The Effects of an Endocrine Disruptor on Courtship Behaviors in

Drosophila Melanogaster

John Henry Cruz

, Ya’seen Ellison

, Michael Pursley

, Nafiul Huda

, Faith Boyer

and Rita M. Graze

Computational Biology REU 2019

University of Texas at Austin, Austin, TX.

Morehouse College, Atlanta, GA.

Department of Biological Sciences, Auburn University, Alabama.

Abstract

Experimental Method

Introduction

Acknowledgements: We would like to thank Dr. Kimberly Mulligan-Guy , Dr. Cordelia Brown, and all involved in

the Collaboration Among Scientists and Engineers (CASE) REU and Dr. Goertzen from the Auburn Diversifying

Undergraduate Participation in Computational Biology REU, as well as the Graze Lab for hosting us and assisting

us this summer. This work was additionally supported by the Department of Biological Sciences, and the

National Science Foundation (NSF) CAREER award (NSF DEB 1751296) and the Auburn Diversifying

Undergraduate Participation in Computational Biology REU program (NSF DBI 1560115).

Drosophila are exposed to an array of environmental factors which can influence their behavior. Common

examples are pesticide exposures or low resource availability leading to metabolic stress. This study focuses

on factors that disrupt endocrine signaling, namely, Endocrine disruptors (ED’s) . ED’s can have desirable

effects, e.g. when used in food production as insecticides, or undesirable effects, e.g when insecticides affect

non-target organisms. If endocrine disruptors fail to affect target insects, they may negatively impact crops. If

they affect non-target organisms, ecosystems may be disrupted. Endocrine disruptors can also affect humans

via water contamination and their widespread presence in plastics, as well as in cosmetic products.

Therefore, understanding environmental and genetic variation in the affects of ED’s on endocrine function

has widespread importance in the natural environment, in agriculture, and for human health. In our

experiment the PI 3-kinase inhibitor ‘Wortmannin’ is used to understand the effects of pharmacological

perturbation of insulin signaling on fruit fly behavior. Our focal question pertains to whether or not an

exposure to this endocrine disruptor has any effect on courtship latency and duration in Drosophila. i.e. the

amount of time it takes to court, and the length of time courting transpires, respectively. We found that

there is no significant effect of Wortmannin on courtship behavior.

Results

Objectives

• Understand the potential implications of endocrine disruptor exposure on adult

fruit fly behavior.

• Troubleshoot behavioral assay and machine learning software for use in future

experiments.

• Test whether or not endocrine disruption will cause flies treated with

Wortmannin to be less/ more likely to engage in courtship activity.

Feeding Assay

• BARCODE Feeding Assay

was used to determine

how much treatment was consumed, later used

to compare amount eaten in each treatment.

BORIS

• BORIS software was used to manually count for

chosen behaviors within specified region in

arenas.

Statistical

Analysis

• Two-sample t tests compared Chase, Circling,

Total Behaviors and CI between Control and

Wortmannin treatment groups.

• Endocrine disruptors are a class of chemicals that interfere with hormone

signaling, which include BPA and Wortmannin

• Endocrine disruptors are found both naturally and synthetically, and are used

in industry, making understanding their implications on target/non-target

organisms important

• The ED Wortmannin interferes in the insulin signaling pathway, phenocopying

starvation stress in Drosophila

• Orientation of the male toward the female (“circling”) and chasing are

activities associated with Drosophila courtship

• The intensity and duration of these activities, included in courtship index and

courtship latency, can provide insights into how ED’s impact the Drosophila

reproductive behaviors

Media

Results

Acknowledgements

Day 1

Day 2

Wort.

1 hr.

4 hrs.

R = Replicate #

Wort./Cont. = Treatment Type

5 per group per sex

Fig 2: Total Behavior Counts in each chamber per

treatment.

Fig 3: Courtship Indexes of each chamber per

treatment.

Conclusions

Fig 8. The parametric two sample t test results.

Fig 9: The nonparametric two sample t test results.

Fig 4: The two-sample t test results comparing the consumption

levels of two treatments in males and females.

Fig 5: Boxplots of each treatment, comparing

the male (cyan) and female (plum)

consumption levels after standardization.

• Comparing circling, chase, total behavior, and courtship index, No significant

difference was seen between the control and wortmannin treatments.

• There was no significant difference in the amount of treatment consumed by

the subjects. Males and females both consumed yeast paste with wortmannin

in the same amount as the control yeast paste.

• Going forward, the experiment will be re-run, factoring in room for

improvement in; lighting, sample size, and arena setup/ analysis.

Wort.

Cont.

Fig 1: Phone

camera, stand

and arenas used

in Behavioral

Assay Setup.

QR Codes: Gifs displaying target

courtship behavior

References

1. Rubin BS (2011): Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. https://www.ncbi.nlm.nih.gov/pubmed/21605673

2. Anuradha A. Shastri, Vijay Hegde, Swetha Peddibhotla, Zahra Feizy, Nikhil V. Dhurandhar (2018) E4orf1: A protein for enhancing glucose uptake despite impaired proximal insulin signaling,

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208427

3. Park A, Tran T, Atkinson NS (2018) Monitoring food preference in Drosophila by oligonucleotide tagging. Proceedings of the National Academy of Sciences of the United States of America, 115(36):9020–9025.

https://doi.org/10.1073/pnas.1716880115

Fig 6: Distribution of the counts of chase

behavior(Left: Control, Right: Wortmannin). The

mean(green triangle) and median(orange line) are

shown. The whiskers show the minimum and

maximum values.

Fig 7: Distribution of the counts of circling behavior

(Left: Control, Right: Wortmannin). The

mean(green triangle) and median(orange line) are

shown. The whiskers show the minimum and

maximum values.

Cont.

Fruit Fly Mating: https://www.youtube.com/watch?v=58pQsVeADj8. Drosophila Courtship Song – Genetic –University

of Leicester: https://www.youtube.com/watch?v=KzWIuhXMUko