Circuit and Behavioral Basis of Egg-Laying Site
Circuit and Behavioral Basis of Egg-Laying Site Selection in Drosophila Melanogaster
Edward Yun Zhu
Department of Pharmacology and Cancer Biology
Chung-Hui Yang, Co-Supervisor
Cynthia Kuhn, Co-Supervisor
Dissertation submitted in partial
fulfillment of the requirements for the degree
of Doctor of Philosophy in the Department of
Pharmacology and Cancer Biology in the Graduate School of
Circuit and Behavioral Basis of Egg-Laying Site Selection in Drosophila Melanogaster
Edward Yun Zhu
Department of Pharmacology and Cancer Biology
Chung-Hui Yang, Co-Supervisor
Cynthia Kuhn, Co-Supervisor
An abstract of a dissertation submitted in partial
fulfillment of the requirements for the degree
of Doctor of Philosophy in the Department of
Pharmacology and Cancer Biology in the Graduate School of
Edward Yun Zhu
One of the outstanding goals of neuroscience is to understand how neural
circuits produce context appropriate behavior. In an ever changing environment, it is
critical for animals to be able to flexibly respond to different stimuli to optimize their
behavioral responses accordingly. Oviposition, or the process of choosing where to lay
eggs, is an important behavior for egg-laying animals, yet the neural mechanisms of this
behavior are still not completely understood. Here, we use the genetically tractable
organism, Drosophila melanogaster, to investigate how the brain decides which substrates
are best for egg deposition. We show that flies prefer to lay eggs away from UV light
and that induction egg-laying correlates with increased movement away from UV. Both
egg-laying and movement aversion of UV are mediated through R7 photoreceptors, but
only movement aversion is mediated through Dm8 amacrine neurons. We then identify
octopaminergic neurons as being potential modulators of egg-laying output.
Collectively, this work reveals new insights into the neural mechanisms that govern
Drosophila egg-laying behavior.
List of Figures ............................................................................................................................. vii
Acknowledgements .................................................................................................................. viii
1. Introduction ............................................................................................................................... 1
1.1 Drosophila egg-laying preferences ................................................................................ 1
1.2 Mechanisms of egg-laying control ................................................................................. 6
1.3 Neural mechanisms of oviposition ................................................................................ 8
2. Egg-laying induces aversion of UV light in Drosophila...................................................... 11
2.1 Drosophila prefer to lay eggs away from UV ............................................................ 12
2.2 Egg-laying induces behavioral aversion of UV.......................................................... 17
2.2.1 Drosophila spend less time on UV during egg-laying ........................................... 20
2.2.2 Drosophila move away from UV during egg-laying ............................................. 22
2.3 Neural substrates for UV aversion ............................................................................... 25
2.3.1 UV aversion is mediated through the visual system ........................................... 25
2.3.2 R7 photoreceptors are required for both egg-laying and movement aversion of
UV ......................................................................................................................................... 27
2.3.3 Dm8 neurons are required for movement aversion, but not egg-laying
aversion, of UV ................................................................................................................... 31
3. Central mechanisms of egg-laying site selection ................................................................ 38
3.1 Role of octopaminergic neurons during oviposition................................................. 38
3.1.1 Octopamine modulates egg-laying preference and egg-laying rates ................ 39
3.1.2 Candidate OA cell clusters for regulating egg-laying rate .................................. 42
4. Discussion and Future Directions......................................................................................... 50
4.1 Switching the behavioral valence of UV ..................................................................... 51
4.2 Delineating the visual circuit during UV aversion .................................................... 53
4.3 OA and egg-laying control ............................................................................................ 54
4.4 Concluding Remarks...................................................................................................... 55
5. Materials and Methods........................................................................................................... 57
5.1 Fly stocks ......................................................................................................................... 57
5.2 Egg-laying preference assay ......................................................................................... 57
5.2.1 Egg-laying chambers and LED holders.................................................................. 57
5.2.2 Behavior setup and analysis .................................................................................... 58
5.3 UV light setup ................................................................................................................. 58
5.3.1 Light source ................................................................................................................ 58
5.3.2 LED driver and intensity control ............................................................................ 58
5.4 Behavioral analysis ......................................................................................................... 59
5.4.1 Video recording setup .............................................................................................. 59
5.4.2 Tracking position of flies .......................................................................................... 60
5.4.3 Positional PI analysis ................................................................................................ 60
5.4.4 Return PI analysis ...................................................................................................... 61
5.5 Mosaic Analysis .............................................................................................................. 61
References .................................................................................................................................... 63
Biography ..................................................................................................................................... 73
List of Figures
Figure 1: Egg-laying chambers to assess egg-laying preferences......................................... 13
Figure 2: Drosophila prefer to lay eggs away from UV........................................................... 15
Figure 3: Drosophila also prefer to lay eggs away from wavelengths of light..................... 16
Figure 4: Setup to record and track positions of flies during egg-laying ............................ 19
Figure 5: Egg-laying demand induces positional aversion of UV ....................................... 21
Figure 6: Egg-laying demand induces movement aversion of UV ...................................... 24
Figure 7: Egg-laying and movement aversion of UV is mediated through the eye .......... 26
Figure 8: R7 photoreceptors are required for egg-laying and movement aversion of UV29
Figure 9: Dm8 amacrine neurons are required for movement aversion but not required
for egg-laying aversion of UV ................................................................................................... 34
Figure 10: Role of PRs during UV-driven egg-laying behavior ........................................... 36
Figure 11: Movement attraction uses redundant PR pathways ........................................... 37
Figure 12: Octopamine neurons modulates egg-laying preference and rates .................... 40
Figure 13: Mosaic analysis to determine which OA neurons regulate egg-laying ............ 44
Figure 14: ASM and VM mx are candidate OA neurons that regulate egg-laying............ 47
I would like to first thank my mentor, Dr. Chung-Hui Yang, for her guidance and
for providing me with the opportunity to work in her lab. She was the primary driver
behind our projects, and she gave me the chance to work in a completely new research
environment. She recognized my weaknesses and pushed me to face them rather than
avoid them. Without her effort, I would not have learned or grown as much as I did
during my time here at Duke.
I would also like to thank Dr. Ulrich Stern, who was very generous with his time
towards my project. His contribution towards our project cannot be overstated, as he
designed, built, and programmed all of the equipment I used during my project. It goes
without saying that my work would not have advanced as far as it did without Uli
helping me every step of the way.
Next, I would like to thank my lab mates, for simply being the best lab to work
with. Dr. Ananya Guntur, Dr. Bin Gou, Dr. Stephanie Stagg, Ruo He, and Hsueh-Ling
Chen are amazing colleagues, mentors, and friends.
Finally, I would like to thank my family and friends for all their support and
The fruit fly, Drosophila melanogaster, is a powerful model organism to
understand the neural basis of behavior. In addition to having only ~100,000 neurons in
its relatively simple nervous system (Chiang et al., 2011), the fly has an arsenal of genetic
tools that allow researchers to manipulate specific sets neurons to test their role in
specific behaviors (Luo et al., 2008, Venken et al. 2011). Recent work has used the fly as
model system to understand how neural circuits are constructed to produce various
behaviors including aggression, courtship, and grooming (Zwarts et al. 2012, Manoli et
al., 2005, Stockinger et al., 2005, Yamamoto and Koganezawa, 2013, Seeds et al., 2014).
Choosing a site for egg-laying, or oviposition, is an important behavior for
Drosophila. Ideally, eggs should be deposited in areas that optimize offspring survival.
Accordingly, Drosophila have been shown to be sensitive to many different stimuli when
selecting for an egg-laying site. However, understanding how the fly transforms
sensory information into egg-laying decisions remains elusive. In this dissertation, I will
review the current understanding of Drosophila egg-laying behavior before describing
some novel discoveries that provide greater insight as to how the female fly chooses
where the lay her eggs.
1.1 Drosophila egg-laying preferences
Prior to egg deposition, Drosophila exhibit a stereotypical “searching” behavior
when looking for a suitable egg-laying site. This behavior can sometimes last from just a
few minutes to as long as a few hours. During this time period, females will walk
around and survey its’ environment by probing substrates with their proboscis and
ovipositor, presumably to evaluate the chemo and mechanosensory quality of possible
substrates (Yang et al., 2008). Once a favorable substrate has been found, the female will
bend her abdomen downward to insert the ovipositor into the substrate and expel a
single egg, a process that only takes about 6 seconds to complete (Yang et al., 2008).
Through its lifetime, adult Drosophila will encounter a variety of rotting fruits
that can serve as egg-laying substrates (Jaenike 1983, Hoffmann and McKechnie, 1991).
Moreover, the landscape of the rotting fruit can also be heterogeneous (Miller et al.,
2011), making it critical for flies to be able to assess the various chemosensory quality of
oviposition sites to determine which patches are the best for their eggs. Recent work has
begun to examine how Drosophila respond to different stimuli for egg-laying. For
example, acetic acid (AA) has been shown to be an attractive cue for egg-laying, though
this attraction is concentration dependent, as higher concentrations of AA results in egglaying away from acetic acid (Gou et al., 2014, Joseph et al., 2009, Eisses, 1997).
Interestingly, attraction to AA is not mediated through the olfactory system, as flies with
their antennae cut still prefer to lay eggs on acetic acid substrates, but is rather mediated
through the gustatory system. Moreover, attraction is specific to AA and cannot be
generalized to other acids (Joesph et al., 2009). AA is produced by fermenting fruit and
is thought to help direct flies to food sources (Eisses, 1997). As such, AA attraction
during egg-laying may reflect a decision to lay eggs close to food sources. Flies have
also been shown to be attracted to lobeline for egg-laying (Yang et al., 2008, Joseph and
Heberlein, 2012). Oviposition attraction to lobeline is mediated through Gr66aexpressing gustatory cells (Joseph and Heberlein, 2012). It is worth nothing that both
acetic acid and lobeline are normally aversive stimuli yet are attractive for egg-laying,
suggesting egg-laying can change the behavioral valence of certain stimuli for
Egg-laying sensitivity to sweet compounds is complex. Yang et al. showed that
flies prefer to lay eggs away from sucrose which is mediated through Gr5a-expressing
sugar sensing neurons (Yang et al., 2008). This phenotype appears to contradict the
hypothesis that flies prefer to deposit eggs next to food sources described earlier.
However, oviposition away from sucrose decreases as distance increases between the
two substrates. Therefore, it is possible that flies may be less inclined to lay eggs directly
on sucrose, but rather somewhere nearby in order for larvae to be close enough to feed.
Other reports have suggested experimental setup can influence how flies behave
towards sugar during egg-laying. When using larger egg-laying substrates or larger
chambers to house egg-laying females, sugar becomes preferable as an egg-laying
substrate (Schwartz et al., 2012). Combined, these results highlight the challenge of
recapitulating native egg-laying preferences of Drosophila in laboratory settings, as flies
would typically encounter numerous egg-laying substrates with complex environmental
cues, rather than a single stimulus between two egg-laying substrates.
Drosophila are also sensitive to olfactory cues during egg-laying. Recently, it has
been shown that flies have been found to prefer citrus fruits, such as oranges and
grapefruit, for egg-laying (Dweck et al., 2013). Interestingly, they prefer unpeeled,
rather than peeled oranges. Further investigation showed that attraction to citrus fruits
is not through sugar or acid detection, but rather through limonene, a volatile molecule
that is detected by ai2A olfactory neurons that express the olfactory receptor, Or19a
(Dweck et al., 2013). Moreover, ai2A neurons are both necessary and sufficient for
conferring oviposition attraction to limonene, suggesting this attraction is innately wired
within the nervous system. Interestingly, endoparasitoid wasps that use fly larvae as
hosts for their eggs and developing larvae are repelled by limonene. Thus, egg-laying
attraction towards limonene may have evolved to help flies avoid parasitization from
However, not all olfactory stimuli are attractive for oviposition. For example,
geosmin is an olfactory cue that inhibits oviposition (Stensmyr et al., 2012). Geosmin is
produced from Penicillum fungal molds and Streptomyces bacteria and serves as a cue for
harmful microbes that decrease fly survivability (Mattheis and Roberts, 1992, Gerber
and Lechevalier 1965). Addition of geosmin to egg-laying substrates also decreases
Drosophila attraction to acid-containing substrates and activates ab4B olfactory sensory
neurons that express Or56a and Or33a odorant receptors.
Visual stimuli can also influence oviposition behavior in flies. Flies show no
difference in number of eggs laid on substrates with or without color. However, when
forced to choose between a colored substrate and non-colored substrate, they will prefer
to lay eggs on the colored substrate. Moreover, they prefer green substrates over red,
blue, and yellow substrates (Solar et al., 1974). This attraction may be another
mechanism to help direct flies to food sources, as green could represent foliage
Flies also use mechanosensory and social cues to determine suitable egg-laying
substrates. Roughness and texture of substrates can influence oviposition as they prefer
to deposit eggs in grooves rather than smooth surfaces (Atkinson, 1981). It has also been
shown that flies previously exposed to a neutral stimulus results in more eggs being laid
on the stimulus-containing substrate (Sarin and Dukas, 2009). Moreover, naive females
that interact with stimulus-experienced females also lay more eggs on the stimuluscontaining substrate, suggesting that preferable egg-laying substrates may also be
learned through social interaction (Sarin and Dukas, 2009, Battesti et al., 2012). Feeding
larvae has also been shown to serve as a learning cue for oviposition (Durisko et al.,
2013). Finally, females tend to exhibit aggregated or gregarious oviposition, preferring
substrates where eggs have previously been laid (Ruiz-Dubreuil and Solar, 1993, RuizDubreuil et al., 1994).
1.2 Mechanisms of egg-laying control
Although the egg-laying preferences of Drosophila have been extensively studied,
the neural mechanisms that mediate the conversion of sensory information into
oviposition choice remain unclear. Only recent work has begun determining some of
the central circuits required for this behavior. The neural circuits that regulate motor
control of egg-laying include neurons that send neurites all along the reproductive
system including the ovary, oviduct, and uterus (Middleton et al., 2006). There are
several lines of evidence that suggest the biogenic amine octopamine (OA) is required
for egg-laying (Monastirioti 2003). OA biosynthesis in the nervous system requires two
enzymes: tyrosine decarboxylase (Tdc2) and tyramine β-hydroxylase (Tβh). Mutants for
both Tdc2 and Tβh cannot lay eggs, which can be rescued when females are
supplemented with OA in their food (Monastirioti, 2003, Monastirioti et al., 1996, Cole et
al., 2005), suggesting the neurotransmitter itself is required for egg-laying. The OA
neurons that are required for egg-laying are clustered at the ventral tip of the VNC,
which send processes down towards the reproductive tract and overlaps with neurons
that express the transcription factor doublesex (Monastirioti, 2003, Rodriguez-Valentin et
al., 2006, Rezaval et al., 2012, Rezaval et al., 2014, Rideout et al., 2010). Physiologically,
OA is necessary for relaxing the oviduct and increasing muscle contraction to help move
eggs from ovary to oviduct (Middleton et al., 2006). In addition, mutants for the two OA
receptors, OAMB and Octβ2R, also show a reduction in egg-laying further suggesting
that the OA system in the VNC is critical for successful egg deposition (Lim et al., 2014,
Li et al., 2014, Lee et al., 2003, Lee et al., 2009).
Another set of neurons that can influence egg-laying include a set of
glutamatergic neurons that innervate the oviduct. These neurons co-express the peptide
Insulin-like peptide 7 (Ilp7) (Gronke et al., 2010) and are motor neurons that can regulate
the contraction of the muscle surrounding the oviduct (Gou et al., 2014, Castellanos et
al., 2013). Moreover, inhibition of these neurons leads to eggs being jammed in the
oviduct, suggesting that these neurons are required for motor control/egg expulsion
(Yang et al., 2008), though the peptide itself is not required for egg-laying as Ilp7
mutants can still lay eggs (Gronke et al., 2010).
Finally, there are a set of sensory neurons that express the mechanosensory
channel pickpocket (PPK) that tile the reproductive tract in Drosophila. Inhibiting these
PPK-expressing neurons also leads to a failure to lay eggs with eggs being jammed in
the oviduct (Gou et al., 2014). These neurons are thought to sense the expansion of the
oviduct when an egg is present and relay to the central brain the readiness of an egg to
be laid, thereby representing the first neural substrate instructing the female to begin
oviposition searching behavior (Gou et al., 2014).
1.3 Neural mechanisms of oviposition
Although the egg-laying preferences of Drosophila have been extensively studied,
the neural mechanisms that mediate the conversion of sensory information into
oviposition choice remain unclear. Only recent work has begun determining some of
the central neural circuits required for this behavior. Joseph and colleagues showed that
oviposition preference for acetic acid requires the mushroom body (MB) neurons.
Interestingly, they also find that positional avoidance of acetic acid does not require the
MB, but rather another set of central neurons known as the central complex (CC) (Joseph
et al., 2009). Moreover, since both positional avoidance and oviposition preference for
acetic acid require olfactory and gustatory input, respectively, this suggests that both
behaviors (movement vs. egg-laying) are mediated through separate sensory and central
circuits. Whether the behavioral output for avoidance or attraction in either behavior
requires overlapping circuitry outside of the MB or CC remains to be seen, but these
results identify one of the first neural substrates for egg-laying behavior in flies.
Similarly, the bitter tasting compound lobeline also leads to positional avoidance
and oviposition preference. However, both behaviors are mediated through different
sets ofGr66a-expressing sensory neurons of the gustatory system, and both are regulated
by the MB (Joseph et al., 2012). At face value, this appears to contradict the previous
report that suggests positional avoidance is mediated through the CC and oviposition
preference is dependent on the MB. However, since both position and oviposition
towards lobeline is mediated through the gustatory system, it is possible that the MB is
an important structure for processing gustatory information across many behaviors and
therefore inhibiting MB function affects both position and egg-laying during these
Another article has suggested that there are subsets of neurons within the
dopaminergic (DA) circuit that also play an important role in determining oviposition
preference. Azanchi et al. show that Drosophila are attracted to ethanol-containing
substrates for egg-laying (Azanchi et al., 2013). Moreover, they suggest that the two DA
cell clusters, PAM and PPM3, are important for mediating attraction for ethanol during
egg-laying. However, they also inhibited function of DA neurons in the PPL1 cluster,
which led to more eggs being laid on ethanol substrates, suggesting PPL1 neurons
mediate avoidance of ethanol substrates. Combined, these data suggest that different
DA neurons can act in opposing fashion when assessing the attractiveness of egg-laying
substrates. The authors also show that the B’ neurons of the MB and R2 neurons of the
CC are also important for attraction to ethanol, which are downstream of the PAM and
PPM3 DA neurons, respectively.
Finally, neuropeptide F (NPF) has also been shown to modulate how Drosophila
behave towards ethanol-containing substrates during oviposition (Kacsoh et al., 2013).
As mentioned earlier, flies normally have a preference for ethanol when egg-laying, but
they will increase the relative number of eggs laid on ethanol when visually exposed to
female parasitic wasps. Interestingly, overexpression of NPF in NPF neurons and global
neuronal expression of NPF receptors leads to no increase in egg-laying on ethanol after
exposure to wasps. Moreover, knockdown of NPF in NPF neurons and global
knockdown of NPF receptors results in increased egg-laying on ethanol even in female
flies that have not been exposed to wasps (Kacsoh et al., 2013). Combined, this data
suggests the ability to lay more eggs on ethanol when Drosophila see parasitic wasps is
mediated, at least in part, by a decrease in NPF activity.
2. Egg-laying induces aversion of UV light in Drosophila
Recent reports have shown that flies are sensitive to many different cues for
oviposition. Gustatory, olfactory, and mechanosensory stimuli can all influence how
females assess potential egg-laying substrates. We wanted to expand our analysis to see
how other stimuli can change the attractiveness of egg-laying sites. Ultraviolet light
(UV) is a well documented phototactic cue for adult Drosophila (Jacob et al. 1977,
Fischbach 1979, Paulk et al. 2013, Yamaguchi et al. 2010, Yamaguchi et al. 2011, Gao et al.
2008, Karuppudurai et al. 2014). Light attraction is thought to reflect the “escape”
response for flies, signifying open space and possibly to evade potential predators
(Benzer 1967). Flies also prefer UV when placed in spectral preference assays. Here,
flies are asked to move towards one of two light sources that are placed at opposite ends
of a T-maze. In these conditions, UV is preferred over blue and green wavelengths,
suggesting UV is the most attractive light wavelength for Drosophila. In contrast, fly
larvae find UV to be an aversive cue, preferring to move away from UV during
behavioral assays (Sawin-McCormack et al. 1995, Xiang et al. 2010, Kane et al. 2013).
Given this dichotomy in behavior between adults and larvae, we sought to understand
how egg-laying females would respond UV when selecting for an egg-laying substrate
and the neural mechanisms that regulate this behavior.
2.1 Drosophila prefer to lay eggs away from UV
To determine how Drosophila respond to UV for egg-laying, we first constructed
egg-laying chambers that could house up to 30 female flies (Fig 1A-B). Each egg-laying
arena consists of two egg-laying substrates composed of 1% agarose (Fig 1C). A small
amount of grape juice (5 μl) is placed in the center of the arena to serve as a food source
during behavioral experiments. This set up allowed us to assay the egg-laying
preferences of Drosophila at a much higher throughput compared to previous behavioral
experiments (Yang et al., 2008). When choosing between two 1% agarose egg-laying
sites, flies show no preference and lay eggs equally on both sites (Fig 1C).
We then designed custom lids that could hold light emitting diodes (LEDs) and
be attached to the top of the egg-laying chambers. UV LEDs (λ = 380 nm) were inserted
to illuminate one of the two substrates with UV (Fig 2A-D). We then tested the egglaying preferences of flies between UV-illuminated (UV) and non-illuminated (dark)
sites and found that they prefer to lay eggs away from UV (Fig 2E). To see how sensitive
Drosophila were to UV during egg-laying, we varied the strength of our UV LEDs during
egg-laying assays. Even at our lowest testable UV intensity (.16 μW/mm2), wild type
flies robustly lay away from UV (Fig 3A-B), suggesting preference to lay away from UV
is a very robust and reproducible behavior.
Figure 1: Egg-laying chambers to assess egg-laying preferences
(A-B) Photographs of assembled (A) and disassembled (B) egg-laying chambers. Each
chamber contains 30 arenas to assess egg-laying preferences of flies.
Representative photograph and quantification of wild type (w1118) flies when
choosing between 2 non-illuminated (dark vs. dark) egg-laying sites made of 1%
agarose. Grape juice is placed in the middle of the arena to serve as a food
source during experiments. Egg-laying preference index (Egg-laying PI) is
calculated as (Nleft – Nright)/(Nleft + Nright) where Nleft and Nright represent the
number of eggs on the left and right site, respectively. The white arrow points to
fly in the arena. Egg-laying substrates are outlined in black. All error bars
represent SEM and number of flies assayed is indicated above each bar. &, p >
.05, one-sample t-test from 0.
To test whether flies were sensitive to other wavelengths of light, we also used
blue, green, red, infrared, and white (visible) LEDs during egg-laying assays. We found
that females lay away from blue and green light but did not lay away from red and
infrared light (Fig 3C-D). Given that females lay away from blue and green light, it is
unsurprising to see females also lay away from white light. Females still prefer to lay
away from UV when choosing between UV-illuminated and white-illuminated sites (Fig
3E, G). Collectively, these data suggest flies prefer to lay eggs away from light, but are
most sensitive to UV when choosing for an egg-laying substrate. However, UV does not
inhibit egg-laying in general, as flies lay comparable number of eggs between dark-only
and UV-only conditions (3F, H).
Figure 2: Drosophila prefer to lay eggs away from UV
(A-B) Schematic of assay used to test egg-laying preferences of Drosophila. A UV LED
is placed above one of the two substrates in UV vs. dark egg-laying assays.
(C-D) Photographs of UV LED setup used to illuminate egg-laying sites with UV. (C)
LEDs in LED holder (1) are connected to LED driver (2). LED intensity is
controlled by a microcontroller (3). (D) LEDs attached to top of egg-laying
Representative photograph and egg-laying PI of wild-type flies when one of the
two sites is illuminated with UV. Egg-laying PI is calculated as (NUV –
Ndark)/(NUV + Ndark) where NUV and Ndark represent the number of eggs on the UV
and dark site, respectively. p < .0001, one sample t-test from 0.
Figure 3: Drosophila also prefer to lay eggs away from wavelengths of light
(A-B) Egg-laying PI of w1118 (A) and CS (B) females with different intensities of UV in
UV vs dark assays. Unless otherwise noted, we used 2 μW/mm2 (dark gray bar)
for other egg-laying experiments.
(C-D) Egg-laying PI of w1118 (C) and CS (D) females for different wavelengths of light.
Blue (467 nm), green (525 nm), red (631 nm), infrared (940 nm) LEDs were set at 2
μW/mm2. White light was set at maximum LED intensity (109 μW/mm2). UV vs.
blue, p < .0001. UV vs. green, p < .0001.
(E,G) Egg-laying PI of w1118 (E) and CS (G) females in UV vs. white assays. Both UV
and white LEDs were set to 2 μW/mm2. p < .0001, one sample t-test from 0.
(F,H) Egg-laying rate of w1118 (F) and CS (H) females in dark vs. dark and UV vs. UV
assays. p > .05, t-test.
2.2 Egg-laying induces behavioral aversion of UV
Given that flies prefer to lay eggs away from UV but are normally phototactic
towards UV, this suggests that egg-laying may temporarily turn UV into an aversive cue
that leads to egg-laying away from UV. We hypothesized that there are at least three
behavioral strategies that flies could use to reconcile phototactic attraction and
oviposition avoidance of UV. First, it is possible that Drosophila maintains UV attraction
throughout egg-laying, but only move towards the UV substrate for egg deposition
immediately prior egg deposition. Conversely, it is also possible that Drosophila prefer
to spend all their time away from UV during egg-laying, which contributes to the
preference to lay eggs away from UV. The last strategy could be the medium of the two
extremes, where flies maintain UV phototaxis but go through periods of UV aversion
that correlates with egg-laying.
To get a better understanding of the behavioral strategy of egg-laying away from
UV, we refitted our egg-laying chambers with cameras in order to video record the
animals as they explored and laid eggs during egg-laying experiments. In order to
attach cameras to the top of chambers, the UV LEDs were moved to illuminate the egglaying substrate from the bottom, rather than from the top for previous egg-laying
experiments (Fig 4A-B). This change in LED position did not affect egg-laying
preferences of wild type flies (Fig 4F). We then manually annotated the timestamp of
individual egg-laying events (ELE) and tracked the position of each female using a
modified version of the open-source tracking software Ctrax (Fig 4C-E, Branson et al
2009, Stern and Yang 2014). Flies were recorded for 8 hours and their positional data
was used for behavioral analysis.
Figure 4: Setup to record and track positions of flies during egg-laying
(A-B) Side and angled view of (1) egg-laying chambers with (2) cameras and (3) UV
LEDs attached. Not shown: red lightpad used to illuminate chambers during
Representative frame from recording video showing 2 mated w1118 females. Dark
spots on top substrates are eggs.
Representative frame after videos are converted and traced with Ctrax.
Red/green lines represent recent path of flies up until the given frame.
Two 1-hour long trajectories of two mated females tracked using Ctrax.
Wild type females (w1118) lay away from UV in recording setup (UV
illuminated from below). p < .0001, one-sample t-test from 0.
2.2.1 Drosophila spend less time on UV during egg-laying
To determine whether egg-laying demand induces UV aversion, we first
analyzed the relative amount of time females spent on UV and dark sites during egglaying (Fig 5A-B). Interestingly, virgin females spend equal amounts of time between
UV and dark sites but mated females spend more time on dark sites, suggesting UV has
become less attractive (Fig 5D). Given that mated females lay significantly more eggs
than virgin females, this data correlates with the hypothesis that egg-laying induces UV
To get a better understanding of whether egg-laying leads to more time spent
away from UV, we next compared the relative time spent between UV and dark sites
when they are versus are not laying eggs. Because the temporal pattern of ELEs is
unevenly distributed during each 8 hour experiment, we separated each video into hour
long segments to identify periods of no egg-laying as well as periods with high egglaying (Fig 5C). We then compared the relative time spent between no and high egglaying periods. We found that during periods of no egg-laying, mated females prefer to
spend equal amounts of time on both sites but during periods of high egg-laying,
females tend to spend more time on dark (Fig 5E). These results recapitulate the
phenotype observed between virgin and mated females, suggesting that egg-laying
induces UV aversion in egg-laying females.
Figure 5: Egg-laying demand induces positional aversion of UV
A representative frame of a video where the position of an egg-laying fly is being
tracked. The bright spot in the chamber is the UV LED illuminating the substrate
from below. The red line that follows the animal is part of the trajectory
generated by Ctrax. The dark specs on the dark site are eggs.
Schematic of how trajectories were analyzed as they explored and laid eggs in
UV vs. dark assays. The y-axis denotes y position within the video frame, and
the x-axis denotes time. Panel depicts time spent on UV site (timeUV) and time
spent on dark site (timedark), which were used to calculate the index for relative
time spent on UV vs. dark (positional PI).
Temporal pattern of ELEs from two wild-type flies. Blue lines represent
individual ELEs. Red bars depict a 1 hour period of no egg-laying (0 eggs laid)
whereas green bars depict a 1 hour period of high egg-laying (7+ eggs laid).
Positional PI of virgin and mated flies. Positional PI was calculated as (TUVTdark)/(TUV+Tdark), where TUV and Tdark represent time spent on UV or dark sites.
*** p < .0001, t-test. & p > .05, one-sample t test from 0.
Positional PI of mated flies during periods of no egg-laying and high egg-laying.
*** p < .0001, t-test.
2.2.2 Drosophila move away from UV during egg-laying
Although positional PI suggests egg-laying correlates with UV aversion, this
analysis has its limitations. Namely, each ELE is often followed by a minute or two of
“rest” on the substrate where an egg was laid (Fig 6E, red arrows). Therefore, for wild
type females that deposit their eggs on dark sites, these rest periods after egg-laying
could artificially inflate the positional aversion we are measuring when comparing time
spent between UV and dark sites. To overcome this caveat, we reexamined the
trajectories and found instances which females exhibited attractive turns towards or
aversive turns away from UV. We defined these attractive turns towards UV (UV
return) as instances where the fly would leave and return back towards the UV site
without entering the dark site (Fig 6A). Similarly, we defined aversive turns away from
UV (dark return) as instances where the fly would leave and return back towards the
dark site without entering the UV site (Fig 6A). We propose that these returns reflect
UV attraction and aversion and used the relative amount of UV and dark returns to
determine whether a fly is exhibiting UV attraction or aversion during a given time
We first examined the return preference indices (returns PI) of virgin and mated
females. Virgin females exhibit more UV returns relative to dark returns, suggesting
that these flies are attracted to UV, whereas mated females show neither attraction nor
aversion of UV (Fig 6B). However, the reduction in relative UV vs. dark returns
suggests mated females avoid UV more often than virgin females. Like our positional PI
analysis, we then examined the relative returns towards UV and dark sites in mated
females between no and high egg-laying periods. Here, we found that during periods of
no egg-laying, wild type flies exhibit more attractive UV returns, similar to the
phenotype observed in virgin females (Fig 6C, D). However, females exhibit more turns
away from UV during high egg-laying periods, suggesting UV is aversive in this context
(Fig 6C, E). Furthermore, analysis of the return index during the minute immediately
before each ELE suggests UV aversion is present prior to egg deposition (Fig 6C).
Together, these results suggest that egg-laying demand turns UV into an
aversive cue for Drosophila. We show that females (1) avoid laying eggs on UV sites and
(2) tend to turn away from UV sites prior to egg-laying. We refer to the former
phenotype as “egg-laying aversion of UV” and the latter phenotype as “movement
aversion of UV.”
Figure 6: Egg-laying demand induces movement aversion of UV
Schematic depicting a UV return and a dark return within a trajectory, which
were used to calculate the index for relative returns towards UV and dark sites
Return PI of virgin and mated flies. Return PI was calculated as (RUVRdark)/(RUV+Rdark), where RUV and Rdark represent the number of UV or dark returns
in a given trajectory. *** p < .0001, t-test. & p > .05, one-sample t test from 0.
Return PI of mated flies during periods of no egg-laying and high egg-laying.
*** p < .0001, t-test.
(D-E) Representative 30 min trajectories of a period with no egg-laying (D) and a
period with high egg-laying (E). The x-axis denotes time and the y-axis denotes
the y position of the fly. Purple boxes outline UV returns, whereas black circles
outline dark returns. Red arrows point to the stereotypical rest periods that
follow individual ELEs. Black, vertical lines on trajectories represent the
occurrence of an ELE.
2.3 Neural substrates for UV aversion
2.3.1 UV aversion is mediated through the visual system
We next sought to determine the neural substrates that regulate both egg-laying
and movement aversion of UV. Our first goal was to identify the sensory system that
was required for egg-laying away from UV. As such, we tested the egg-laying
preferences of different mutants with an impaired visual system. Mutants that lack
physical eyes (GMR-hid, Grether et al. 1995), the phototransduction molecule,
phospholipase C (norpA36, Bloomquist et al. 1988), or histamine (hdcJK910, Burg et al. 1993),
the neurotransmitter of photoreceptors (PRs), all lay equal number of eggs between UV
and dark substrates, suggesting that these mutants can no longer distinguish the
differences between these two sites (Fig 7A). We then took advantage of the GAL4/UAS
binary expression system (Brand and Perrimon 1993) and used the GMR-GAL4 driver to
express the synaptic inhibitor tetanus toxin (UAS-TNT), which eliminated egg-laying
away from UV (Fig 10A, Sweeney et al. 1995). We also used GMR-GAL4 to express
norpA (UAS-norpA) in all PRs in norpA36 mutants. These norpA rescued flies also lay eggs
away from UV (Fig 7A). To test whether movement of aversion of UV was also
mediated through vision, we recorded, tracked, and analyzed the trajectories of norpA36
mutants. We found that these mutants do not exhibit either UV attraction or aversion
during no and high egg-laying periods (Fig 7B). Collectively, this data suggest that the
visual system is required for both egg-laying and movement aversion of UV.
Figure 7: Egg-laying and movement aversion of UV is mediated through the eye
Egg-laying PI of structural eye mutants (GMR-hid), phototransduction mutants
(norpA36), histadine decarboxylase mutants (hdcJK910), necessary for histamine
production), and norpA36 mutants with norpA rescued in all PRs
(norpA36;GMR>norpA). & p > .05, one-sample t-test from 0.
Return PI of norpA36 mutants during periods of no and high egg-laying. & p >
.05, one-sample t-test.
2.3.2 R7 photoreceptors are required for both egg-laying and
movement aversion of UV
Next, we aimed to identify the photoreceptors (PRs) that regulate egg-laying and
movement aversion of UV. The PRs in the Drosophila eye can be divided into two
anatomically and functionally distinct groups. The two inner PRs R7 and R8 can be
further divided into two subgroups (Paulk et al. 2013). The dorsal area of the eye
contains both R7 and R8 PRs that express the short UV wavelength sensitive rhodopsin
Rh3. This region is designed to detect polarized light (Labhart and Meyer 1999, Wernet
et al. 2003, Wernet et al. 2012). For the rest of the eye, R7 and R8 PRs can be found in
pale (p) or yellow (y) ommatidia. P-type ommatidia include R7 PRs that express the UV
sensitive Rh3 and R8 PRs that express the blue sensitive Rh5. Y-type ommatidia contain
R7 PRs that express the longer wavelength UV sensitive Rh4 and R8 PRs that express the
green sensitive Rh6 (Salcedo et al. 1999, Montell et al. 1987, Zuker et al. 1987). In
addition, p- and y-type ommatidia are distributed as a 30/70 ratio and are stochastically
patterned across the retina. The outer PRs R1-6 express the UV/blue sensitive Rh1 and
are important for motion detection and optomotor responses (O’Tousa et al. 1985,
Heisenberg and Buchner 1977, Yamaguchi et al. 2008, Wardill et al. 2012).
Given that both R7 and R1-6 express UV sensitive rhodopsins, both groups of
PRs could contribute to UV aversion. We first removed R7 function to test their
necessity in this behavioral paradigm. The R7 mutant, sevenless (sev14, Harris et al. 1976,
Yamaguchi et al. 2010), laid fewer eggs away from UV (Fig 8A). We also used the R7GAL4 driver to synaptically inhibit (UAS-TNT, Sweeney et al. 1995) or hyperpolarize
(UAS-Kir2.1, Baines et al. 2001) R7 PRs and found that these flies also laid fewer eggs
away from UV (Fig 8A, 10B). We then rescued norpA in R7 (R7>norpA) in norpA36
mutants which rescued egg-laying away from UV (Fig 8A), suggesting that R7 is both
necessary and sufficient for egg-laying aversion of UV.
We then tested the role of R1-6 PRs in egg-laying aversion of UV. Rh1 mutants
(ninaE17, O’Tousa et al. 1985) still lay eggs away from UV similar to wild type flies (Fig
8C). Moreover, inhibition of R1-6 (Rh1-GAL4 expressing UAS-TNT or UAS-Kir2.1) also
does not reduce egg-laying aversion of UV (Fig 8C, 10C). Interestingly, rescue of R1-6
function (Rh1>norpA) in norpA36 mutants led to flies preferring UV substrates for egglaying. This suggests R1-6 promote egg-laying attraction to UV that is normally
suppressed in the presence of R7 PRs in wild type flies. Indeed, rescuing both R7 and
R1-6 function in norpA36 mutants results in egg-laying aversion of UV (Fig 8C).
Figure 8: R7 photoreceptors are required for egg-laying and movement aversion of UV
Egg-laying PI of flies with defective R7 photoreceptor function (sev14 and
R7>TNT) and flies with only functional R7 photoreceptors (norpA36;R7>norpA).
*** p < .0001, t-test. One-way ANOVA, Bonferroni post-hoc for R7>TNT.
Return PI of flies with defective R7 function (R7>TNT) and flies with only
functional R7 (norpA36;R7>norpA). One-way ANOVA, Bonferroni post-hoc for
R7>TNT. *** p < .0001, t-test. & p > .05, one-sample t-test from 0.
Egg-laying PI of flies with defective R1-6 photoreceptor function (ninaE17 and
Rh1>TNT), flies with only functional R1-6 photoreceptors (norpA36;Rh1>norpA),
and flies with both functional R1-6 and R7 photoreceptors
(norpA36;Rh1+R7>norpA). *** p < .0001, t-test. One-way ANOVA, Bonferroni
post-hoc for Rh1>TNT.
Return PI of flies with defective R1-6 function (Rh1>TNT) and flies with only R16 (norpA36; Rh1>norpA). & p > .05, one-sample t-test from 0.
We next assessed the roles of R7 and R1-6 in movement aversion of UV. We
found that inhibition of R7 eliminates movement aversion of UV during high egg-laying
periods, whereas rescue of norpA function in R7 rescues movement aversion (Fig 8B). In
contrast, inhibition of R1-6 does not impair movement aversion during high egg-laying
periods (Fig 8D). It has been suggested that UAS-TNT may not be effective at blocking
R1-6 function (Rister and Heisenberg, 2006), so we also analyzed the behavior of
Rh1>Kir2.1 flies. These flies also do not have impaired movement aversion (Fig 10F). In
addition, movement aversion of UV cannot be rescued with only functional R1-6 PRs
(Fig 8D). Together, our results suggest egg-laying and movement aversion of UV are
both mediated by R7 and not R1-6 PRs.
Inhibition of R7 or R1-6 did not reduce movement attraction during no egglaying periods (Fig 11A). We hypothesize that this may be due to redundant UV
attractive pathways from all sets of PRs. If this hypothesis were true, then we would
expect single sets of PRs to be able to rescue UV attraction. Indeed, rescue of R7
function alone is sufficient to rescue movement attraction similar to wild type flies
during no egg-laying periods (Fig 8B). Rescue of R1-6 function leads to a different form
of UV attraction. These females spend more time on UV during both no and high egglaying periods (Fig 10E). They also tend to exhibit less locomotion compared to wild
type and R7 rescued animals alone. Therefore, if R1-6 rescued flies spend more time on
UV and do not move away from the UV substrate, then there would be fewer
opportunities to “return” back towards the UV substrate for our returns analysis, which
may explain why we do not observe “returns” attraction in these animals. Given the
extensive literature describing the roles of R7 and R1-6 for light attraction in different
behavioral experimental setups, the UV attraction observed from independent rescue of
R7 and R1-6 PRs in our paradigm suggests that the UV attraction may be hardwired
through the majority of the Drosophila visual system and is difficult to isolate from a
single group of PRs.
2.3.3 Dm8 neurons are required for movement aversion, but not egglaying aversion, of UV
Next, we wanted to determine how egg-laying and movement aversion of UV
are regulated by the circuit components downstream of R7. Drosophila photoreceptors,
including R7, are histaminergic (Elias and Evans 1983, Sarthy 1991, Burg et al. 1993).
Since histamine is required for egg-laying away from UV (Fig 7A), downstream neurons
must express histamine receptors to receive R7 information. There are currently two
identified histamine-gated ionotropic channels in Drosophila: ort (ora transientless) and
HisCl1 (Gengs et al. 2002, Gisselmann et al. 2002, Witte et al. 2002, Zheng et al. 2002,
Pantazis et al. 2008). Because these two channels were simultaneously discovered by
three groups, ort has also been referred to as hclA and HisCl2, whereas HisCl1 has also
referred to as hclB. Ort is required for motion detection and is required for
neurotransmission between R1-6 and the large monopolar cells (LMCs), L1 and L2, in
the lamina (Gengs et al. 2002, Pantazis et al. 2008). Ort is also required for R7 mediated
spectral preference (Gao et al. 2008). However, eliminating ort function alone is not
sufficient to remove spectral preference altogether. Spectral preference is eliminated
only in double null mutants for ort and HisCl1 (Gao et al. 2008), suggesting both Ortexpressing and HisCl1-expressing neurons contribute to spectral preference.
To test whether ort was required for egg-laying away from UV, we assayed the
egg-laying preferences of two ort null mutants, ort1 and ort5. Both mutants lay fewer
eggs away from UV compared to wild type flies (Fig 9A), suggesting this behavior is
mediated, at least in part, through ort. However, they still exhibit egg-laying aversion of
UV. Therefore, we also tested the HisCl1 and ort double null mutant, HisCl1134, ort1, in
UV vs. dark assays. HisCl1134, ort1 double mutants exhibit even less egg-laying aversion
compared to ort1 and ort5 single mutants (Fig 9A). Although these double mutants still
exhibit egg-laying avoidance, their phenotype is on par with what we observe in sev14 R7
PR mutants (sev14 mean: -.36 vs. HisCl1134, ort1 mean: -.39) in UV vs. dark assays.
Gao et al. showed that the ort-expressing Dm8 amacrine neurons are the synaptic
targets of R7 that mediate spectral preference in Drosophila (Gao et al. 2008). In addition,
Dm8 neurons pool information of roughly 16 R7 PRs to Tm5c transmedulla neurons
(Karuppudurai et al. 2014). It has been proposed this R7
Tm5c form a hard-
wired glutamatergic circuit that mediates UV spectral preference. If UV spectral
preference and egg-laying aversion of UV use the same visual circuits, then we would
expect Dm8 neurons to also be a critical neural substrate for egg-laying aversion. To test
the role of Dm8s in UV aversion, we used the split-GAL4 combination, ortc2∩vGlut, to
specifically inhibit their output (Gao et al. 2008). Inhibiting Dm8 neurons did not reduce
egg-laying aversion of UV (Fig 9B). This result is recapitulated when we use a GAL4
driver (ortC1-4-GAL4) that marks ort-expressing neurons to inhibit Dm8 neurons (Fig 9B).
However, females with inhibited Dm8s no longer exhibit movement aversion to
UV during high egg-laying periods (Fig 9C). Interestingly, they still show movement
attraction during periods of no egg-laying (Fig 11A). Visual inspection of their
trajectories (Fig 9D-E) reveals that although Dm8-inhibited females lay eggs away from
UV, they still exhibit frequent “UV returns” during high egg-laying periods. In contrast,
wild type females primarily exhibit “dark” returns during high egg-laying periods (Fig
6E). These results suggest egg-laying and movement aversion of UV do not use
overlapping circuits in the Drosophila visual system, but are rather under separate circuit
control downstream of R7 PRs.
Figure 9: Dm8 amacrine neurons are required for movement aversion but not required
for egg-laying aversion of UV
Egg-laying PI of ort histamine receptor mutants (ort1 and ort5) and HisCl1/ort
double mutants (HisCl1134,ort1). ** p < .001, *** p < .0001, t-test.
Egg-laying PI of flies with inhibited Dm8 amacrine neurons (vGlut∩ortC2>TNT)
and inhibited ort expressing neurons (ortC1-4>TNT). p > .05, one-way ANOVA,
Bonferroni post hoc.
Return PI during periods of high egg-laying in flies with inhibited Dm8 neurons
(vGlut∩ortC2>TNT). * p < .05, *** p < .0001, one-way ANOVA.
(D-E) Representative 30 min trajectories of a period with no egg-laying (C) and a
period with high egg-laying (D) of flies without functional Dm8 neurons. Note
that these flies still lay eggs on dark sites but exhibit frequent UV returns.
Model of the contribution of R7 and Dm8 neurons during egg-laying. When flies
are not laying eggs, they exhibit spectral preference (Gao et al., 2008,
Karrupudurai et al., 2014) and movement attraction towards UV in our
paradigm. Once egg-laying begins, flies exhibit both egg-laying and movement
aversion of UV. The R7-Dm8 pathway is required for movement aversion but is
not required for egg-laying aversion.
Figure 10: Role of PRs during UV-driven egg-laying behavior
Egg-laying PI of flies with all PRs inhibited with UAS-TNT. *** p < .0001, oneway ANOVA, Bonferroni post hoc.
(B-C) Egg-laying PI of flies with R7 or R1-6 PRs inhibited with UAS-Kir2.1. *** p <
.0001, one-way ANOVA, Bonferroni post hoc.
Egg-laying PI of flies with rescued R8 function in norpA36 mutants. *** p < .0001,
t-test. & p > .05, one-sample t-test from 0.
Positional PI for periods of no and high egg-laying in w1118, norpA36, R7 rescued,
and R1-6 rescued flies. *** p < .0001, t-test. & p > .05, one-sample t-test from 0.
Figure 11: Movement attraction uses redundant PR pathways
Return PI for periods of no egg-laying for flies without R7 (R7>TNT), R1-6
(Rh1>TNT), or Dm8 (vGlut∩ortC2>TNT) function. p < .0001, one-sample t-test from
(B-E) Expression pattern of R7-GAL4 (B), Rh1-GAL4 (C), ortC1-4-GAL4 (D), and
vGlut∩ortC2-GAL4 (E) crossed to UAS-mCD8::GFP. Scale bar = 50 μm.
3. Central mechanisms of egg-laying site selection
In addition to characterizing egg-laying behavior in flies, we also sought to
elucidate how the brain evaluates information from different sensory systems to
determine the suitability of an egg-laying substrate. Previously, Yang et al. showed that
Drosophila prefer to lay eggs away from sucrose when choosing between a sucrosecontaining and plain egg-laying substrate (Yang et al 2008, Yang et al 2015). We used
this platform to screen different sets of neurons and found a candidate neuromodulatory
system that can influence the egg-laying preferences in sucrose vs. plain assays.
3.1 Role of octopaminergic neurons during oviposition
Octopamine (OA) is a biogenic amine that is thought to be the invertebrate
homolog of norepinephrine. Described as a neurotransmitter, neuromodulator, and
neurohormone, OA can be found in both neuronal and non-neuronal tissues and has
been reported to be involved with many physiological processes including the
neuromuscular junction, energy mobilization, and modulation of the neuroendocrine
system (Roeder, 1999). It can also modulate sensory processing and is required for
associative learning in insects (Roeder, 1999). OA biosynthesis occurs in two steps.
First, tyrosine is converted to tyramine by tyrosine decarboxylase (Tdc), which is then
modified by tyramine-β-hydroxylase (Tβh) to produce octopamine.
OA is also intimately tied to Drosophila reproductive physiology. Monastirioti et
al. cloned the Drosophila gene Tβh and generated the TβhnM18 null mutant (Monastirioti et
al., 1996). These mutants lack OA and exhibit no obvious external defects. However,
while TβhnM18 females mate normally, they are sterile and unable to lay eggs. Female
mutants that are fed food supplemented with OA are able to restore egg-laying,
suggesting that egg retention is OA-dependent (Monastirioti et al., 1996). Further
examination suggests the OA neurons at the posterior tip of the VNC are required and
sufficient for ovulation (Monastirioti 2003).
3.1.1 Octopamine modulates egg-laying preference and egg-laying
To test whether central OA neurons are involved with determining the suitability
of an egg-laying substrate, we utilized a tdc2-GAL4 driver to selectively mark and
manipulate OA neuron function (Cole et al., 2005). Because this GAL4 driver also labels
the VNC OA neurons that regulate contraction of the oviduct, we used the VNC specific
tsh-GAL80 to repress GAL4 expression in the VNC (Clyne and Miesenbock, 2008).
Indeed, comparison of GFP expression in tdc2>GFP flies with or without tsh-GAL80
suggests significantly less tdc2-GAL4 expression in the VNC, particularly the cluster of
OA neurons at the VNC tip that send neurites towards the reproductive tract (Fig 12AB). Although there are still some labeled tdc2 cells left in the VNC, GAL4 expression is
repressed in the OA neurons at the furthest tip of the VNC ganglion (white boxes),
allowing us to manipulate central OA neurons to interrogate their role in egg-laying.
Figure 12: Octopamine neurons modulates egg-laying preference and rates
(A-B) Fluorescent micrographs of octopamine (OA) neurons. (A) tdc2>GFP expresses
GFP in the VNC tip neurons that are required for ovulation (white box). (B) tshGAL80 represses GAL4 expression in VNC tip neurons (white box).
Egg-laying PI of flies with inhibited OA neurons (tdc2>TNT). Note that tdc>TNT
has tsh-GAL80 in the background to inhibit GAL4 expression in the VNC tip
neurons. *** p < .0001, one-way ANOVA, Bonferroni post hoc.
Egg-laying rate of flies with activated OA neurons (tdc2>dtrpA with tsh-GAL80 in
the background). Blue bars represent flies tested at 22° C (inactive temperature
for dtrpA). Red bars represent flies tested at 32° C (active temperature for dtrpA).
*** p <.0001, two-way ANOVA, Bonferroni post hoc.
We first tested whether OA neurons were required for choosing between sucrose
and plain egg-laying sites. Inhibition of OA neurons with UAS-TNT resulted in fewer
eggs being laid away from sucrose, suggesting OA neurons are required for proper
evaluation of sucrose and plain sites (Fig 12C). Given that inhibition of OA neurons
leads to less egg-laying away from sucrose, this raises the possibility that OA might
signal to lay away from sucrose. To address this question, we used the tdc2-GAL4 driver
to express the temperature gated cation channel, dtrpA1 (Hamada et al., 2008). dtrpA1
is the mammalian homolog of trpA1 and can be used to manipulate the activity of
neurons at higher temperatures (Pulver et al., 2009). Under low temperatures (22° C),
dtrpA is closed, but upon raising the temperature (32 ° C), dtrpA channels open and
neural activity is invoked. We expressed UAS-dtrpA in OA neurons with the tdc2-GAL4
driver. Surprisingly, when we raised the temperature to increase activity of OA
neurons, rather than laying more eggs away from sucrose, tdc2 activated flies completely
shut down egg-laying (Fig 12D). This suggests that OA neurons are sufficient to
modulate the egg-laying motor program.
3.1.2 Candidate OA cell clusters for regulating egg-laying rate
We next sought to identify which OA neurons were responsible for regulating
egg-laying output. The tdc2-GAL4 driver marks roughly 100 OA neurons which can be
anatomically segregated into 4 main clusters (Busch et al., 2009). The anterior superior
medial (ASM) cluster contains 6-7 OA cells per hemisphere that are located above the
antenna lobes in the anterior portion of the fly brain with processes that extend
throughout the dorsal protocerebrum, the penduncle of the mushroom body, and the
fan shaped body of the central complex. The ventral lateral (VL) cluster contains 2 OA
cells per hemisphere that are located in the lateral protocerebrum and have processes
projecting down into the subesophageal ganglion (SOG). The antennae lobe (AL2)
cluster contains 7-8 OA cells per hemisphere that are located proximally to the antenna
lobe. These neurons contain processes that project to different neuropil in the optic lobe
and the superior posterior slope. Finally, the largest cluster of OA neurons is the ventral
medial (VM) cluster. VM cells are located in the subesophageal ganglion (SOG) near the
most inferior portion of the brain (Fig 13A-B). In addition, the VM cluster can be further
subdivided into three subclusters based on the anteroposterior axis: the mandibular
(md), maxillary (mx), and labial (lb) subclusters (Fig 13C-F).
Mosaic analysis with a repressible cell marker (MARCM) is a powerful tool for
neural circuit tracing. This technique stochastically labels a subset of neurons within a
GAL4 driver (Lee and Luo, 2001, Wu and Luo, 2006), after which MARCM flies can be
tested for behavior. Presumably, neurons of interest will be labeled in flies that exhibit
the phenotype whereas unimportant neurons will not, thereby allowing researches to
correlate labeled neurons with a given behavior. As such, this technique can be
employed to determine which OA neurons are required for inhibiting egg-laying in
Figure 13: Mosaic analysis to determine which OA neurons regulate egg-laying
Schematic of OA cell clusters in Drosophila. ASM: anterior superior medial
cluster, VL: ventral lateral cluster, AL2: antenna lobe cluster, VM: ventral medial
cluster. VM cluster can be subdivided into 3 subclusters: md (mandibular), mx
(maxillary), lb (labial).
OA cell clusters outlined in a fluorescent micrograph (tdc2>GFP). αTH antibody
(red) is used to help distinguish different VM subclusters.
Magnified image of VM OA cluster. Subclusters md, mx, and lb are outlined in
(D-E) Z stacks of individual VM subclusters from (C). αTH is used to distinguish the
VM mx and VM lb subclusters. Typically, 2-3 large TH SOG soma can be seen in
between mx and lb subclusters (arrows in E)
Schematic of MARCM technique. MARCM randomly labels OA neurons in
different flies. After testing egg-laying, flies can be separated based on
behavioral phenotype (i.e. lays eggs vs. does not lay eggs) and neuron expression
pattern can be examined. Expression pattern that correlates with lack of eggwould suggest these neurons are important for egg-laying output.
Unlike their temperature and genotype controls, flies with activated OA neurons
lay at most 2 eggs, with the majority of the flies laying 0 eggs (Fig 14A). Therefore, we
used 2 eggs as the threshold for MARCM flies to be considered for expression analysis.
To create mosaic flies, we heat shocked the flies under two separate conditions, 15 and
30 minutes (see Materials and Methods for detailed explanation of MARCM
methodology) and tested them for egg-laying rate. The majority of flies that were not
heat shocked lay many eggs and do not show any labeling of OA neurons (Fig 14B, E).
However, mosaic flies that were heat shocked had some flies lay 0-2 eggs like tdc2>dtrpA
flies. We compared the expression pattern between flies that laid 0-2 eggs and flies that
laid many eggs (40+ eggs laid) and found that ASM, VM md, and VM mx clusters to be
enhanced in flies that laid 0-2 eggs (Fig 14C-D, F).
Figure 14: ASM and VM mx are candidate OA neurons that regulate egg-laying
Distribution of the egg-laying rates of tdc2>dtrpA flies. Note that tdc2>dtrpA flies
only lay between 0-2 eggs, therefore our cutoff for MARCM analysis was 0-2
eggs to determine which neurons were important for egg-laying.
Distribution of the egg-laying rates of MARCM flies. Flies that are not heat
shocked (no OA neurons expressing dtrpA) lay eggs. Flies that are heat shocked
have a proportion of flies that exhibit the no egg-laying phenotype, and are
subjected to expression analysis.
(C-D) Expression pattern of 2 flies that laid 0-2 eggs. (C) Fly with one labeled ASM
neuron. (D) Fly with one labeled VM mx neuron.
Fly with no heat shock shows no labeled neurons and lays 40+ eggs.
Frequency of labeled cells in flies that lay 40+ eggs (light yellow bars) and flies
that lay 0-2 eggs (yellow bars). * p < .05, *** p < .0001, Fisher’s exact test.
Regression analysis of MARCM data. Top table is the regression results of
MARCM data, whereas bottom table is the regression results of MARCM data
after data is changed into binary form (i.e. data is converted to whether or not a
cell cluster is mark (yes/no), this conversion does not take into account if
multiple cells are marked within one cell cluster). ASM and VM mx cell clusters
are marked with yellow as they both have a high correlation value to no egglaying, particularly in the binary regression analysis.
We then performed regression analysis to see if we could further refine our data
to give us the most likely correlates for egg-laying control. Our first model was a best-fit
model of the data as is, where if one fly had 3 cells expressed in AL2, 2 in VM mx, and 1
in VM lb, then it was entered that way into the data set. Our second model converted all
the data into binary fashion where we inputted the data set simply as whether or not the
cell cluster was labeled in the fly. Both models suggest ASM to have high correlation
with the no egg-laying phenotype (Fig 14G). The binary model also suggested VM mx
to be correlated with no egg-laying (Fig 14G). Combined, we think that the most likely
OA neural candidates for egg-laying control lie somewhere within the ASM and/or VM
mx cluster. Further experiments will have to be done to support this hypothesis.
4. Discussion and Future Directions
Egg-laying is an important behavior for fruit flies. Appropriately, their brain has
been hardwired to search and find substrates that are best for oviposition. While the
substrate preferences of Drosophila have been extensively studied, the neural
mechanisms that contribute to egg-laying preference are still relatively unknown. Here,
we expanded our knowledge of Drosophila oviposition behavior and identified several
sets of neurons that are important for determining the attractiveness of egg-laying
First, we discovered and characterized how Drosophila respond to UV for egglaying. We showed that not only do flies prefer to lay eggs away from UV, but they also
tend to move away from UV, suggesting UV switches from being attractive to aversive
in the behavioral context of egg-laying. Both egg-laying and movement aversion are
mediated primarily by R7 photoreceptors. Interestingly, the R7 synaptic target Dm8
neuron is not required for egg-laying but is required for movement aversion, suggesting
egg-laying and turns away from UV use different circuits in the Drosophila visual
We also identified a set of neuromodulatory neurons that influence how flies
assess sucrose during oviposition. Inhibiting octopaminergic neurons leads to less egglaying away from sucrose, suggesting these neurons are important for sucrose
oviposition preference. Moreover, activating OA neurons leads to a complete shutdown
of egg-laying. Further work will need to be done to refine our understanding of how
these neurons function within the circuits that control egg-laying.
4.1 Switching the behavioral valence of UV
The switch from UV attraction to UV aversion during egg-laying is likely to
provide evolutionary advantages for Drosophila. This behavior may be an attempt to
minimize predation or dehydration of eggs prior to larval hatching. Alternatively, egglaying away from light may parallel the innate avoidance behavior of larvae towards
light (Sawin-McCormick et al., 1995, Xiang et al., 2010, Kane et al., 2013). It is also
conceivable that egg-laying preferences reflect optimal survival conditions of the egglaying female in order for them to lay more eggs during their lifespan. Deposition of an
egg only requires a few seconds, but each egg-laying event is often followed by a minute
of rest (Fig 6E; Yang et al., 2008), which could expose females to danger. However, it has
not been tested whether flies’ escape response is dampened during this time period.
From a circuitry perspective, how does the fly “know” it should switch to begin
avoiding UV for egg-laying? One possibility is that this behavioral switch occurs when
the female fly has mature eggs ready to be laid out its ovipositor. Recently, Gou and
colleagues identified the neural substrate that signifies when egg-laying demand arises
in Drosophila. They performed behavioral and circuit analysis to expand our
understanding of how flies respond to acetic acid during egg-laying. Similar to the
aversive turns away from UV, they show that flies prefer to turn towards acetic acid
during egg-laying (Gou et al., 2014). Moreover, they identify a set of neurons that
express the mechanosensory DEG/ENaC channel, pickpocket (ppk), that innervate the
reproductive tract of female flies (Gou et al., 2014). These neurons are thought to be able
to sense the “stretch” of the oviduct when an egg passes through, thereby informing the
fly that an egg is ready to be laid. If these oviduct-innervating ppk neurons are the first
neural substrate for egg-laying demand, then they should also be required for UV
aversion during egg-laying. That is, the switch from UV attraction to UV aversion
occurs after ppk-tract neurons sense the stretch of the oviduct and tell the fly that it
should begin searching for a site to deposit her eggs.
The second order neurons that relay of “readiness” of an egg to be laid remain
unknown. Because egg-laying is intimately tied to mating status in females, it is
possible that egg-laying circuitry utilizes the same neurons that help determine the
receptivity of females. One candidate includes a set of neurons that co-express the sex
determining gene, doublesex (dsx), the transcription factor important for developing
courtship circuitry fruitless (fru), and ppk. These VNC neurons have been shown to be
important for post-mating behavior and project upward into the brain (Rezaval et al.,
2012), making them possible candidates for relaying egg-laying signals from the
reproductive tract up to higher-order circuits critical for selection of egg-laying sites.
4.2 Delineating the visual circuit during UV aversion
Since inhibiting R7 function does not completely eliminate egg-laying aversion,
there is likely to be additional PRs that contribute this behavior. Although R8 PRs are
primarily sensitive to blue or green light, they can still detect UV wavelengths, albeit at a
much weaker level (Yamaguchi et al., 2010). Indeed, rescue of R8 function in norpA36
mutants leads to mild egg-laying UV aversion (Fig 10D). Alternatively, egg-laying
aversion of UV may be an extremely robust behavior, such that even minimal signaling
from R7 is sufficient to induce oviposition away from UV.
Given that UV aversion is mediated through histamine, it is somewhat puzzling
that inhibiting all ort-expressing neurons fails to reduce egg-laying away from UV. This
suggests neurons expressing the HisCl1 receptor also contribute to egg-laying aversion
of UV. However, HisCl1 is primarily expressed in glial cells in the optic lobe (Gao et al.,
2008), and HisCl1134,ort1 double null mutants still exhibit egg-laying UV aversion. Failure
to eliminate UV aversion with HisCl1134,ort1 mutants suggests there is a third,
unidentified histamine receptor in Drosophila. Other scientists studying vision in other
behavior paradigms have also hypothesized that there may be additional histaminergic
receptors in Drosophila (personal communication with Dr. Chi Hon Lee, 2014). This
putative histamine receptor would be expressed in other second order neurons outside
of ort and HisCl1 neurons in the optic lobe. Recent reports have identified additional
targets of R7s, including Mi9, Dm2, Rh3TmY, and Rh4TmY (Takemura et al., 2013,
Jagadish et al., 2014), each of which could contribute to egg-laying aversion of UV.
Unlike egg-laying aversion, movement aversion of UV is mediated primarily through
Dm8 neurons downstream of R7 PRs. Because Dm8 also promote UV spectral
preference (Gao et al., 2008, Karrupudurai et al., 2014), these neurons may promote
orientation or movement behavior towards or away from UV depending on behavioral
Lastly, movement attraction (returns towards UV) during no egg-laying periods
was not impaired during any of our experiments. This result may simply be a reflection
of fruit flies’ robust innate attraction towards light, such that inhibiting any single set of
photoreceptors is not sufficient to reduce their ability to move back towards UV during
periods of no egg-laying. Both R1-6 and R7 PRs have been reported to contribute to UV
attraction in other paradigms (Gao et al., 2008, Yamaguchi et al., 2010, Fischbach, 1970),
which is mirrored in our own experiments, where R7-only flies exhibit movement
attraction and R1-6-only flies spend more time on UV.
4.3 OA and egg-laying control
Flies need to be able to evaluate its entire environment in order to determine
which areas are optimized for egg-laying. Where this occurs in the central brain remains
enigmatic. While octopamine neurons modulate both egg-laying preference and rate,
they are not the neurons that determine which sites are most preferable during
oviposition. Given that activating OA neurons led to a complete shutdown of egg54
laying, perhaps the OA neurons are one of the pathways that can be recruited to prevent
egg-laying when flies are on less desirable substrates. For example, in sucrose vs. plain
egg-laying conditions, when flies are sampling the sucrose substrate, OA neurons are
activated to help inhibit egg-laying on sucrose. This would explain why inhibition of
OA neurons led to flies laying fewer eggs away from sucrose, as the normal OA neural
signal is eliminated and so the system no longer has that inhibition signal coming from
the OA circuit.
Whether this system is recruited to inhibit egg-laying in other contexts remain to
be seen. Preliminary experiments suggest inhibiting OA neurons does not reduce egglaying away from UV (data not shown). The OA system may simply be a unique system
for sucrose evaluation during egg-laying. OA neurons have previously been identified
to be a critical component of sucrose reward during associative olfactory learning
(Waddell 2013, Liu et al., 2012, Burke et al., 2012). It is possible that OA is a modulatory
system that is tied specifically to sucrose and/or gustatory stimuli for the rest of the
4.4 Concluding Remarks
Determining how the fly decides where to lay its eggs is a difficult and
challenging problem. Although Drosophila melanogaster is a powerful genetic model
organism, its greatest research tool is also its greatest flaw. Without identifying a GAL4
driver that labels neurons that are important for egg-laying preference, scientists are
severely limited, as there are little, if any, viable alternatives to interrogate the function
of specific neurons to determine their function in behavior. Cognizant of this limitation,
Jenett et al. created an expansive library of 7000 transgenic GAL4 lines from unique
fragments of the Drosophila genome (Jenett et al., 2012). This GAL4 library could be used
to examine the potential role of otherwise previously unidentified neurons during egglaying site selection. Since there are a finite number of neurons in the fly nervous
system, it is only a matter of time before we determine the identity and function of the
neurons that are important for this simple decision making behavior.
5. Materials and Methods
5.1 Fly stocks
CSWU , w1118, GMR-hid (Grether et al. 1995), norpA36 (BL 9048), hdcJK910 (Bloomquist
et al., 1988), GMR-GAL4 (BL 8605), UAS-norpA (BL 26267), sev14 (BL 5691), R7-GAL4 (BL
8604), UAS-TNT (BL 28838), ninaE17 (BL 5701), Rh1-GAL4 (BL 8691), ort1 (Gao et al., 2008),
ort5 (BL 29637), HisCl1134,ort1(Gao et al., 2008), ortC1-4-GAL4 (Gao et al., 2008), vGlutdVP16AD;ortC2-GAL4DBD (Gao et al., 2008), norpA36;Rh5-norpA (Gift from Dr. Chi-Hon
Lee), UAS-Kir2.1 (Sweeney et al., 1995), tdc2-GAL4 (Cole et al., 2005), tsh-GAL80 (Clyne
and Miesenbock 2008), UAS-dtrpA (Hamada et al., 2008), hs-flp, tub>GAL80>, UASmCD8::GFP
5.2 Egg-laying preference assay
5.2.1 Egg-laying chambers and LED holders
Egg-laying chambers and LED holders were custom made in conjunction with
the Duke Physics Shop. Chambers are made of acrylic and measure 20.75 cm x 7.5 cm x
3 cm in size. The dimensions of one egg-laying arena are 1.6 cm x 1 cm x 1.75 cm
(height). Each chamber houses 30 egg-laying arenas that can assay up to 30 flies
simultaneously. LED holders are designed to position an LED above an egg-laying
substrate. The designs of the chamber and LED/LED holder are available upon request.
5.2.2 Behavior setup and analysis
Newly eclosed adult females were collected into groups of 20-30 flies and housed
with 15-20 males in yeasted vials. After 4-5 days, females were loaded into egg-laying
chambers and allowed to lay eggs for 14-16 hours overnight. The following morning,
egg-laying chambers were disassembled and eggs on both egg-laying substrates were
quantified. In earlier sets of experiments, eggs were directly counted under a dissecting
microscope. In later experiments, we took pictures of the egg-laying results and counted
the eggs on a computer monitor. Egg-laying preference indices were calculated as
follows: (NUV – NDark)/ (NUV + NDark), where NUV and NDark represent the numbers of eggs
on the UV site and dark site, respectively.
5.3 UV light setup
5.3.1 Light source
For UV experiments, we used 5 mm UV LEDs (380nm, radiant power 30mW,
2θ½ = 15°; part number: RL5-UV0315-380, superbrightleds.com). For other wavelengths,
the part numbers are as follows: blue (RL5-B5515), green (RL5-G8020), red (RL5-R5015),
and white (RL5-W6030) (superbrightled.com). The infrared LEDs are from jameco.com
(part no. 106526).
5.3.2 LED driver and intensity control
The LEDs are powered by a 20 mA LED driver using ON Semiconductor’s
CAT4104 chip. LED intensity is controlled by pulse-width modulation using a
microcontroller (Arduino Uno, http://arduino.cc). The UV intensity was set at 5%,
illuminating the egg-laying site (area 10x5 mm2) with about 0.1 mW of UV, yielding a
UV intensity of about 2 µW/mm2 on the UV site. (LED power was measured using a
Thorlabs PM100USB power meter). In comparison, the power of UV between 300 and
400 nm in sunlight at the equator when illuminating an area the size of one egg-laying
site is about 1.25mW (25 µW/mm2), thus ~ 12.5 times stronger. 1.25mW from sunlight
was obtained by multiplying the sun’s irradiance between 300 and 400 nm – estimated
from the spectra chart (Reference Solar Spectral Irradiance: Air Mass 1.5) as .5 X
.5W/m2/nm X 100 nm = 25 W/m2 – by the area of one egg-laying site. We measured the
UV intensity for the middle of the chamber and the dark site to be 0.24 µW/mm2 and
0.03 µW/mm2, respectively.
5.4 Behavioral analysis
5.4.1 Video recording setup
4 Microsoft LifeCam Cinema cameras are attached to the top of chambers. Each
camera is positioned above 2 egg-laying arenas to record 2 flies. Because the cameras
are placed on top of the egg-laying chambers, the UV LEDs are moved and placed
underneath the egg-laying chambers, thus illuminating egg-laying sites from the
bottom, rather than the top. This change in LED position does not affect egg-laying
preferences. Videos are recorded using CamUniversal and converted using Avidemux.
Note that to better see eggs in the videos, we record at a higher resolution than what is
required for tracking; the conversion reduces resolution but significantly speeds up
5.4.2 Tracking position of flies
Fly position is tracked used a version of Ctrax that we extended to allow tracking
in the difficult lighting conditions. In particular, the LEDs often cast shadows of flies on
the sidewalls, and we could not tune Ctrax, or our lighting, to avoid the mistake of
tracking these shadows as flies. Therefore, we added a heuristic to Ctrax to
automatically eliminate shadows (Stern and Yang, 2014). Our Ctrax extensions are
available as project yanglab-ctrax on Google Code (https://code.google.com/p/yanglabctrax). After tracking, we use a MATLAB script (also included in the yanglab-ctrax
project in https://code.google.com/p/yanglab-ctrax) to analyze the positions of the flies in
order to produce parameters such as time spent or trajectory reversals (UV and dark
returns) exhibited by the flies.
5.4.3 Positional PI analysis
To compare periods of no and high egg-laying for behavioral analysis, the times
of egg-laying events were manually annotated by viewing the videos. We used two
features to identify egg-laying events. First, females exhibit a unique and stereotypical
ovipositor motor program when they are laying eggs. Second we also looked for the
appearance of an egg on the video screen. 8-hour videos were then divided into hourlong periods, with each period being labeled with the number of eggs laid within that
period. No egg-laying periods were defined as hours with 0 eggs laid within that period
while high egg-laying periods were defined as hours with 7 or more eggs laid within
that period, which represents roughly 66.7% of total egg-laying events (ELEs) annotated.
Positional PI was calculated as (TUV – TDark)/(TUV + TDark), where TUV and TDark represent
times spent on each egg-laying site.
5.4.4 Return PI analysis
To calculate attraction or aversion of UV, we defined “returns” as trajectories
where flies would leave one site and return back to the given site without visiting the
opposite site. Therefore, UV returns are events where flies would leave the UV site and
return back to the UV site without visiting the dark site. We propose such UV returns
indicate UV attraction. Similarly, dark returns are events where flies would leave the
dark site and return back to the dark site without visiting the UV site. We propose such
dark returns indicate UV aversion. Return PI was calculated as (RUV – RDark)/(RUV + RDark),
where RUV and RDark represent the numbers of returns to UV and dark sites in a given
5.5 Mosaic Analysis
MARCM flies were created by crossing two stocks. The genotype of the first
stock is tdc-GAL4,tsh-GAL80/cyo;tub>GAL80>/TM6B. The genotype of the second stock is
UAS-dtrpA,UAS-mCD8::GFP/cyo;mKRS,hs-flp/TM2. These two stocks were crossed to
create the genotype: tdc2,GAL4,tsh-GAL80/UAS-mCD8::GFP,UAS61
dtrpA;tub>GAL80>/mKRS,hs-flp. Under normal conditions, GAL80 is expressed
throughout the animal under the tubulin promoter. GAL80 is a repressor of GAL4,
thereby inhibiting expression of dtrpA and GFP in tdc2-GAL4 cells. However, flies were
heat shocked for 15 or 30 minutes, which causes flippase to be stochastically expressed
under the heat shock promoter (hs-flp). Flippase is a recombinase that recognizes FRT
DNA sequences to induce recombination and is analogous to the Cre/Lox recombinase
system. The transgene, tub>GAL80> has FRT sites flanking GAL80, so once flp is
expressed in cells, it can excise GAL80 and now GAL4 is expressed in these cells, leading
to expression of GFP (for expression analysis) and dtrpA (for behavior testing).
After mosaic flies were tested for egg-laying rate, flies that laid 0-2 eggs or 40+
eggs were saved for expression analysis. Flies were dissected, stained with αTH, and
brains were imaged using a Zeiss LSM 700 confocal microscope. Neurons that
expressed GFP were quantified and categorized according to cell cluster, which was
then used for statistical analysis.
Reference Solar Spectral Irradiance: Air Mass 1.5. (National Renewable Energy
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Edward Zhu was born on February 24, 1987 in Portland, Oregon, but spent much
of his childhood in Lafayette, Colorado before moving to San Jose, California in 1996. In
2009, Edward obtained his Bachelor of Arts in Molecular and Cellular Biology with an
emphasis in Neurobiology from the University of California, Berkeley. While at
Berkeley, he played the clarinet in the University of California Marching Band (Cal Band
Great!) and also completed minor studies in Music. His research in the biomedical
sciences began in the lab of Dr. Lance Kriegsfeld at UC Berkeley. Under the guidance of
Dr. Sheng Zhao, Edward studied the role of RF-amide peptide and its relationship with
regulation of the spermatogenesis cycle in Syrian hamsters. In 2010, this work was
published in the article, “RFamide-related peptide and messenger ribonucleic acid
expression in mammalian testis: association with the spermatogenic cycle” in
Endocrinology. In August of 2010, Edward moved to obtain his doctoral degree in
Pharmacology at Duke University in the department of Pharmacology and Cancer
Biology. There he joined the lab of Dr. Chung-Hui (Rebecca) Yang where he studied the
neural and behavioral mechanisms of egg-laying site selection in the fruit fly, Drosophila
melanogaster. His work centered around characterizing neurons in the fly visual system
that are important for mediated egg-laying and movement away from UV light, which
was published in the report, “Egg-laying demand induces aversion of UV light in
Drosophila females” in Current Biology in 2014.