UNIVERSITY OF CALIFORNIA SAN DIEGO

Suppression of Escape Behavior during Mating in Female Drosophila Melanogaster

A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science

in

Biology

by

Yan Huang

Committee in charge:

Professor Ralph Greenspan, Chair

Professor Chih-ying Su

Professor Jing Wang

2019

©

Yan Huang, 2019

All rights reserved.

iii

The thesis of Yan Huang is approved, and it is acceptable in quality and form for publication on

microfilm and electronically:

_____________________________________________________________

_____________________________________________________________

_____________________________________________________________

Chair

University of California San Diego

2019

iv

TABLE OF CONTENTS

Signature Page............................................................................................................................... iii

Table of Contents........................................................................................................................... iv

List of Figures................................................................................................................................. v

Acknowledgements........................................................................................................................ vi

Abstract of the Thesis .................................................................................................................. vii

Introduction..................................................................................................................................... 1

Results and Discussions.................................................................................................................. 3

Methods and Materials.................................................................................................................... 9

References......................................................................................................................................11

v

LIST OF FIGURES

Figure 1. Drosophila visually driven escape neuronal pathway..................................................... 2

Figure 2. Wild type fly response towards looming stimulus.......................................................... 5

Figure 3. Optogenetic driven escapes in female flies..................................................................... 7

Figure 4. Escape responses of mating female flies were suppressed compared to single female

flies………………………………………………………………………………………………...8

vi

ACKNOWLEDGEMENTS

I would like to acknowledge Professor Ralph Greenspan for giving me the opportunity to

do research in his lab and supporting me as the Chair committees for my thesis.

I would like to acknowledge my supervisor Ruichen Sun, who guided me and taught me

to be a critical researcher with patience and optimism.

I would like to acknowledge my other supervisor Takeo Katsuki, who introduced me to

this fly project, and continued to provide advises even after he left the lab.

I would like to acknowledge Frank Cardone for providing technical support for our

experiment setups.

I would also like to acknowledge undergraduate students that helped me out with

collection and experiments. Without them, I wouldn’t be able to collect as much data as I have

for this dissertation.

This thesis contains material that is coauthored with Yan Huang, Ruichen Sun, Takeo

Katsuki, Dennis Tran, Yuqing Wang, Yuxuan Wang, Ralph Greenspan. The thesis auther was

the primary author of this material.

vii

ABSTRACT OF THE THESIS

Suppression of Escape Behavior during Mating in Female Drosophila Melanogaster

by

Yan Huang

Master of Science in Biology

University of California San Diego, 2019

Professor Ralph Greenspan, Chair

Animals face higher predation risk during mating due to temporary conspicuous

appearance, physical restriction and lowering of vigilance. Studies were done on animals to

investigate mating-related risks and escape strategies. However, few has dug into the internal

state of mating animals, and how mating lowers the awareness of danger. In this study, we used

Drosophila melanogaster as a model to study the mechanism behind the effect of mating on

viii

escape behaviors. We created an environment that allows flies to exhibit both escape and mating

behavior, and established a reference showing escapes of wildtype female flies during mating are

suppressed. In an attempt to understand the suppression location in the brain of mating on escape

behaviors, we optogenetically activated transgenic flies during mating. Our results suggest that

activating lobular columnar cells LC6, or giant fiber neurons (GF) do not affect the suppressed

escape behavior during mating, indicating that short takeoff escapes are suppressed at or

downstream of GF neurons, and for long takeoff escapes, the suppression site is at or

downstream of LC6.

1

Introduction

Mating behaviors of Drosophila have been extensively studied in as an important social

behavior. When a male fly senses pheromone from a female fly, it approaches the female fly by

taping her, extending wing to produce a courtship song, licking the genital area of the female fly,

and attempting to hop on top of the female flies. Countering the approach of male flies, female

flies can choose to accept or reject the proposal (Spieth 1974; Bastock et al., 1955; Dickson,

2008; Ferveur, 2010).

The Giant fiber neurons (GF) and the optic lobes are essential for visually driven escapes

of flies. There are two kinds of escapes in flies, short takeoff and long takeoff escapes. The GF is

responsible for short takeoff escapes. It is an interneuron responsible for transmitting visual

stimuli to motor neurons that innervate tergotrochanteral jump muscles (TTMs) and dorsal

longitudinal flight muscles (DLMs), which are major forces for escape movement (Tanouye and

Wyman, 1980; Von Reyn and Card 2012; Von Reyn et al., 2014). The cell bodies of the GF are

located in the central brain, and they terminate in the ventral nerve cord (Fig. 1AB). Lobular

columnar (LC) cells LC4 and LC6, and Foma-1 are neurons located in the optic lobe that detect

visual signals (Otsuna and Ito, 2006; Katsov and Clandinin, 2008) (Fig. 1B). LC4 provides

retinal angular velocity cues to the GF to induce short take off escapes (Von Reyn and Card

2012; von Reyn et al., 2017, Williamson et at., 2018; Ache et al., 2019). LC6 do not have

terminal overlap with GF (Fig. 1B), and it is involved in long takeoff escapes (Wu et al., 2016;

Keleş et al., 2017; Williamson et at., 2018). Foma-1 neurons have expression in the optic lobes

and the mushroom bodies, and activation of Foma-1 neurons also evokes escape the response (de

Vries and Cladinin, 2012, de Vries and Cladinin, 2013).

2

Figure 1. Drosophila visually driven escape neuronal pathway

A. The GF pathway. VNC, ventral nerve cord. TTMn, tergotrochanteral motor neuron. DLM,

dorsal longitudinal motor neuron (von Reyn et at., 2014)

B. GFP expression in the GF, LC4, LC6 and Foma-1 neurons. Scale bar represents 200um

LC4

A

GF

LC4

LC6

Foma-1

B

3

To address the mechanism of how mating behavior can affect escapes, we used

optogenetic techniques to activate neurons in the aforementioned escape pathways to determine

where suppression sites are located. Our results show both short and long takeoff escapes were

suppressed during mating. Short takeoff escapes are suppressed at or downstream of the GF, and

for long takeoff escapes, the suppression site is at or downstream of LC6.

Results and Discussion

A digital visual looming stimulus, which mimics a colliding stimulus, can elicit robust

escape responses in flies. ((Fotowat and Gabbiani 2007; Oliva et al. 2007; Preuss et al. 2007,

Yamamoto et al. 2003). To observe the effect of mating on the escape response, a looming

stimulus was presented either to a single fly or to a couple of flies at different stages of sexual

behavior: 20sec into wing vibration, 1min into mating, 0sec post mating, and 1minute post

mating. Flies that showed a jump or a run immediately following the stimulus were scored as

positive escapes, and flies that showed less robust response, such as backward walking, flinching

and freezing, were not scored as escapes.

Our results show that both female and male flies showed less escape response to the

looming stimulus when they were mating as compared to when they were alone in the arena (Fig.

2). The looming stimulus was able to evoke escape behavior in 65% percent of female flies and

59% of male flies. During mating, the female flies’ escape response decreased significantly to

18% (Fig. 2A). Among the 18% of the flies that responded to the stimuli, none were jumping,

and all were running. During mating, we consider all male flies as non-responsive to looming

object as they stayed on top of female flies (Fig. 2B). When male flies started courtship (i.e.

when male flies are performing courtship songs with one wing extended), their response to

4

looming stimulus decreased, while the response of female flies showed no difference during this

stage compared to the response rate of single female flies. After mating is finished, both female

and male flies showed decreased escape responses compared to single flies (Fig. 2). The

decreased escape response rate seen in males before and after mating, and in female after mating

indicates that the suppression was not due to physical restraint, but neural activity as they were

physically alone (as were the single flies).

5

Figure 2. Wild type fly response towards looming stimulus

A. The escape response of female flies was suppressed during mating, and immediately after

mating was finished (multiple comparison Benjamini & Hochberg procedure, χ2 test, *P < 0.05,

***P << 0.001, n on each bar, error bars show 95% confidence interval)

B. The escape response of male flies was suppressed after courtship (multiple comparison

Benjamini & Hochberg procedure, χ2 test, *P < 0.05, *P< 0.005, ***P << 0.001, n on each bar,

error bars show 95% confidence interval)

single wing extension mating 0sec post mating 1min post mating

Resp

onse R

ate

0.0

0.2

0.4

0.6

0.8

1.0

single wing extension mating 0sec post mating 1min post mating

Re

spon

se R

ate

0.0

0.2

0.4

0.6

0.8

1.0

***

***

**

***

*

female

male

282 21 92 55 52

21 61

1

92 55 52

B

x

* A

x

6

To determine the suppression site in the escape pathway, we expressed csChrimson, a

red light activated Ca+ channel, targeted to either the GF, LC4, LC6 or Foma-1 through GAL4

driver. We activated these targeted neurons using red light to elicit an escape response in both

single and mating female flies (Klapoetke et al., 2014). If mating female flies show less escapes

than single female flies when any of these neurons is activated, the suppression site is either at or

downstream of the activated neuron. If the escape response rates are comparable in both single

and mating flies, then the suppression site is likely to be upstream of the activated neuron.

We first determined the appropriate levels of intensity to activate the neurons in flies.

Light intensities that elicits a 50% escape response rate in single female flies were chosen to test

on mating flies of the same genotype. We observed that at stimulus intensity of 4.35 mW/cm2,

51% of GF activated female flies showed escape response. 57% of LC4 activated flies showed

response at intensities of 2.12mW/cm2 and 56% of LC6 activated flies showed escape response

at 1.20mW/cm2. Notably, less than 30% of flies responded to the activation of Foma-1 even at

the highest intensity (7.69mW) of the power supply (Fig 3A). As successful activation of the

neurons in these flies requires the flies to be fed on food supplied with all-trans retinal (ATR)

dissolved in EtOH prior to the experiments, we also tested flies of the same genotype that were

fed with on EtOH-supplemented food (with no ATR) as our control group. Under the highest

light intensity, ATR-fed flies, except Foma1-GAL4>UAS-CsChrimson, showed significant

higher response rates than EtOH-fed flies. Supplemented with ATR, GF activated flies showed an

89% response rate, both LC4 and LC6 activated flies showed 100% response, and only 23%

Foma-1 activated flies showed response. For EtOH-fed flies, 15% of GF activated flies and LC4

activated flies responded to the stimulus, 16% of LC6 activated flies showed response. And

7

Foma-1 activated flies showed a response rate of 9% (Fig. 3B). This indicates that the jump

response seen during the experiment are due to the light activation of the targeted neurons.

Figure 3. Optogenetic driven escapes in female flies

A. Response increased as the intensity of stimulus increased (error bars show 95% confidence

interval)

B. The escape responses of GAL4-UAS flies were significantly decreased when not fed with all-

trans retinal. The intensity of the stimulus was 7.69mW/cm^2(χ2 test, ***P << 0.001, n on each

bar, error bars show 95% confidence interval)

2 4 6 8

0.0

0.2

0.4

0.6

0.8

1.0

Stimulus Intensity (mW/cm^2)

Re

sp

osn

e R

ate LC6>UAS-csChrimson

GF-split-GAL4>UAS-csChrimson

Foma1-GAL4>UAS-csChrimson

LC4>UAS-csChrimson

(1.2, 0.56)

(4.35, 0.51)

(2.12, 0.57)

GF LC4 LC6 Foma-1

Resp

on

se R

ate

0.0

0.2

0.4

0.6

0.8

1.0

ATREtOH

A

n.s.

B

19 20

10

14 11

17

17

53

11

*** *** ***

8

Figure 4. Escape responses of mating female flies were suppressed compared to single female

flies (χ2 test, *P < 0.05, *P< 0.005, n on each bar, error bars show 95% confidence interval)

We then applied the 50% escape-evoking stimuli to compare escape responses between

single and mating female flies. We observed that under activation of GF, LC6 and LC4, mating

female flies showed a reduced escape response compared to single female flies (Fig. 4). GF

single mating

Re

sp

on

se R

ate

0.0

0.2

0.4

0.6

0.8

1.0

GF split GAL4>UAS-csChrimson

*

single mating

Re

sp

on

se R

ate

0.0

0.2

0.4

0.6

0.8

1.0

LC4>UAS-csChrimson

*

single mating

Re

sp

on

se R

ate

0.0

0.2

0.4

0.6

0.8

1.0

*

LC6>UAS-csChrimson

74 75 72 73

84 74

**

9

activated mating female flies showed a 22% decrease in response rate compared to single female

flies. LC4 activated mating flies also showed a 25% decrease in response rate. Since LC4

synapses onto the GF, the suppression site is also located downstream of LC4, and it is

reasonable to see fewer mating flies showing escapes when LC4 was activated. The result of GF

and LC4 suggests that short takeoff escape is suppressed during mating, and the suppression site

can be either at or downstream of the GF. LC6 is involved in the long takeoff escapes, and the

lower escape rate of LC6 activated mating flies suggests that long takeoff escape is suppressed at

or downstream of LC6.

In conclusion, both long and short takeoff escapes are suppressed by mating. Short

takeoff escapes are suppressed at or downstream of the GF, and for long takeoff escapes,

suppression happens at or downstream of LC6. It is possible that the suppression is global such

that multiple sites of the escape neuron pathways are suppressed. It is also possible that a specific

point is suppressed. Future work, such as examining TTMn, a downstream neuron of GF, and

testing neuron modulators, can add more details to the mechanism of the suppression.

Methods and Materials

Fly strains

We obtained GF-split-GAL4, LC6-GAL4 and LC4-GAL4 from Janelia Farm Research

Campus, Foma-1-GAL4 as a gift from Thomas Clandinin at Stanford, UAS-CsChrimson and

UAS-myr-EGFP from Bloomington.

10

Looming stimulus experiment:

Looming stimulus setup was developed by Diana Lam, a former lab technician in the

Greenspan lab. A custom-made stage was machined out of acrylic with a main stage with a

diameter of 30mm surrounded by a 20mm wide moat that is 2mm deep. The Digital Looming

Object is displayed on a BeagleTouch monitor connected to a Pandaboard computer running

Android ICS. Both the monitor and computer are from Liquidware Engineering. Using Thorlabs

parts, BeagleTouch monitor is held at a 47⁰ angle from the horizontal, centered to the stage, with

the base of the monitor only 40mm away from the closest edge of the stage. The Digital

Looming Object code was written in Java for Android Developers. The code draws a 100mm2

black square at a center of the display to expand to a 3025mm2 square within 300ms, modeling a

realistic looming object. Experiments were conducted in the dark and illuminated with an 850nm

IR LED backlight. An IR sensitive camera was installed on top of the stage to record fly

behaviors.

Female and male virgin flies were selected under CO2 anesthesia, and each fly was

isolated in a 10x50mm polystyrene test tube loaded 200ul corneal agar food. Flies were placed in

a 25°C incubator in 12h dark: 12h dark cycle, and 50% humidity for 3-8 days before

experiments. During experiments, flies were transferred to the experiment stage by an aspirator.

Stimuli were presented to single flies 1min after they were transferred to the stage. For flies

underwent mating process, stimuli were presented to flies 20 minutes after first wing extension

of male flies, 1min after mating started, right after mating finished, and 1min after mating

finished.

11

Optogenetic experiments:

The stage, IR LED backlight and the camera were identical as the looming object

experiment setup. Experiments were also conducted in darkness. A 627nm red LEDs panel was

placed 5cm underneath the experiment stage. The LEDs are tiled in the center 90 x 90 mm area

at 5 mm intervals. The red panel was coded to flash 50ms red lights 10 times with an interval of

50ms between each flash, producing a flashing stimulus that lasted for 1sec. The panel was

connected to a power supply and was controlled by a trigger button.

CsChrimson expressing virgin female flies and wild type virgin male flies were selected

under CO2 anesthesia. Each female fly was isolated in a 10x50mm polystyrene test tube loaded

200ul corneal agar food with 3ul of 116mmol ATR solution added onto the surface. 116mmol

ATR solution was prepared by dissolving 32.9mg of ATR power in 1000ul of 95% EtOH. For

control group, each test tube was loaded 200ul corneal agar food added 3ul of 95% EtOH. Male

were put into test tubes loaded 200ul corneal agar food. All flies were placed in a 25°C incubator

in 12h dark: 12h dark cycle, and 50% humidity for 3-8 days before experiments. During

experiments, flies were transferred to the experiment stage by an aspirator. Stimuli were

presented to single female flies 1min after they were transferred to the stage. For mating

experiments, csChrimson expressing female flies and wild type male flies were transferred to the

experiment stage, and stimuli were presented to the couples 1min after mating started.

This thesis contains material that is coauthored with Yan Huang, Ruichen Sun, Takeo

Katsuki, Dennis Tran, Yuqing Wang, Yuxuan Wang, Ralph Greenspan. The thesis auther was

the primary author of this material.

12

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