simple
associative learning can be used to explain much of insect social behavior
(Dawson et al. 2013 ) or other kinds of visual learning (Darmaillacq et al.
2008 ; Horn 1998 ; Giurfa 2012 ). Indeed, the visual learning described here is
modulated by the state of the food and larval waste. This then may be a
simple mechanism by which larval social behavior is modulated by food: too
many larvae could make these animals non­social.
Learning likely plays a key role in survival for larvae in unpredictable
environmental conditions (Dukas 2008 ). While Drosophila larvae can show
social behavior, they also survive well in isolation in the lab. However,
survival in isolation may not be the case in a field setting (Rohlfs et al. 2005 ;
Venu et al. 2014 ). It is intriguing that larvae can combine visual recognition
with an assessment of food conditions. While there are advantages for larval
clustering in both food processing and curbing of fungal growth (Wertheim et
al. 2002 ; Stamps et al. 2012 ), overcrowding produces toxic ammonia
(Mueller et al. 2005 ). It would be advantageous to balance out decision
making between clustering and remaining alone. Why such a decision is made
in an irreversible critical period is unclear. Many sensory systems and
complex behaviors develop with critical periods and this might be an efficient
way to combine distinct environmental experiences and the formation of
circuits in the brain (Bateson 1979 ; Hensch 2004 ). Future molecular and
cellular studies of this relatively simple system may shed light on this
question.
AQ4
Acknowledgments
We thank Bloomington Stock Center for providing fly stocks, C. Deppmann,
A. Guler, D. B. Bone and T. Dobrzelewski for reading and commenting on the
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manuscript, and J. Levine (U. Toronto) for initially encouraging these
experiments. Special thanks to Jeremy Louissaint for making the initial
observations. Fly strains were obtained from Bloomington Stock Center (NSF
Grant No. DBI­0,841,154).
Compliance with ethical standards
Conflict of interest The authors declare no competing interest.
Funding This work was supported by NSF Grants CRCNS 1010333, ABI
1062433, and the University of Virginia.
Electronic supplementary material
Supplementary Figure S1. S1A. Specificity of visual attraction Same as 4B
except that tracks of larvae were digitized as described for Fig. 3 a. Twenty
samples were used for each data point and heat­plot. S1B. Heat plots and
representative traces are shown for each sample. Responses were compared
between the different targets at 0 and at 80 % tubby (**p < 0.01; ***p < 0.001).
Statistical analysis was performed using one­way ANOVA, followed by
Tukey’s multiple­comparison posttest. (TIFF 1141 kb)
Supplementary Figure S2. S2AB. A more complete plot of times and group
sizes with attraction to larval targets S2A and plastic targets S2B. (TIFF
987 kb)
Supplementary videos. S3­ L3F2 attraction to a white plastic control (MOV
898 kb)
Supplementary videos. S4­ L3F2 attraction to a tethered larval target (MOV
761 kb)
Supplementary videos. S5­ L3F1 grouped 10x6 h in old food from a 20x8 h
grouping. No attraction is seen. (MOV 674 kb)
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