Effects of Age, Diet, and Sex on the Gustatory and Olfactory

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EFFECTS OF AGE, DIET, AND SEX ON THE GUSTATORY AND OLFACTORY

SENSING CAPABILITIES OF THE FORENSICALLY IMPORTANT BLOW FLY,

LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Dissertation

Submitted to

The College of Arts and Sciences of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree of

Doctor of Philosophy in Biology

Allissa Marie Blystone

UNIVERSITY OF DAYTON

Dayton, OH

May, 2015

i EFFECTS OF AGE, DIET, AND SEX ON THE GUSTATORY AND OLFACTORY

SENSING CAPABILITIES OF THE FORENSICALLY IMPORTANT BLOW FLY,

LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Name: Blystone, Allissa Marie

APPROVED BY:

______Karolyn M. Hansen, Ph.D. Faculty Advisor Assistant Professor, Department of Biology University of Dayton

______Mark Nielsen. Ph.D. Amit Singh, Ph.D. Committee Member Committee Member Department Chair Graduate Program Director Professor, Dept. of Biology Associate Professor, Dept. of Biology University of Dayton University of Dayton

______Thomas M. Williams, Ph.D. Committee Member Assistant Professor, Dept of Biology University of Dayton

______M. Eric Benbow, Ph.D. Aaron M. Tarone, Ph.D. Committee Member Committee Member Assistant Professor, Dept. of Entomology Assistant Professor, Dept. of Entomology Michigan State University Texas A&M University

Allissa Marie Blystone

2015

iii ABSTRACT

EFFECTS OF AGE, DIET, AND SEX ON THE GUSTATORY AND OLFACTORY

SENSING CAPABILITIES OF THE FORENSICALLY IMPORTANT BLOW FLY,

LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Name: Blystone, Allissa Marie University of Dayton Advisor: Karolyn M. Hansen, PhD Blow flies are members of the family Calliphoridae, specifically Lucilia sericata, are important to forensic investigations by aiding in the determination of a post-mortem interval, or the time elapsed since the expiration of a living organism. Decomposing organic material is a source of nourishment and is key to the normal development of the blow fly; without a nutritive source the eggs will often fail to hatch, and the larvae will fail to pupate. Nutrition is not only vital for proper larval development, but also for physiological maintenance in adults. It is known that a protein meal is essential for sexual maturation in female blow flies, although it is typical to find both males and females near and on decomposing material. Seeking to understand effect of sex, age, and diet on the gustatory and olfactory response attraction of blow flies, four studies were undertaken.

First, to establish common practices with regard to raising and breeding blow flies, nine

variations of the three most prevalently used diets were compared for increased fecundity

and lifespan. The goal of this study was to not only help establish a common culture

iv practice for forensic entomology, but also to aid in colony establishment for medical

entomological colonies used for maggot debridement therapy, and to have a basis of

knowledge from which to perform behavioral studies. The diet was then tested to

examine whether protein supplementation could affect the macrostructure of both the male and female adults. Six decomposition-related amino acids and two sugar sources

(in water) were tested on the gustatory response of L. sericata males and females fed either a diet of honey water only, or one supplemented with protein, utilizing the proboscis extension reflex (PER) assay to determine differences in the behavior of the

sexes associated with the stimulus. Also examined was the olfactory response of both

male and female flies fed either a diet of honey water only, or one supplemented with

protein, utilizing the electroantennogram to establish whole neural depolarizations in

response to a challenge with one of five decomposition-related volatile organic

compounds.

A number of important hypotheses and applications have been proposed from the

results of this research that will impact forensic, medical, and classical entomology.

Utilizing a common diet consisting of honey-water and a broad-spectrum protein source,

such as bovine liver, will allow for continuous and replicable culture of laboratory

colonies of Lucilia sericata in conditions similar to those found in the natural

environment. Details of the post-eclosion adult female and male time line as affected by

the consumption of protein should help inform post-mortem interval estimation.

Combining data from the diet and morphometric studies with the olfactory and gustatory

sensing findings allows insight into whether Calliphorid sensing occurs at the local level

of the sensilla or at a higher processing level in the antennal lobe of the cephalic ganglion.

This Dissertation is Dedicated To:

My best friend, college sweetheart, confidante, lover, husband, and baby-daddy. Captain Matthew S. Blystone, USAF Without your enduring love, persevering support, and unwavering belief in me, I would never and could never have both started and finished this. Thank you for everything. I love you.

My sweet “Big Boy,” Bubba, and (not-so) Bitty, whom I love dearly. Master Matthew Bennett Blystone May your love for, and curiosity in bugs (pronounced “buh-GUH”) continue to grow. And may you please, continue to leave them outside.

and

My precious Daughter, Baby Girl, Little Flower, and Sweet Miracle, Miss Violet Marie Blystone Welcome to the family! We love you tons and look forward to introducing you to the science and nature we all enjoy so much.

vi ACKNOWLEDGEMENTS

Formally, I would like to thank the University of Dayton Department of Biology for the provision of facilities, and the many undergraduate students who helped in the gathering of data for this project. As well, I have a great appreciation for the UD

Graduate School as they awarded my research three Student Summer Fellowships, and the University of Dayton Dissertation Year Fellowship which partially funded this project.

Dr. Karolyn Hansen gave me the freedom to follow my ideas and pursue my hypotheses, supporting me both financially and scientifically throughout the course of my project; for this I am extremely grateful. When I needed to learn a new technique, she sent me to the USDA facility in Miami, FL and to Notre Dame in South Bend, Indiana.

When I needed to learn more about my model organism, she sent me to Texas for a month. Whatever I needed to be successful, she supplied, including helping me believe in my scientific abilities and myself. I am also proud to say that my advisor is not only a successful scientist, making new roads in her field as a bioengineer, but also a woman in a traditionally difficult scientific community, a loving mom, and devoted wife. While these qualities may never make it to a CV, it was wonderful to have a successful example to follow, as during this time I became a mom and desired to learn how to balance work and home life. Our journey hasn’t always been without difficulty, but I am ever so

vii grateful to have traveled it with her. Thank you.

A special thanks to Dr. Aaron Tarone for hosting me at Texas A&M for the entire

month of May in 2010, during which time I gained the knowledge to begin, and the ideas

to design my doctoral research program. As a Baylor grad, it was difficult at first for me

to “like” one of our rival schools, but thanks to the tutelage of the F.L.I.E.S. lab (Dr.

Jeffery Tomberlin, Adrienne Brundage and Micah Flores, in particular) and the extensive time Dr. Tarone spent with me during my visit helping me learn about Lucilia sericata

and how to culture Calliphorids, not only was I able to design and complete my project,

but I now no longer have a strong “distaste” for their university. Sic em Bears!

I would also like to acknowledge and thank the members of my committee: Dr.

Mark Nielsen, who was always complimentary and encouraging; Dr. Tom Williams, who

asked really good, probing questions about my research and the bigger scope into which

it fit; Dr. Eric Benbow, who first introduced me to the blow fly, and helped me develop a

deep, working knowledge of statistics; and Dr. Amit Singh who never doubted my capabilities, despite me having a rather rough start in his class and at UD.

Also, a large (HUGE, MASSIVE, ENORMOUS, GARGANTUAN) debt of

gratitude is due to my encouraging, loving, poking, and prodding parents, who gave of

their time, money, and energy in support of this project. “Muggy” (Grandma Char) and

Poppa (Grandpa Jim) spent three summers, and most recently some time this Fall,

playing, romping with, and generally wearing out Bennett, so that I could get my research

and writing done in a timely manner. Knowing that my kids were in capable and loving

hands gave me the freedom to finish. And the dishes! They even did the dishes!! (I hate

doing the dishes.) I appreciate you more than you will ever know. Thank you so much.

viii PREFACE

Biosensor — BīōˌSensər

“Any biotic factor that has the inherent ability to sense a particular compound.

A device that detects, records, and transmits information regarding a physiological

change or process. A device that uses biological materials to monitor the presence of

various chemicals in a substance.”

– www.thefreedictionary.com, August, 2009

Armed with the basic knowledge that a biosensor can be described as simply as a human nose smelling brownies, or as complex as a device designed with microfluidics to detect a

single molecule of a pollutant, I started my almost five years at the University of Dayton

as a graduate student under Karolyn Hansen. My interest in pursuing biosensor research

began with one of the Biology Department seminar speakers, Dr. Jeffery Tomberlin of

Texas A&M University. He presented some of his research that involved training and

using live wasps to exhibit feeding behaviors in response to trinitrotoluene (TNT) and

dinitrotoluene (DNT) vapors. After his talk, I approached Dr. Tomberlin and asked him

why he had trained the wasps to create a mechanical biosensor when what was needed

was to isolate the molecular components that allowed the wasps to sense those vapors,

then mount those on a sensing device. He replied that such research had not yet been

done. With this reply, I had the nucleus of a research project and chose an organism with

a finely honed sensing capability – a fly that responds to the odor of dead carrion.

“When choosing an experimental animal, therefore why settle for anything so

prosaic as the laboratory rat, so giddy as the guinea pig, so phlegmatic as the frog, so

reptilian as the chicken, so cousinly as the chimpanzee? Why not choose an excitingly

different creature like the aardvark or the dugong?

Why not choose the fly?”

- Vincent G. Dethier, in “To Know a Fly”

I selected the blow fly, Lucilia sericata, for my research organism based on the distinct odor profile to which it responds: the volatiles from flesh of decaying carrion. In assembling my literature review it soon became clear that there was little to no genetic or genomic data available on this particular organism. The more I read, the less I found.

The less I found, the more questions that I formulated. The more questions I formulated, the broader my dissertation became. Thankfully, I had a wonderful graduate advisory committee that continually challenged the focus and scope of my research. After a number of revisions to my dissertation proposal, a thorough and broad literature review, and some hard conversations with professors, it was determined that a contribution to science that would most help in the future design of a biosensor device, was a broad understanding of what affected the blow fly’s sensing.

The Texas A&M’s F.L.I.E.S. laboratory graciously provided the training necessary to culture my experimental organism and I established a Lucilia sericata colony at the University of Dayton in June 2010 with stock organisms provided by Dr.

Aaron Tarone of Texas A&M. The UD culture ‘facility’ underwent three iterations over the period of my dissertation research and now exists as a modern climate-controlled walk-in room.

I had noticed while in Texas, that one fly facility fostered the growth of a number of different species, aided by two different labs, which had a number of various feeding and breeding protocols. I began to wonder if these different feeding and breeding protocols could affect the overall growth and development of both the larvae and adults.

While the main objective of my dissertation research was to better understand the sensing capabilities of Lucilia sericata, in order to someday utilize this information to build a biomimetic sensor, understanding how something as simple as the breeding and rearing practices affected the fly as a whole could better inform my future olfactory and gustatory experiments. This initial observation became one of my first experiments: subjecting flies to the common diets and measuring the most common morphometrics—wing length, width, and area; thoracic length; abdominal length, width, and area—on the adult blow flies as they aged. This seemingly simple study of adult blow fly morphometrics suggested that different diets did affect the growth of the flies and led me to wonder if the difference in diet also affected colony continuity—lifespan, ovipositioning events, egg number, etc. This question became the focus of my next set of experiments and the results strongly suggested that diet indeed affected both lifespan and fecundity of blow flies.

xi I was now able to apply the knowledge gained from these two “holistic” experiments to my study of gustatory and olfactory behavior. During my training at

Texas A&M, Dr. Hubert Amrein trained me how to perform the proboscis extension reflex assay (PER) on my flies. Dr. Paul Kendra and Mr. Wayne Montgomery, at the

USDA Sub-Tropical Horticultural Facility in Miami, Florida, painstakingly taught me the fine art of fly electroantennography (EAG). As well, Dr. Helene LeBlanc, from the

University of Ontario Institute of Technology, and Dr. Zain Syed at The University of

Notre Dame helped inform the analysis of my EAG experiments. I am grateful to each of these researchers for the contributions they made to my education and research training.

While being trained in Dr. Amrein’s lab, I made an interesting discovery—while his Drosophila melanogaster would not respond to his 100M amino acid solutions, preferring only the sugar sources, my Lucilia sericata did respond. Taking this initial discovery back to Dayton with me, I embarked on a series of experiments to determine if sex and diet could affect the gustatory behavior of the fly. Surprisingly, females responded more than males to the amino acid solutions, while those fed a protein supplemented diet responded less than those fed only honey-water. These findings supported my initial belief that the feeding and breeding practices were extremely important. So important, in fact, that something so simple could affect not only the fly as a whole, but potentially the fly physiology and behavior.

I applied the same experimental design used in the gustatory experiments to testing both the diet and sex effects on the olfactory response as measured using the electroantennogram (EAG). While the results of the EAG experiments were more varied than those from the PER, when combined with the information gathered from all of my

xii previous experiments, the conclusion was powerful, suggesting a specific timeline for the adult blow fly life cycle as regarding seeking, feeding, and breeding behaviors.

The results of my dissertation research collectively provide a strong foundation on the basic culture and gustatory and olfactory responses of Lucilia sericata and set the stage for future research on my original question – how can we utilize the unique and highly evolved sensing capability of this organism to develop a biomimetic sensor?

xiii TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………...…iv

DEDICATION…………………………………………………………………………....vi

ACKNOWLEDGEMENTS…………………………………………………………...... vii

PREFACE…………………………………………………………………………...……ix

LIST OF FIGURES……………………………………………………………………..xvi

LIST OF TABLES…………………………………………………………………….xviii

SPECIAL NOMENCLATURE………………………………………………………….xx

1. INTRODUCTION AND LITERATURE REVIEW……………………………...1

2. SIGNIFICANCE AND RESEARCH OBJECTIVES……………………………19

3. A COMPARISON OF COMMON DIETS FOR THE CONTINUOUS CULTURE OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE) FOR FORENSIC AND MEDICAL ENTOMOLOGICAL APPLICATIONS………………………………………...... 23

4. THE EFFECT OF AGE, SEX, AND DIET ON THE FITNESS AND FORENSICALLY RELEVANT MORPHOMETRICS OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE) ……………………………………………….45

5. SCANNING ELECTRON MICROGRAPHS OF THE SENSING ORGANS OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)………………………...65

xiv

6. THE EFFECT OF AGE, SEX, AND DIET ON THE GUSTATORY SENSING CAPABILITIES OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)……………………………………………………………..85

7. THE EFFECT OF AGE, SEX, AND DIET ON THE OLFACTORY SENSING CAPABILITIES OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)……………………………………………………………111

8. SUMMARY AND CONCLUSIONS…………………………………………..131

9. PROPOSED POST-DOCTORAL RESEARCH……………………………….142

REFERENCES………………………………………………………………………....154

APPENDIX 1: Supplementary Tables For Chapter 3…………………………..……....177

APPENDIX 2: Supplementary Tables For Chapter 4……………………………...…...181

APPENDIX 3: Supplementary Tables For Chapter 6………………………………...... 190

APPENDIX 4: Supplementary Tables For Chapter 7………………………...………...198

APPENDIX 5: A Phylogenetic Tree Of Diptera……………….………………….…...213

VITA……………………………………………………………………………………217

LIST OF FIGURES

FIGURE 3.1: Experimental Design…………………………………………………………..…40

3.2: Survival Curves…………………………………………………………………..41 3.3: Oviposition Events………………………………………………………...... 42 3.4: Egg Area and Egg Number………………………………………………………43 3.5: Egg Number According to Female Density……………………………………...44 4.1: Diagram of Wing Area Measurement……………………………………………59 4.2: Morphometric Comparisons of Male and Female Flies Fed HWP………………60 4.3: Morphometric Comparisons of Male and Female Flies Fed HWO……………...61 4.4: Linear Regression Models of Wing and Abdominal Area to Age………….……62 4.5: Linear Regression Model of Weight Measurements…………………………….63 4.6: Linear Regression Model of Ovary Area and Day Post-Eclosion……………….64 5.1: SEM of the Male Antenna……………………………………………………….73 5.2: SEM of the Female Antenna……………………………………………………..75 5.3: SEM of Female Arista……………………………………………………..….....77 5.4: SEM of Female Pedicel……………………………………………………..…...79 5.5: SEM of Female Tarsi……………………………………………………..……...81 5.6: SEM of the Blow Fly Mouthparts………………………………………………..83 6.1: Proboscis Extension Stages…………………………………………………….102 6.2: Day 5-7 Proboscis Extension Reflex to Amino Acids and Sugars……………..103 6.3: Mean Proboscis Extension of HWO fed Flies………………………………….104

xvi

6.4: Mean Proboscis Extension of HWL fed Flies…………………………………..105 6.5: Comparison of HWO- and HWL fed Female PER……………………………..106 6.6: Linear Regression Model of Ovary Area and Day Post-Eclosion……………...107 6.7: Comparison of Ovary Stages…………………………………………………...108 6.8: Proposed Lucilia sericata Adult Time Line…………………………………….109 7.1: Two-way ANOVA of Mean EAG Response of Male and Female Flies……….127 7.2: Two-way ANOVA of the Age Effect of Mean EAG Response………………..129 8.1: Proposed Lucilia sericata Adult Time Line………………………………….....140 8.2: Proposed Framework for Lucilia sericata Sensing………………...…………...141 A5.1 A Phylogenetic Tree of Diptera………………………………………………...214

xvii

LIST OF TABLES

TABLE 3.1: Diet Descriptions………………………………………………………………...39 6.1: Ovary And Ovariole Stages…………………………………………………….110 A1.1: Log-Rank Survival Curve Analysis…………………………………………….177

A1.2: Linear Regression Model of Female and Egg Number………………………...178

A1.3: Two-Way ANOVA of Fly Age and Egg Area…………………………………179

A1.4: Two-Way ANOVA of Fly Age and Egg Number……………………………...180

A2.1: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and Weight of HWP fed Flies…………………………………………………………………………………181 A2.2: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and Weight of HWO fed Flies…………………………………………...... 183 A2.3: Linear Regression Model Statistics for HWP Flies…………………………….185

A2.4: Two-Way ANOVA of the Age Effect of HWP Flies…………………………..186

A2.5: Two-Way ANOVA of the Age Effect of HWO Flies………………………….188

A3.1: Two-Way ANOVA of the PER for HWO fed Male and Female Flies………...190

A3.2: Two-Way ANOVA of the PER for HWL fed Male and Female Flies…………192

xviii

A3.3: Two-Way ANOVA of the PER of HWO- and HWL fed Females……………..194

A3.4: Age Effect Two-Way ANOVA of the PER of HWO fed Males……………….196

A3.5: Age Effect Two-Way ANOVA of the PER of HWO fed Females…………….197

A4.1: Two-Way ANOVA of the EAG Response of HWO- and HWL fed Female Flies…………………………………………………………………………………….198 A4.2: Two-Way ANOVA of the EAG Response of HWO- and HWL- fed Male Flies…………….……………………………………………………………………….200 A4.3: Two-Way ANOVA Analysis of the EAG Response of HWL-fed Flies...... 202

A4.4: Two-Way ANOVA Analysis of the EAG Response of HWO-fed Flies……….204

A4.5: Two-Way ANOVA of the Age Effect of the EAG of HWO-fed Females……..206

A4.6: Two-Way ANOVA of the Age Effect of the EAG of HWL-fed Females……..207

A4.7: Two-Way ANOVA of the Age Effect of the EAG of HWO-fed Males……….209

A4.8: Two-Way ANOVA of the Age Effect of the EAG of HWL-fed Males………..211

xix

SPECIAL NOMENCLATURE

ANOVA Analysis of Variance BA Butyric acid CA Corpus allatum DMDS Dimethyl Disulfide DNT DiNitroToluene EAG Electroantennogram GR Gustatory Receptor GRN Gustatory Receptor Neuron HWL Honey-water plus Liver HWO Honey-water Only HWP Honey-water plus Protein JH Juvenile hormone OBP Odorant Binding Protein OR Odorant Receptor ORN Odorant Receptor Neuron PER Proboscis Extension Reflex Assay PMI Post-Mortem Interval RT-PCR Real Time Polymerase Chain Reaction SEM Scanning Electron Microscopy TARDIS Time and Relative Dimension in Space

TNT TriNitroToluene VOC Volatile Organic Compound

xxi

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Almost the minute an organism’s vital functions cease—the beating of the heart, the breathing of the lungs, and the function of the brain—abiotic post-mortem phenomena such as the loss of consciousness, muscle tone, and circulation can be observed in a corpse (Clark and Pless, 1997). Almost the moment breath leaves the body, the transformation and creation of putrefaction commence as anaerobic bacteria begin to degrade the organic matter of the corpse, and release putrid volatile organic compounds

(VOCs) (Madea, B., et al., 1995). These, as well as other transformative and consequential phenomena, are useful in the practice of forensic medicine to determine the post-mortem interval (PMI) and approximate time of death.

Forensic Entomology

The popularity of current television shows such as “CSI: Crime Scene

Investigation,” “NCIS,” and “Law & Order” have allowed the general public access to

and familiarity with the basic proceedings of a forensic investigation. Characters “Gil

Grissom” and “Ray Langston” impart narrow insight into the data collected by forensic

pathologists, among which the most familiar are body (or liver) temperature and degree

1 of rigor mortis. While these indices are helpful, they are highly variable and lose validity

after only 24 to 36 hours post-death (Greenberg, 2004). A much more accurate post- mortem interval (PMI) can be established through the use and analysis of arthropod evidence found on the body at the scene of death.

The first use of arthropods as investigative tools occurred in China in the thirteenth century (Amendt, et al., 2004; McKnight, 1981). Entomological evidence, however, was not introduced into a courtroom until the middle of the 19th century, despite

the fact that contemporary understanding of the blow fly life cycle—that eggs needed twelve months of time to develop into adults—was incorrect according to today’s standards (Bereget, 1855; Amendt, et al., 2004). Not too many years later did

Yovanovich (in 1888) and Megnin (in 1894) attempt to “properly establish… the science of forensic entomology” (Amendt, et al., 2004). Currently arthropods are used in forensic criminal cases of murder, human neglect, and animal poaching.

Arthropod colonization of carcasses has been studied for just under 250 years;

Carl von Linne in 1767 was the first to characterize the succession of insects that contribute to the decomposition of carrion (Campobasso, et al., 2001). Insect succession

on and visitation to the carrion is essential to current forensic science, as each new wave

of arthropods with known life history traits, corresponds to a specific stage of decay.

Based on this knowledge, a relative post-mortem interval can be established upon

observation of the developmental stages of the insects present on the body, the level of

decay, amount of material present on the decaying carcass, and the VOC profile of decaying carrion.

2 Bolstering von Linne’s early work, Megnin, in the late 1890’s identified eight

stages of decomposition for a human body exposed to natural elements, which include

“barometric pressure, light intensity, wind speed, ambient temperature, relative humidity and rainfall” (Megnin, 1894; Greenberg, 1991; George, et al., 2013). Currently utilized are the five major stages of decomposition (fresh, bloated, active decay, advanced decay, and dried remains) identified by Rodriguez and Bass (1983). They also identified the two major types of arthropods that contribute to these stages: Diptera (flies) and Coleoptera

(beetles) (Benecke, 1998; Campobasso, et al., 2001). Many Dipteran taxa have been found to associate with decomposing remains, including Brachysera (Calliphoridae,

Muscidae) and Nematocera (Psychodidae) (Arnaldos, et al., 2005). Of these taxa,

Calliphorids are typically the first to reach, feed on, and colonize the body. Within two to four hours of elemental exposure on the body, Calliphorids (blow flies) sense the release of decomposition VOCs, visit the carcass and deposit eggs on the carrion (Reibe and Madea, 2010). Six to forty hours after oviposition, hatching occurs and the larval feeding stage commences. Blow fly activity continues around the carcass until the middle to end of the dry stage of decomposition (Campobasso, et al., 2001).

Seeing the importance of such arthropod data to law proceedings and criminal prosecution, the Daubert v. Merrell Dow Pharmaceuticals decision mandated that scientific evidence be testable, have a known error rate, to be peer-reviewed and accepted practice within the scientific community. Following this decision, the National Research

Council in 2009 expressed the need for the forensic sciences, including the specialty of forensic entomology, to improve its standards and accuracy (Tomberlin, et al., 2011;

Tomberlin, et al., 2006). Forensic entomology currently relies heavily on fieldwork; the

3 collection of larvae from carcasses, as well as visual identification of species and larval stage (Campobasso, et al., 2001). One major way of increasing precision is to rely less on the currently accepted standards of observation and measurement, and to begin performing genetic or hormone titer tests to uncover markers unique to each larval and adult age, which would allow the establishment of a more accurate PMI (Boehme, et al.,

2013; Boehme, et al., 2012; Park, et al., 2013; Tarone and Foran, 2008; Tomberlin, et al.,

2011; Tomberlin, et al., 2006; Tourle, et al., 2009; Wells and Stevens, 2008). Still

wholly unsequenced, Calliphorid genomes continue to be a mystery, despite a large

current focus, both genetic and behavioral on these particular organisms (DeBry, et al.,

2013; Frederickx, et al., 2012a; Sze, et al., 2012). As a pioneer colonizer of decomposing remains, Lucilia sericata has become a focus for these forensic entomological studies.

Lucilia sericata

Commonly known as “the Green Bottle Fly” for its metallic blue-green coloring,

Lucilia sericata or Phaenicia sericata was first characterized in 1826, then documented in 1894 by J. P. Megnin in his classic “La fauna des cadavres application de l'entomologie a la medicine legal.” This fly is a facultative ectoparasiste that derives adaptive advantage from its ability to survive in a wide range of climates and regions

(Fisher, et al., 1998). Endogenous to regions in the southern hemisphere such as

Australia, New Zealand, and Africa, L. sericata prefers and thrives in warm moist climates, although can be found all over the world, particularly in urban areas.

Lucilia sericata follows the typical Calliphorid life cycle: eggs hatch into first

instar larvae within 24 hours of being laid; larvae pass through three feeding instar stages

before entering into a prepupal stage; after pupation, the flies will emerge into adults.

4 While adults are not particularly picky about their diet, the larva need a high protein diet

typical of decomposing or necrotic tissue, to complete the three molts of the larval stages.

The entire life cycle, depending on feeding and climate conditions, can take between two

to three weeks to complete. Interestingly, the larval stage and growth rate, which is a contributing factor to the PMI estimate, can also be affected by any prescription or

recreational drugs, or toxins that remain in the victims’ tissues post-mortem (Gosselin, et al., 2011; Tomberlin, et al., 2011; Zou, et al., 2013). Sex can also affect the growth of the larvae, as third instar L. sericata males feed earlier than do females, resulting in different growth rates (Picard, 2013). This closely studied and well-documented life cycle allows forensic entomologists to use the insect’s developmental stage to help estimate the post- mortem interval. Due to the fact that L. sericata is one of the first calliphorids to reach a decomposing body, its presence or absence, normal or abnormal development, allows investigators to determine if a body has been moved or tampered with post-mortem.

Current forensic research surrounding L. sericata includes behavioral, genetic,

hormone, and volatile studies. Recently Butcher, et al., published a study that focused on

the use of larval cuticular hydrocarbons in establishing a more accurate post-mortem

interval (2013). To better understand the swarming behavior surrounding carrion,

Tomberlin, et al., performed behavioral studies on L. sericata which determined that age,

diet, and ovarian status affects the oviposition response of bow flies (2012). Genetic

material from a larval crop can be analyzed for investigative purposes, but gene

expression during larval development, the size of the larval genome, and mitochondrial

DNA (cytochrome oxidase I) can be analyzed to help establish a more accurate PMI

(Boehme, et al., 2012, 2013; Picard, et al., 2013; DeBry, et al., 2013).

5 Not only is L. sericata of primary importance in forensic investigations, but in

New Zealand and Australia Lucilia cuprina, a very closely related sister species, is more

commonly known as an agricultural pest (Dallwitz, 1984; Wall, 2000). “Fly Strike,” or

myiasis of livestock dermal tissue, poses a sizable problem to the wool industry, which in

Australia alone, accounts for $2.4 billion in exports (Dallwitz, et al., 1984; Tellam and

Bowles, 1997). During seasons with particularly heavy rain and flooding, it is normal for the fleece to become moldy and to rot, which causes an infection of the underlying tissue,

a condition called dematophilosis (Colditz, et al., 1984; Wall, et al., 2000). While fleece

rot can heavily affect regions of the lower limbs and underbelly, the hindquarters (known

as the “breech”) with feces-matted fleece are the most common infected areas (Phillips,

2009). The secretions from these infections mixed with the fleece rot exude scents similar to those of decaying flesh, which in turn attracts blow flies to the contaminated areas where eggs are laid, larvae hatch, and myiasis results.

Megnin first discovered the medical importance of Lucilia sericata in 1826 when he removed larva from a human myiasis infection involving the ocular, nasal, and oral

cavities of a patient. Today human myiasis is fairly uncommon even in developing

countries. Lucilia sericata larve can be used as a biosurgery agent in a controlled

hospital environment for debridement and removal dead or necrotic tissue of wounds, in

maggot debridement therapy (MDT) (Dumville, et al., 2009; Gupta, 2008; Whitaker, et

al., 2007). These larvae aid in medical situations where surgery is not feasible, but it has

also been shown that larval secretions have antimicrobial properties, and can aid in tissue regeneration (Parnes and Lagan, 2007). Specifically, Pseudomonas aeruginosa and

Methicillin Resistant Staphylococcus aureus (MRSA) infections have been reduced by

6 the application of L. sericata larval therapy. As well, L. sericata derived chymotrypsin can be used to disrupt Staphylococcus epidermidis and aureus biofilms associated with chronic infection (Harris, et al., 2013). Recently, Seraticin is an antimicrobial agent created and patented from the larval secretions of this blow fly.

While research on the blow fly life cycle was originally undertaken to further the field of Forensic Entomology, Veterinary and Human Medicine clearly also continue to benefit from the strides made in the understanding of L. sericata.

Physiological and Physical Development

Unlike the controlled, “clean” laboratory environments used to raise colonies of

Lucilia sericata for both medical and scientific applications, the natural environment in which these blow flies are found is completely unconstrained. While an MDT colony will be provided with a fresh, hormone-free, antibiotic-free, “organic” protein source, it is from a decaying carcass that blow flies naturally acquire nutrients essential for both larval and adult growth and development. A natural bio sensor for decomposition, blow flies are attracted to the necessary protein source by the volatile organic compounds

(VOCs) released by carrion. Without access to an open environment for release of the

VOCs, insect attraction to the carrion is delayed thereby providing a connection between the presence of the volatile compounds and the attraction of blow flies.

Before a blow fly lands on and colonizes a potential food source, the location of the decomposing or necrotic material must be determined by sensing the accompanying suite of volatile organic compounds (VOCs). Even from a distance of close to 2km

Dipteran species can sense these odors and make the long flight with the purpose of

7 finding a viable food source, as well as oviposition (egg-laying) within the carcass

(Greenberg, 2004, 1991; Browne, 1993; Campobasso, et al., 2001).

Both the larval and eclosed adult rate of development is highly dependent on the

type of nutrient source, as well as the rate at which ingestion occurs (Hobson, 1935;

Browne, 1993). In a laboratory setting blow flies are typically raised on a carbohydrate

based diet, such as 0.1M sucrose (Hobson, 1932, 1935), 1:1 Honey-water, or sugar

granules; however, in order for females to reach sexual maturity, dietary protein is required (Browne, 1993; Huntington and Higley, 2010). The preference of females for different types of food is highly influenced by their reproductive state, which in turn affects the food-seeking behaviors (Browne, 1993). During ovarian development, females exhibit food-seeking behaviors specific for the protein necessary for vitellogenesis, the process by which eggs are formed within the ovary (Adams and Hintz,

1979; Browne, 1993; Browne, and Vangerwen, 1992; Browne, et al., 1976; Belzer, 1978;

Hayes, 1999).

In L. cuprina, a sister species to L. sericata, the ingestion of protein-rich material is a prerequisite for sexual receptivity (Browne, et al., 1976). When organisms are fed carbohydrate or protein-rich diets, respectively, they tend to shift that organism’s preference for a diet rich in the lacking element (Simpson and Carlson, 1990). This shift in food preference is linked to the organism’s physiology. Preference of female blow flies for a protein-rich diet is correlated with the organism’s stage of ovarian development

(Strangways-Dixon, 1962; Dethier, 1976; Roberts and Kitching, 1974; Belzer, 1978;

Rachman, 1980).

8 Calliphorids are known to be anautogenous, thus requiring protein to reach full sexual maturity. A strain of L. cuprina, however, can be both anautogenous and autogenous, not requiring a protein-enriched diet to reach sexual maturity and to perform vitellogenesis (Browne, et al., 1976). Despite the fact that dietary protein is essential to the sexual development of most female blow flies, and more specifically L. sericata, it is undocumented as to whether or not protein is essential for male sexual development. It has been shown, however that blow fly males do consume protein, most likely for somatic growth, and for the full development of the accessory sex organs (Stoffolano,

1974). Phormia regina males tend to consume the most protein within the first 15 and 24 hours post-eclosion, L. cuprina males, however, do not show a marked increase in protein ingestion during that same post-eclosion period (Roberts and Kitching, 1974).

Sugar consumption for male blow flies remains constant throughout the adult blow fly life (Belzer, 1978a). For females, protein is not required for previtellogenic somatic growth, but is necessary and preferentially consumed, however, both during vitellogenesis, to provide material for egg and yolk development, as well as directly after egg production to replenish depleted bodily stores (Belzer, 1978a; Strangways-Dixon,

1962). In P. regina females, the corpus allatum (CA) is stimulated within 12 hours of a protein meal to produce juvenile hormone (JH) (Liu, et al., 1988; Zou, et al., 1989). Over the next 48 hours post-protein meal, JH production reaches its maximum, which then exerts either partial of full control over sexual behavior (Zou, et al., 1989; Yin and

Stoffolano, 1997). Just prior to oviposition, the Locusta migratoria female preference for protein decreases dramatically (McCaffrey, 1975). In both sexes of blow flies, consumption of protein is essential to “turn on” mating activity (Stoffolano, et al., 1995;

Tobin, 1979).

9 Some sexual behavior occurs as a response to sex pheromone released by females

as a type of calling behavior (Browne, 1993). Sex pheromones present on the cuticular

surface of the female organisms act as an olfactory and gustatory stimulus for males

(Blomquist, 1987). The production of sex pheromones in the female is dependent on age and development of the organism (Blomquist, et al., 1987), which in turn is dependent

upon dietary protein intake. Successful mating in P. regina is affected by the presence or

absence of protein in the diet (Yin, et al., 1999); feeding S. calcitrans a carbohydrate-only

diet, suppresses mating behaviors of both male and female, regardless of any pheromones

present (Meola, et al., 1977).

Volatile Organic Compounds

A natural biosensor, blow flies can not only sense sex pheromones, but also

volatiles released by necrosis and decay, and alter behavior accordingly. While there are

only five main stages of decomposition, there is a broad spectrum of volatiles released

during the process of bodily decay (Sharonowski, et al., 2008). As previously mentioned,

each decomposition level has a distinct subset of visiting and ovipositioning arthropods.

It postulated that a distinct spectrum of volatile organic compounds (VOCs) attracts a

specific subset of arthropods, and directly affects the order of insect succession. Multiple

studies have been performed to establish the identity of these volatiles: Vass, et al. (2004)

identified 424 airborne chemicals released during the process of burial decomposition;

Statheropoulous, et al. (2005) identified another 80 volatiles released from bodies

decaying in bags; Dekeirsschieter, et al. (2009) identified an additional 104 compounds

released in the headspace of decaying pigs.

10 Over the course of the decomposition stages, nine different categories of volatiles

are released: acids, esters, ketones, aldehydes, alcohols, nitrogenous compounds,

sulfurous compounds, cyclic hydrcarbons, and non-cyclic hydrocarbons (Dekeirsschieter,

et al., 2009). Initially, during the “fresh decomposition” stage, no volatile organic

compounds were detected. Alcohols, sulfurous compounds, and a few nitrogenous

compounds were detected during the second “bloated” decomposition stage. Most

volatiles are released during the stage of active decay; the stage most commonly

associated with the putrid, acrid smell of decomposition (Dekeirsschieter, et al., 2009).

Frederickx, et al. have established that L. sericata does, in fact, have an antennal response to decomposition-related VOCs which include Butanoic Acid, Phenol, Indole, Putrescine, and Cadaverine, among many others (2012).

Olfactory and Gustatory Sensing

The array of volatile organic compounds (VOCs) associated with fleece rot and decomposition may not be distinguishable by the human nose, but Lucilia sericata, one of the most widely examined species of blow fly, easily discriminates between fecal odors and the volatile profile of decaying or infected organic material through pattern recognition (Jacquin-Joly and Merlin, 2004; Morita, 1992). Similar to the way in which musical chords are played on a piano, a certain set of neurons triggered in the fly brain is

translated as a particular odor, and if this odor is associated with decomposition, behavior is then altered (Stocker, 1994).

Literature states that blow flies can sense a decaying carcass as early as two to

fours hours post-mortem, anecdotal evidence, however, describes the presence of calliphorids as early as within a few minutes of a carcass’ environmental exposure,

11 suggesting that blow flies can sense and respond to decomposition-related volatiles

almost immediately. This sensing involves highly specialized organs, such as the

antennae, and its composing sensilla, which allow the organism a level of sensation

which most readily equates to the sense of smell (Jacquin-Joly and Merlin, 2004).

Chemical and other odorant messages are received on the antennae through pores in the

cuticle; the chemical messages are then translated into electrical neuronal impulses, and relayed to the brain centers where a physiological/behavioral response is initiated

(Hansson, 1995; Golebiowski, et al., 2007). It is this sense-response connection that has fascinated scientists for decades—how an external environmental signal (volatile organic compound) can elicit such a specific behavioral response within an insect.

Many olfactory receptor neuron- (ORN) containing sensilla are present on a single antenna, allowing the reception and translation of a wide variety of chemical messages (Golebiowski, et al., 2007). Once received, a chemical compound undergoes a number of early olfactory processing steps, called the perireceptor events, which begin with the binding of the specific volatile to an odorant binding protein and end with the activation of a neuronal receptor (Jacquin-Joly and Merlin, 2004; Mustaparta, 1996). The late 1980’s saw a shift in the understanding of insect sensing, from that of the odorant traveling through antennal pore tubules and activating the odorant receptor neuron (ORN), to the current knowledge of the shuttling odorant binding proteins (OBPs) (Vogt, et al.,

1985; Steinbrecht, 1997). This initial perireception event requires three main classes of proteins: the odorant binding proteins (OBPs), the odor-degrading enzymes (ODEs), and the olfactory receptors (ORs) on the local sensory neurons (Jacquin-Joly and Merlin,

2004).

12 In both Drosophila (the current Dipteran “model organism”) and Calliphorids, the

VOC molecule must be bound by an odorant binding protein, before sensing can begin.

This protein binding aids the typically hydrophobic molecules in crossing the cuticle and

extracellular boundary of sensilla cells (Jacquin-Joly and Merlin, 2004). Once across the cell membrane, the OBP remains attached to the molecule until it reaches the transmembrane odorant receptor complex (Krieger and Breer, 1999). There are a number of different odorant receptors, each with specificity for a class of odorant molecules, making these proteins essential to the connection between the sensing of a chemical and the behavior elicited in the insect, without which certain species would not respond to specific odors (Vogt, et al., 1991; Xu, et al., 2009).

After the OBP-odorant complex binds to the OR, the odorant receptor undergoes a conformational change. This shape alteration allows the transfer of the chemical message from the extracellular membrane surface to the intracellular surface by initiating a chemical cascade through which the end result is the activation of olfactory neurons,

and the resultant behavior (Jacquin-Joly and Merlin, 2004). Once the ORN is activated,

the chemical message is quickly degraded by odorant degrading enzymes (ODEs), to

prevent constant stimulation and to allow for new chemical messages to be received.

Odorant receptors across many different species share the same seven-

transmembrane region of a G-protein coupled receptor structure, although sequence

homology may be poor. Each odorant receptor neuron (ORN) expresses a unique a

unique odorant receptor (OR), which allows for odorant signal specificity. As well, all

ORNS expressing a specific OR converge into the same antennal lobe of the glomerulus

13 within the Dipteran “brain,” which is very similar to the structure of the vertebrate olfactory system (Vogt, et al., 1991; Xu, et al., 2009).

Although these genes are not shared with vertebrates, Krieger, et al. (2003) reported the conserved amino acid sequences of odorant receptors between insects of different orders, including Diptera (blow fly), and Drosophilidae (fruit fly); specifically

Odorant Receptor 83b (Or83b) and Odorant Receptor 43a (Or43a) (Fuss and Smith,

2009). Expression of LSOr83 was confirmed in Lucilia sericata throughout all life stages

(Wang, et al., 2012). Currently this particular OR is believed to be a non-specific co- receptor, aiding a second receptor specific for certain volatiles. Knowing that odorant receptor genes are conserved between species of flies allows the probing of other species for known DOR genes, with the hope of not only uncovering further homologies, but also to gain a deeper understanding of why the blow fly responds to the specific VOC profiles of decomposing carrion.

As with olfaction, Drosophila melanogaster is also considered “the classic insect model” for studying gustation in flies, with most of the system conserved between different species (Amrein and Thorne, 2005). D. melanogaster is considered ideal for understanding the perception of taste due to its ability to sense chemicals within a range of 1 mM down to the micromolar range. The complexity and sensitivity of the

Drosophila gustatory sensing system, unlike mammal systems, is not limited to a single organ (Dethier, 1976; 1978; 1971). Consisting of two labial palps covered in 62 taste bristles, with another 260 taste bristles interspersed between mechanosensory bristles on the forelimbs and anterior regions of the wings, this large number of body-covering

14 sensilla emphasizes the importance of chemosensory stimuli to the fly and its ability to

survive, feed, and reproduce (Stocker, 1994; Nayak and Singh, 1983).

“The fly equivalent to the human tongue,” the two labial palps, distally located at

the end of the proboscis, are each covered with taste bristles (or sensilla), allowing initial

food tasting and contact before entrance to the pharynx (Amrein and Thorne, 2005;

Stocker, 1994). While undocumented in blow flies, a sexual dimorphism in the number

of forelimb taste bristles has been noted in Drosophila, with males having an average of

50, whereas females only have 37 (Nayak and Singh, 1983; Possidente and Murphey,

1989). The increased number of taste bristles is attributed to the necessary ability of

males to taste pheromone chemicals secreted by the female to attract courtship (Bray and

Amrein, 2003).

Taste bristles and pegs (i.e. hairs) in both Drosophila and Calliphorids are similar

to the olfactory system in that they are composed of sensilla. This hair like projection is

covered with waxy cuticle enclosing a lymph-filled interior, and pierced by a terminal

pore allowing direct access of food molecules to the dendritic process of the gustatory

neuron (GN) (Stocker, 1994; Morita, 1992). Typically there can be between two to four

neurons, and just as many receptors, within the sensillium that can be activated simultaneously by different types of chemical messages (Rodrigues and Siddiqi, 1981).

Due to its innervations, the sensillar cells allow a response to multiple chemical

messages—whether it may be to water, sugars, high or low salt concentrations—

simultaneously. While the contents of the taste lymph have yet to be characterized, it is

believed to act similarly to the olfactory lymph allowing chemical messages to permeate

the cuticle; it is even believed that olfactory binding proteins are expressed in taste lymph

15 to allow both volatile and soluble environmental chemicals to access the gustatory receptors (GR) on the dendritic process of the gustatory neurons innervating the sensilla

(Shanbahag, et al., 2001; Nayak and Singh, 1983; Galindo and Smith, 2001). Before arriving at the GN, the chemical signal must activate the GR, which, similar to the olfactory receptors, are believed to also be G-coupled protein receptors (Scott, et al.,

2001).

A dipteran’s gustatory and olfactory abilities are vital to the association between behavior and sensing (Jaquin-Joly and Merlin, 2004). While many strides toward deeper understanding these areas have been made in Drosophila melanogaster, very little has been done to advance the knowledge base of these processes in a Calliphorid organism, such as Lucilia sericata. Seeing as though D. melanogaster is not one of the primary

Dipterans colonizing carrion, it is assumed that decomposition-related volatiles are either sensed at the perireception level and degraded, not triggering a neuronal response; or that a neuronal response is triggered, but simply does not elicit a behavioral response.

Phylogeny

Drosophila melanogaster is often considered a “model organism” of Brachycera and Alcalyptratae within the Dipteran order due to the extensive amount this particular species has been studied with regard to genetics, behavior, and physiology (Yeates, et al.,

2003). Currently, biological information relative to all three of these research areas is scarce for Lucilia sericata, thus it was necessary to reference previously published reports on D. melanogaster, and when available, other species of Calliphorids. A phylogenetic tree diagramming the relationships between Dipterans is located in

Appendix 5, and derived from the extensive review of literature published in 1999 by

16 Yeates and Wiegmann. This review is considered to be a seminal paper on the subject

and widely referenced, despite focusing on morphological relationships and not taking

into account the more current phylogenetic relationships detailed using mitochondrial

genomic DNA as did Cameron (2007) when looking at Aschiza, Brachycera, and

Muscomoprha, and Bernasconi (2000) with Mucoidea, specifically taking into account

relationships between Drosophila, Anopheles, and Calliphoridae. The molecular

phylogeny of a number of species within Calliphoridae have also been examined. Wells

and Sperling (1999) examined the monophyletic lineage of Chrysomya spp., with regard

to relationships with Phaenicia sericata and Phormia regina (Sperling, 1994). A few

years later, Stevens and Wall (2001) looked more specifically at the lsu rRNA gene as a

phylogenetic marker between Calliphora spp., Lucilia spp., and Protophormia spp., and

confirmed the classic morphology-based phylogeny of Calliphorids

Understanding these relationships between organisms within the Dipteran order,

such as Drosophilidae and Calliphoridae, and well as within the Calliphorid family allow

a deeper insight into currently unknown areas of biological research surrounding the

organism of interest in this research, Lucilia sericata.

Deepening the understanding of Calliphorid behavior as it relates to

decomposition is key not only to furthering the fields of forensic, medical and veterinary entomology, but also better understanding both the gustatory and olfactory sensing

cascades in insects. Studying how the behavioral response of L. sericata to

decomposition related volatiles and solutions is affected by the sex, age post-eclosion, and diet of the fly, we, as a scientific community, will be better able to utilize the multiple facets of this organism to create biomimetic systems, manufacture more

17 attractive traps to prevent disease outbreaks, decrease the error rate of post-mortem interval estimates, and to better scientific practice.

18 CHAPTER 2

SIGNIFICANCE AND RESEARCH OBJECTIVES

Significance

Casey Anthony and Anthony Sowell are just two names that bring to mind

lengthy, horrific murder trials, whose outcomes hinged on forensic evidence. Admitted

to the body of proof in both of these cases was testimony of the smells associated with

the crimes. Oak Ridge National Laboratory found that “a portion of the total odor signature from [Casey Anthony’s] trunk ‘is consistent’ with a decomposing body that could be human.” Although this evidence points toward the possibility that a corpse

could have occupied the car, the fact that only five of the possible four hundred

compounds associated with decomposition were discovered did not evince to the jury that

the source of the scents was little Caylee’s body.

For years, Ray’s Sausage Factory in Cleveland, Ohio was blamed for the stench

that permeated the neighborhood, resulting in fines from the Health Inspector and

mandatory replacement of the facility’s interior plumbing and grease trap. Five years later, the public now knows that that stench was not poor practices of the factory, but the eleven rotting and buried bodies of women found on Anthony Sowell’s property next door.

19 As the Sowell and Anthony trials have shown, individuals can be convicted or acquitted based on the validity and availability of forensic data presented in court, for which, as stated by the National Research Council in 2009, collection and processing methods are still lacking. If the officials in both cases could have better discriminated the decomposition-related volatile organic compounds by using a mechanical biosensor utilizing distinct pattern recognition, it is possible that Sowell’s victims could still be alive, and that the Anthony verdict could have been resolved.

Knowing the sense-and-response cascade in the blow fly, and the importance of harnessing this information for applications in the forensic sciences, the main objective of this work is to gain a better understanding of the effect diet, sex, and age have on Lucilia sericata’s gustatory and olfactory capabilities.

Before delving into the nuances of gustatory and olfactory sensing, a proper diet needed to be established, thus a comparison of nine variations of three diets was undertaken. This study, presented in the third chapter, establishes the effects the various diets have on life span and fecundity of the flies. With a diet of honey-water ad libitum and a broad-spectrum protein source, bovine liver, established as providing optimal rearing and breeding conditions for L. sericata, the fourth chapter looks at how, if at all, this diet can affect the physical growth and morphological characteristics, and overall fitness of the organism. Understanding that while a protein-supplemented diet does slightly impact certain morphological ultrastructure characteristics, microstructure of the sensing organs remains unaltered, as is detailed in Chapter 5. Chapter 6 looks at the gustatory response of blow flies, utilizing the proboscis extension reflex to gauge the effect diet, sex, and age have on the gustatory receptors on the tarsi and proboscis.

Chapter 7 follows the same pattern of Chapter 6, by examining the effect of sex, diet, and

20 age of the fly on the olfactory response as evaluated by electroantennography (EAG) of the olfactory receptor neuron (ORN) depolarization.

Recognizing that the age post-eclosion of, the various, specific nutrients consumed by, and the sex of the blow fly can and does affect gustatory and olfactory sensing capabilities informs the understanding of Lucilia sericata’s rapid attraction to decomposing remains. Knowing that flies of a certain age and ovarian status are more attracted than others to the volatile suite released by carrion could potentially affect the way forensic entomologists estimate a post-mortem interval. Appreciating that a protein- supplemented diet promotes optimal blow fly breeding and rearing, allows both medical and forensic entomologists the opportunity to raise healthy colonies for both research and application.

Research Objectives

Main Objective: To understand the effect diet, age, and sex have on both gustatory and olfactory sensing of decomposition-related compounds.

Objective 1: Compare the effect variations of currently utilized diets on the lifespan and fecundity of adult flies.

Objective 2: Evaluate the effect of currently utilized diets on growth and development of adult flies.

Objective 3: Ascertain the microstructure of the sensing organs, so as to better understand future gustatory and olfactory studies.

21 Objective 4: Examine the effect diet, age post-eclosion, and sex of adults on the gustatory response of blow flies to decomposition-related compounds.

Objective 5: Examine the effect diet, age post-eclosion, and sex of adults on the olfactory response of blow flies to decomposition-related compounds.

22 CHAPTER 3

A COMPARISON OF COMMON DIETS FOR THE CONTINUOUS CULTURE

OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE) FOR FORENSIC AND

MEDICAL ENTOMOLOGICAL APPLICATIONS

Published in The Journal of Medical Entomology

December 2014

Abstract Blow fly members of the family Calliphoridae, specifically Lucilia sericata, have applications in the fields of behavioral ecology, forensics, and medicine as agents for assessing ecological succession or decomposition and post-mortem interval (PMI) estimation, and for maggot debridement therapy (MDT), respectively. The lack of standardization of laboratory adult insect breeding and rearing protocols among researchers in behavioral, medical, and forensic fields has become problematic. With the goal of understanding physical and physiological effects of diet as a baseline for future behavioral experiments, this article focuses on determining basic culture requirements for the adult blow fly L. sericata by comparing nine diets and the effects of each on survivorship and fecundity under controlled laboratory conditions. Percent survival, fecundity, and the effect of culture density were analyzed over the course of 120 days.

23 Results indicate that a simple broad spectrum diet of honey water and bovine liver is the

optimum diet for extending the life span of the flies and increasing the number of eggs

laid per female per oviposition event, with 5-20 female flies being the optimum number

per culture vessel. This culture protocol is simple to follow, can be easily incorporated into current behavioral, forensic, and medical entomology research programs, as well, the dietary components are readily available across diverse geographic areas.

Keywords: Lucilia sericata, survivorship, fecundity, culture, nutrition, behavior

Introduction

Principally known for its importance in forensic entomology as a primary

colonizer of decomposing remains, both adults and larvae of Lucilia sericata (Meigen)

are also noted for their medically important roles in human wound debridement and

myasis (Martelet, et al., 2009; Mumcuoglu, et al., 1999; Tomberlin and Adler, 1998).

Recently, however, this particular species of blow fly has become a primary player in

insect behavioral studies focusing on the effect of physical and physiological conditions

on both the behavior of the fly and its endogenous sensing abilities (Sanford, et al., 2013;

Tomberlin, et al. 2013). With this recent research focus, Lucilia sericata is now an

organism of primary interest across a number of disciplines, and the significance of

streamlining breeding and culture conditions, as well as establishing respective effects on

the fly, becomes paramount.

Hobson (1932) is considered the father of blow fly nutrition for both maggot

debridement therapy (MDT) and forensic blow fly colonies, as he developed a complex

24 nutritive food source for colony maintenance. Hobson’s diet was further streamlined in

1991 by Daniels, et al., and in 2001 by Tachibana and Numata. These diets, however, focus mainly on larval rearing, while not addressing the needs of the adults for colony establishment and continuous culture. Some laboratories choose to follow such a recipe to make a semi-synthetic food source for adult flies to decrease food source heterogeneity, while others opt to feed colonies only the essential dietary components, which include sources of sugar and broad-spectrum protein (Rabelo, et al., 2011). In an effort to simplify and standardize the adult blow fly diet, this article compares sugar and protein sources in a permutation of nine diet combinations to determine which natural diet provides for the best feeding, breeding, and rearing outcomes of adult Lucilia sericata blow flies to eliminate variation in behavioral studies, as well as for use in forensic entomology and MDT laboratory colonies.

As an applied science, the emphasis of forensic entomology is to join larger concepts, such as ecology, genetics, and evolution for a concentrated goal, which for this field, is the analysis of crime scene evidence (Tomberlin, et al., 2011). The most common contribution of forensic entomology is in the establishment of a post-mortem interval (PMI) by staging the insect larvae found at the scene (Anderson, 2000; Arnaldos, et al., 2005; Huntington, et al., 2007; Zurwaski, et al., 2009). Establishing the larval stage using life cycle data allows an estimation of the amount of time a victim has been exposed to the elements. Blow fly developmental progression through the three instar stages to pupa is directly affected by specific temperature and environmental conditions

(Browne, 1993; Campobasso, et al., 2001). Adults lay eggs on the body, which then hatch to first instar larvae given a specific amount of degree-days, information that was

25 developed from data collected in controlled laboratory settings, then confirmed in the field (Amendt, et al,. 2007).

Understanding the life cycle of blow flies is not only the key to establishing an appropriate PMI, but also to establishing medically useful blow fly colonies. Nosocomial colonies are established and perpetuated with the express purpose of producing larvae for

MDT. These blow fly larvae, when developed in a “clean” environment, aid in the process of wound healing and cleansing (Martelet, et al., 2009). This application is especially important for the treatment of diabetic and other necrotic ulcers, where festering tissue presents a large health risk for the patient (Martelet, et al., 2009,

Mumcuoglu, et al., 1999). In these cases larvae are applied to the wound on which they consume dead tissue and excrete a saliva-like substance that contains anti-microbial properties; both functions aid in healing (Kerridge, et al., 2005).

Maggot debridement therapy colonies are developed using methods similar to those utilized by forensic entomologists to rear field-collected flies in a laboratory setting

(Lewis and Benbow, 2011). Criminal cases hinge on the work done at the bench, despite the emphasis on fieldwork in this area of study. Seeing the importance of such data in law proceedings, the Daubert v. Merrell Dow Pharmaceuticals decision mandated that scientific evidence be testable, have a known error rate, be peer-reviewed, and be accepted practice within the scientific community. The National Research Council in

2009 also expressed the need for the forensic sciences, including the specialty of forensic entomology, to improve its standards and accuracy. With the introduction of the use of insect genetics in forensic analyses, a renewed importance is now placed on the methods and protocols used in the laboratory to establish and reproducibly rear colonies from

26 field-gathered species. It is essential for consistency to exist between labs performing

any type of molecular or microbial forensic analysis, if specimens are to be analyzed in

more than one laboratory. There are a number of different feeding, breeding, and rearing

techniques currently in use to develop adult blow fly colonies in forensic and medical

entomology laboratories; this diversity of culture protocols has the potential to result in

morphometric and gene expression differences in the adults, and thus the eggs and larvae

(Tarone and Foran, 2001; Tarone, et al., 2011).

To streamline and increase the ease of the laboratory culture of mature flies, we

tested nine diets to see how each affected lifespan and fecundity, two of the most important factors in colony establishment and maintenance. Twenty-seven cages of

between 12-20 L. sericata females and 12-20 L. sericata males were fed one of two

common and easily accessible sugar sources: either dry sucrose ad libitum, or a 1:1

honey:water solution given in a controlled amount or ad libitum. The sugar sources were

then supplemented with a single or double protein source: organically raised bovine liver,

used for its’ broad spectrum of protein and cholesterol, and reconstituted dry milk, as it is

a readily and broadly available source of protein. Liver alone (single protein source) or

liver and milk (double protein source) were added to the cages to determine which

protein source promoted sexual maturation and fecundity in females, and allowed for

optimal colony establishment and perpetuation.

27 Materials and Methods

Source of flies for culture

Adult flies (Lucilia sericata, California strain collected in 2006) for initiating

stock cultures were obtained from the Department of Entomology, Texas A&M

University, courtesy of Dr. A. Tarone. Flies were reared on a 1:1 honey: water plus

bovine liver diet and allowed to oviposition on fresh liver placed into the culture tents.

Eggs were collected, placed into culture jars with vermiculite, and reared on a diet of

bovine liver to pupa stage. Adults and larvae were maintained under constant conditions

at 28C, 40 _ 2% relative humidity (RH), and a photoperiod of 12:12 (L:D) h.

Separation of pupa and caging of flies

After each larval cohort pupated, each of 1200 pupa were separately placed in 1

oz. cups, capped with breathable/air permeable lids, and placed in a Powers Scientific

Incubator (Dot Scientific, Burton, MI), which was maintained at a 28 C, 40 +/- 2%

humidity, on a 12hr light-dark cycle. Newly-eclosed flies were sexed using the

measurement of the occipital sclerite and placed into BugDorm cages (0.028 m3 volume;

BioQuip, Rancho Dominguez, CA) with between12-20 females and 12-20 males, for a total of 24-40 flies per cage, as detailed in Figure 3.1. Flies in each cage emerged within

12 hours of each other and were from the same cohort of pupae. Flies were fed one the respective diets within 2 hours of being separated and caged. Three cages of flies were designated for each of the nine respective diets (Fig. 3.1). All cages were then kept in a walk-in environmental chamber with temperature maintained at 23 +/-3  C, 35 +/- 3% humidity, on a 12:12 hour light:dark cycle. Temperature, humidity, and light cycle culture parameters were monitored using a portable data logger (HOBO® Model U12,

28 Onset Computer Corporation, Bourne, MA) over the course of the experiments.

Diets

Flies were fed one of the nine different diets that contained a sugar source only or sugar source plus protein: controlled honey:water (100uL per fly of a 1:1 by volume honey:water solution only, or in combination with either 100ul per fly non-fat milk plus approximately 5 grams of beef liver introduced at post-eclosion day 14, or beef liver only introduced at post-eclosion day 14); ad libitum honey:water (a 1:1 by volume honey:water solution ad libitum only or in combination with either non-fat milk ad

libitum plus approximately 5 grams of beef liver introduced at post-eclosion day 14, or

beef liver only introduced at post-eclosion day 14); ad libitum dry sugar (table sugar- sucrose- ad libitum only or in combination with either non-fat milk ad libitum plus approximately 5 grams of beef liver introduced at post-eclosion day 14, or beef liver only introduced at post-eclosion day 14). Protein was introduced relatively late in the adult fly life cycle (day 14) to allow assessment of the effects of a sugar source only diet

(Huntington and Higley, 2010). Non-fat milk was prepared from store-bought packaged dry milk (Nestle Carnation Instant Dry-Milk, Fremont, MI) according to the package instructions. Organically grown beef liver (no hormones or antibiotics) was purchased at a local grocery store. Organically produced honey was purchased from a local grocery store, which conformed to the FDA’s requirements for consumable honey (Ball, 2007).

The measured amount of the honey:water solution was applied to half of a tri-fold paper towel then placed on half of a cell-culture petri dish; in the case of the ad libitum honey:water diets, the paper towel was completely soaked in the solution and then placed in a petri dish. Dishes were then inserted into each appropriate cage. The 1:1

29 honey:water solution was made fresh every other week and kept refrigerated to prevent bacterial growth. Those cages receiving the 1:1 honey:water solution either in a controlled amount or ad libitum, received fresh honey:water solutions every-other day

(odd-numbered days), with the older solution-soaked paper towel removed at the time of replacement. Those cages that received either milk and/or liver, would receive the appropriate amount on the days alternating with the honey:water solution replacement

(even-numbered days). Table 3.1 details the diet combinations, number of replicates, and diet abbreviations.

Data Collection

Each cage was assessed every day for live and dead adult ssy counts, and every other day, when the liver was replaced, for the presence of eggs and larvae. When eggs were present, they were counted and photographed for analysis (aerial surface calculation). Eggs were separated from the liver (oviposition medium) and counted individually, then returned to the liver and reared to larvae to assess for egg viability.

Presence of any hatched eggs (live larvae) was noted. Deaths of adult ssies were recorded daily by sex.

Data Analysis

Kaplan-Meier survivorship proportions were entered according to standard protocols. Log-rank (Mantel-Cox) tests were used to compare the survivorship curves and to determine statistical differences. Number of oviposition events were analyzed by one-way analysis of variance (ANOVA), with Bonferroni post-tests to determine statistical differences between the diets. Two-way ANOVA was used to analyze both egg number and egg area according to day, with Bonferroni post-tests used to determine

30 statistical significance between diets. Images for egg area measurements were taken at

5X using a microscope-mounted camera (Canon Rebel Ti3 DSLR, Canon USA), and

analyzed using the length and width of individual eggs Image J (NIH free access Image

Analysis software, http://rsbweb.nih.gov/ij/). Linear regression models were used to

determine the covariance between the number of eggs oviposited and the number of

females. GraphPad Prism version 5.0 (GraphPad Software, San Diego CA, USA,

www.graphpad.com) was used to conduct all statistical analyses.

Results

Survivorship

Survivorship of flies was influenced by the presence of a protein source in the diet,

regardless of sugar source (Fig. 3.2, all P < 0.0001; Supp. Table A1.1). Deaths were not affected by the consistency of the sugar source; the honey-water was not allowed to dessicate enough to become a trap. Fed on a sugar source alone, all flies died by day 40

(honey:water ad libitum), whereas any of the protein-supplemented diets allowed

extension the lives of the flies beyond the initial 30 days, anywhere from 20 days

(sucrose ad libitum) to 70 days (honey:water, controlled). Flies fed sucrose-only died the earliest in the study (day 27), while flies fed a controlled amount of honey:water plus milk and liver lived the longest (112 days; data not shown past day 60). The difference in survivorship between flies fed honey:water ad libitum plus a protein source, and those fed

a controlled amount of honey:water plus a protein source were not significant (Fig. 3.2).

The survivorship differences between both honey:water treatments, and sucrose for all

protein supplements were highly significant (Fig. 3.2, P < 0.0001 for all). The

31 survivorship differences between diets supplemented with protein (liver only or liver and

milk) were not significant for all treatments (Fig. 3.2; Supp Table A1.1).

Fecundity

The number of oviposition events for combined cohorts for each protein-added

diet (average of three replicates) was not significantly different between diets (Fig. 3.3).

Because of the fact that the differences between the honey-water controlled and honey-

water ad libitum diets were not significant, only the honey-water ad libitum data are

shown (Fig. 3.2). The number of eggs laid was influenced by diet and by age of females

(FDiet = 4.21, FDay = 13.88, FInteraction = 39.20, with PDiet < 0.005, PDay < 0.005, PInteraction <

0.0001; Fig. 3,4; Supp. Table A1.2). Regardless of the protein source, flies fed

honey:water diets laid an average of three times more eggs than those given sucrose over the course of 60 days (data not shown). The most significant differences in number of eggs occurred on days 25 and 27 across all diets (P < 0.001 Supp. Table A1.3).

The area of eggs laid was also influenced by both day and age of females (Fig.

3.4; FDiet = 11.58, FDay = 15.28, FInteraction= 56.25, with all P < 0.0001; Supp. Table A1.3).

Flies fed honey:water supplemented with only liver laid eggs having the largest average area, followed by eggs laid by flies fed sucrose and liver only, both of which were larger than eggs laid by flies fed diets supplemented with milk, regardless of the sugar source.

Density

Diet and age of the female flies affected the number of eggs laid and the egg area; however, neither diet nor age significantly affected the average number of eggs laid per female (Fig. 3.5A; FDiet=2.63, FDay=12.80, FInteraction=26.46, with all P > 0.05; Supp.

32 Tables A1.2 and A1.3). Regardless of diet and age, the most eggs were laid per cage

when there were between 4 and 14 females present (Fig. 3.5A), despite all cages

beginning with a 1:1 male:female ration, with egg production peaking with a culture

density of approximately 10 females per 0.028 m3 cage volume (Fig. 3.5).

For diets supplemented with milk and liver, the linear regression model for the

number of females versus the number of eggs oviposited is significant (Fig. 3.5B;

2 2 R HW+M+L=0.1821, PHW+M+L< 0.0106, dfHW+M+L= 33; R Suc+M+L=02591, PSuc+M+L= 0.0184,

dfSuc+M+L= 19; Supp. Table A1.2). The slopes of the lines for number of females versus

the number of eggs oviposited for flies fed diets supplemented with only liver, however,

2 2 were not significantly different (r HW+L=0.04331, PHW+L=0.1470, r Suc+L=0.03218,

PSuc+L=0.3342; Supp. Table A1.2). The differences between the slopes of the lines for

number of females versus the number of eggs oviposited for each diet are significantly

different (P < 0.0001, F = 211.2, R2= 0.8309), with significant differences between all

individual diets (all P < 0.0001), except for those diets supplemented with liver only.

The correlation between number of eggs oviposited and the number of females is

significant only for flies fed sucrose and liver (r2=0.8108, P <0.01).

Discussion

Many laboratories rely solely on honey:water or sucrose as the main component

of a laboratory blow fly diet, given the challenge that an expensive, heterogenous diet

poses for the rearing of larvae retrieved from cadavers, as well as for consistent and clean

MDT cultures. When paired with a protein source, these sugar sources allow continuous

33 culture of fly colonies with females reaching complete ovarian development and

achieving sexual maturity (Browne, 1993). We propose that a simple diet of honey:water

(either ad libitum or controlled) plus bovine liver results in increased longevity and

increased fecundity in adult Lucilia sericata blow flies. The simplicity of this diet allows

an easily reproducible and standard diet for laboratory-reared blow fly cultures for

forensic and medical cultures, as well as for those colonies raised for behavioral research.

The expansive body of work done by Hobson (1932) and Dethier (1962),

demonstrated that a complex protein source is an essential component of the female adult

blow fly diet, as both the amino acids and the cholesterol found in many sources are necessary for vitellogenesis, to produce sex pheromones, and to achieve sexual maturity

(Browne and Vangerwen, 1992). The experiments described here build on that

foundation, strongly suggesting that a protein source is vital for extending the lifespan of

both male and female blow flies and the fecundity of female blow flies, regardless of

sugar source. It has been reported that the mean life expectancy of a blow fly is ~30 days

(Ring, 1973), but here we show that given the proper sugar source and protein

supplementation, flies can be expected to live well beyond 60 days (Fig. 3.2) and

continue to produce viable eggs, as assessed by observing the eggs hatching to larvae

(Fig. 3.3). Other published diets utilize a complex mixture of yeast, refined sugar, agar,

and crude protein, on which flies are reared from the date of emergence (Browne, 1993;

Daniels, et al., 1991; Zhang, et al., 2009). While we have not explored inclusion of a

dietary complex protein source immediately after eclosion, we have established that

protein supplementation beginning at 14 days post-eclosion positively affects

survivorship (Fig. 3.2).

34 Studies using the fruit fly Drosophila melanogaster Meigen (Diptera:

Drosophilidae) suggest that ad libitum diets negatively affect the life span of the flies, and that a diet with controlled amounts of sugar will extend the life of a fly (Partridge et al., 2005). In the present study, however, there were no significant differences between the survival of flies fed the honey:water ad libitum and the honey:water controlled diets

(Fig. 3.2). There was, however, a significant difference in the survivorship of flies fed honey:water versus those fed sucrose, regardless of the protein supplement. Honey, which in the United States is regulated by the FDA, includes a number of dissolved amino acids, a broad spectrum of sugars, and histamines; this complex mix of components most likely contributes to the extended survivorship (Ball, 2007). To consume food, blow flies first regurgitate onto the medium, and then proceed to ingest the nutrients (Hansen, 1978). We hypothesize that diets subsisting mainly of dry sucrose, a common medium for blow fly cultures, have the potential to dehydrate the flies, which could also contribute to their early demise.

Extending the life span of the flies is only consequential if the culture can perpetuate itself. Over the course of 60 days, Lucilia sericata cultures oviposited anywhere from 3 to 10 times depending on the diet (Fig. 3.3). There is a clear trend as to which diet resulted in the highest number oviposition events: all honey:water diets out- performed the sucrose diets, with the sucrose diet supplemented with both milk and liver having the least number of oviposition events during the time period. It is possible that the dissolved amino acids and broad spectrum of sugars contained within the honey also contributed to this difference.

35 Protein was introduced into the diet at post-eclosion Day 14. Five days after the diets were protein-enhanced, the first oviposition event occurred in the milk and liver supplemented honey:water diet, followed two days later by flies fed the other diets, suggesting again that a liquid food source is easier to consume and absorb by the organism (Fig. 3.4). The number of eggs per oviposition event peaked at day 25 and started declining after day 41, suggesting that blow flies need between 2 and 8 days after the initial addition of protein to first achieve sexual receptivity and maturation, and close to 10 days to complete vitellogensis as evidenced by oviposition occurring on days 19-20,

5 days after the introduction of protein (Fig. 3.4). Both honey:water diets followed the same basic pattern in number of eggs produced, whereas there was a large difference between those and the sucrose diets. Because of the large amount of variation, any differences between the diets on each day, with the exception of days 21 and 47, were not significant using a two-way ANOVA (Fig. 3.4; Supp Table A1.4). The day-effect on the number of eggs was found to be not significant by two-way ANOVA; the diet-effect, however, was highly significant (P = 0.0077; Supp Table A1.4), indicating that the frequency of oviposition events and the number of eggs produced at each event are affected by what the flies consume.

The area of eggs produced by flies fed diets supplemented with liver was not significant although the trend loosely followed the pattern of the number of eggs produced at those oviposition events (Fig. 3.4). Diets supplemented with both milk and liver did show significant trends between egg number and area, which was inversely proportional—as the egg number increased, the size of the egg decreased. Exposure to

36 the additional milk protein source not only allows earlier production of eggs, but also

allows the production of larger eggs.

The number of females present in each cage was also expected to affect the number of eggs produced per oviposition event, as in a natural environment the larger the community, the more constrained the resources become (Creighton, et al., 2009). For all diets, as the number of females increased, so did the number of eggs produced, with the exception of milk and liver supplemented sucrose. The most eggs were produced by between 4 to 14 females, with the number of eggs produced peaking at a cage density of

10 females for all diets. As Yang et al. (2012) notes, Chrysomya megacephala (F.) and

Chrysomya rufifaces (Macquart) females tend not to lay eggs on liver that is already populated with larvae. This phenomenon may have contributed to the peak in number of eggs with 4 to 8 females present; it does not, however account for the large amount of eggs laid by high numbers of females. These data also suggest that when more than 12 or

14 females are present in a cage, they are less likely to release larger numbers of eggs due to the effect of competition. The concept of a dense population resulting in decreased offspring production, as well as oviposition increasing as the population decreases has been described in both natural and laboratory environments (Nicholson, 1957; Brillinger, et al., 1980). Our blow fly cultures, even though maintained in a controlled laboratory setting, do tend to reflect principles of natural environments as suggested by Nicholson

(1957).

While all of the diets tested allow for Lucilia sericata to live beyond 20 days post- eclosion, the addition of protein to the diet is essential not only for sexual maturation and vitellogenesis of the females, but also for extending the life span of both sexes. Feeding

37 blow flies a diet of honey:water supplemented with a complex protein source allows the life span of the organism to be extended versus those fed a diet mainly of sucrose.

Rearing fly cultures on this honey:water and protein diet also results in an increased number of oviposition events and a greater number of eggs produced during those events.

The consumption of honey:water and a broad protein source, such as bovine liver, is a loose predictor of egg area, and number. Understanding the differences produced in the physical and physiological development of the adult female blow fly, allows behavioral research to streamline the use of a single diet to eliminate any diet-related variables that could contribute to a change in response. Use of this diet will also allow for successful, reproducible, and optimal culture of blow fly colonies for forensic entomology and maggot debridement therapy applications.

TABLE 3.1 DIET DESCRIPTIONS

Table 3.1 Descriptions of, and abbreviations for each of the nine diets used for these experiments.

FIGURE 3.1 EXPERIMENTAL DESIGN

Figure 3.1 Flow chart showing details of the expperimental design.

40 FIGURE 3.2 SURVIVAL CURVES

Figure 3.2 Kaplan-Meier survival curves of Lucilia sericata over a 60 day period when fed one of three sugar sources (Honey Water ad libitum, Honey Waatter Controlled, or Sucrose ad libitum) with the addition of protein (Non-fat Milk and Liver, or Liver only). Significant differences between survival curves (Mantel-Cox loog-rank test) are indicated by stars (*).

FIGURE 3.3 OVIPOSITTION EVENTS

Figure 3.3 Mean (±SE) oviposition events over the course of 60 days according to diet, combining two cohorts. Significance determined by Bonferronni post-tests after analysis by one-way ANOVA .

42 FIGURE 3.4 EGG AREA AND EGG NUMBER

Figure 3.4 Mean (± SE) egg area (left axis, top curve) for eggs laid by females fed one of four diiets (Honey Water ad libitum plus liver only, or plus liver and milk, or Sucrose ad libitum plus liver only, or plus milk and liver). Mean (± SE) egg number (right axis, bottom curve) for eggs laid by females fed one of four diets. Significant differences derived from Bonferroni post-tests (P<0.05) after two-way

ANOVA, between treatments within sampling days.

43 FIGURE 3.5 EGG NUMBER ACCORDING TO FEMALE DENSITY

Figure 3.5 Mean (±SE) number of eggs laid per number of females present in the cage at time oviposition (A), speparated according to diet. Linear regression models of the mean

(±SE) number of eggs laid per oviposition event per female pressent according to one of four diets (Honey Water ad libitum plus liver only, or plus liver and milk, or Sucrose ad libitum plus liver only, or plus milk and liver). Stars (*) deenote significcance of model. Letters (a, b, c) denote significant differences between slopes of the lines.

44 CHAPTER 4

THE EFFECT OF AGE, SEX, AND DIET ON THE

FITNESS AND FORENSICALLY RELEVANT MORPHOMETRICS OF

LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

C. Gordon Hewitt in his 1914 book “The House Fly,” was one of the first to

describe in fine detail both the external and internal structure of Musca domestica, a close

relative of the blow fly. Regulation of body size, and accompanying structure development is of significant value to insects, and more specifically blow flies, as this impacts adult survival, ability to procreate and produce viable offspring, as well as locating and utilizing nutrient sources. These metrics imply fitness or adaptiveness to the

environment (Chown and Gaston, 2010; Grassberger and Reiter, 2001; Wells and King,

2001). Hobson in the 1930’s, Barton-Browne in the 1950’s, and Dethier in the 1960’s

continued to examine the structure of the fly, noting in all cases the importance of diet in the growth and development of the fly, stopping short of saying that one affects the other.

It is clear, however, from the abundance of literature on the subject, that food availability

and consumption are vitally necessary for development of the larval stages and regulation

45 of adult size (Blystone and Hansen, 2014; Huntington, et al 2007).

There is a surfeit of knowledge surrounding the development of the larval and

pupal stages of the blow fly, however, there remains a dearth of information on any possible development of the adult blow fly for either the forensic or medical entomology fields. While staging of the larvae remains current practice in forensic entomology for the establishment of a post-mortem interval, reports in the literature has show that adult flies can associate with the carrion from between 40 seconds (DeJong, 1994) to up to 2 days (Tabor et al., 2004) before any physical interaction occurs. This interaction, however, can vary with the type of decaying organism, the season, the ambient temperature, and the light cycle (Watson and Carlton, 2003, 2005; Hall and Doisy, 1993;

Grisbaum and Tessmer, 1995; Campobasso, et al., 2001). The use of adult flies in forensic investigations has been encouraged, but only from the perspective of verifying larval age for the establishment of a post-mortem interval (Haskell and Williams, 2008).

Recent studies have attempted to establish the use adult characteristics for determining the age, and potentially, the time a blow fly has associated with the carrion before oviposition occurs (Jonathan Cammack, personal communication). If these characteristics, however, are affected by environmental factors in a manner similar to that of the previous larval stages, then unless this is quantified, it cannot be considered a valid aging technique. Structural metrics under investigation in this study for use in ageing flies are the wing area, ovary size, and possibly abdominal area.

Abdominal area and ovary size of adult female flies, are known to increase with nutrient consumption as dictated by physiological need (Hobson, 1932; Browne, et al.,

1976; Tomberlin et al., 2001). A broad-spectrum protein source is required by female adult flies to provide necessary nutrients for vitellogenesis, ovarian maturation, the

46 production of sex pheromones, and to become sexually receptive (Blystone and Hansen,

2014; Browne, et al., 1976; Hobson, 1932; Blystone and Hansen, 2014). It is believed that males also require a small amount of protein shortly after eclosion to complete

gonadal development, but whether or not this affects the ultrastructure of the fly is yet unknown (Roberts and Kitching, 1974).

This study examines how the fitness of adult blow flies of the species, Lucilia

sericata, and thus larger body sizes as measured by wing area which remains constant

throughout the life of the an adult, can affect nutrient utilization for the purposes of

sexual development and maturation as measured by abdominal and ovary size (Chown

and Gaston, 2010; Grassberger and Reiter, 2001; Wells and King, 2001). Flies were fed

either a diet of honey-water only (HWO), or one supplemented with protein (HWP), and

then compared at five time points over a period of twenty days. Measurements of weight,

abdominal area, wing area, and ovary size were recorded and analyzed. Results suggest

that those flies with larger wing area are more likely to consume larger quantities of nutrients, and thus obtain higher weights, as well as to develop larger ovaries at an earlier age.

Materials and Methods

Separation of Pupa and Caging of Flies

After the larval cohort pupated, each of 800 pupa were separately placed in 1 oz.

cups, capped with breathable/air permeable lids, and placed in a Powers Scientific

Incubator (Dot Scientific, Burton, MI), which was maintained at a 40 +/- 2% humidity,

28 C, with a 12:12 hour light-dark cycle. Adult flies were sexed and placed separately

47 according to sex into Bug Dorms (0.028 m3 volume; BioQuip, Rancho Dominguez, CA), with 12-20 females or 12-20 males placed in each Bug Dorm. Each cage of flies eclosed within 12 hours of each other, from the same cohort of pupae. Newly eclosed flies were fed the respective diets within 2 hours of being separated and caged. One cage of each

sex of flies was designated for each of the two diets. All cages were then kept in a

portable, insulated fly enclosure with temperature maintained at 23 +/-3  C, 35 +/- 3%

humidity, on a 12:12 hour light:dark cycle. Temperature, humidity, and light cycle

culture parameters were monitored using a portable data logger (HOBO data logger,

Onset, Bourne, MA) over the course of the experiments.

Diets

Flies were fed one of two different diets: ad libitum honey:water (a 1:1

honey:water solution ad libitum), or ad libitum honey:water and protein (a 1:1

honey:water solution ad libitum in combination with non-fat milk ad libitum). Non-fat milk was prepared from store-bought packaged dry milk according to the package instructions. The measured amount of the honey:water solution was applied to half of a

tri-fold paper towel, then placed on half of a cell-culture dish to ensure contamination did

not occur. The honey:water solution was made fresh every other week and kept refrigerated to prevent bacterial colonization. Those cages receiving the 1:1 honey:water

solution, would receive fresh honey:water solutions every-other day, with the older

solution-soaked paper towel removed at the time of replacement. Those cages that

received milk as well, would receive the aliquoted amount of on the days alternating with

the honey:water solution replacement.

48 A separate cage of 50-60 females was fed honey-water ad libitum and 5g of

bovine liver in preparation for ovary and ovariole dissection and staging, as these flies

were preserved and killed in 80% ehtanol on the appropriate time points..

Collection of Data

Ten flies were removed from each cage every fifth day, anaesthetized on ice for

5-10 minutes, then measured by hand using micro-calipers for the appropriate

morphometric. Abdominal length was measured down the midline of the underside of the abdominal cavity. Abdominal width was measured across the underside of the third segment. Wing length was measured using the origin of the radial vein to the tip of the

R2 vein; wing width was measured across the largest portion of the wing, typically measured in a straight line vertically from the second branch of the cubitus vein, a diagram of which is shown in Figure 4.1 (Hwang and Turner, 2009).

Three to five females were removed from the honey-water and liver fed cage each day for 10 days, and preserved in 70% ethanol. Ovaries, and ovarioles were dissected and measured according to Adams and Mulla (1969), and Huntington et al. (2007); briefly whole ovary area, and single ovariole length was measured using an Olympus D-

40 microscope and an in-lens micrometer, and staged according to Table 4.1, where 1 is the previtellogenic germarium, 2 is the beginning of vitellogenesis, and 3 is the ovary containing mature eggs.

Data Analysis

Abdominal area, wing area, and weight were analyzed by two-way ANOVA

according to day and sex, with Bonferroni post-tests to determine statistical differences

49 between the sexes at each time point. Correlation models were used to test the linearity

of the relationship between ovary size and day post-eclosion, as well as the relationships

between both wing and abdominal area and day post-eclosion for both male and female

flies. GraphPad Prism version 5.0 (GraphPad Software, San Diego CA, USA,

www.graphpad.com) was used to conduct all statistical analyses.

Results Weight

The weights of whole anesthetized flies fed either a diet of honey-water only or

honey-water and protein, were compared between males and females fed the same diet,

according to age post-eclosion by two-way ANOVA

Sex (F (1,4) = 68.47, P < 0.0001), day or age post-eclosion (F (1,4) = 28.29, P <

0.0001), and the interaction between the two variables (F (1,4) = 7.576, P < 0.0001),

significantly contributed the variation in weight of honey-water and protein (HWP) fed flies (Fig. 4.2). HWO females weighed significantly more than HWP males on Day 10,

15, and 20 (P < 0.001 for all), according to Bonferroni post-test. Male and females emerged without significantly different weights, which increased at the same rate through

Day 5 regardless of diet (Fig. 4.2; 4.3). Day 10 and beyond, however, saw the mean

weight HWP females increase significantly at each time point, while the weight of HWP

males increased at much lower intervals (Fig. 4.2; 4.3). For HWP females only, age post-

eclosion significantly predicted the mean weight of the total fly (b = (1/1922), F (1,3) =

13.93, R2 = .8228, P = 0.0335) (Sup. Table A2.4, Fig. 4.4).

50 Sex (F (1,4) = 21.71, P < 0.0001) and day post-eclosion (F (1,4) = 12.95, P <

0.0001) significantly contributed to the variation in mean weight of honey-water only

(HWO) fed male and female flies, but the interaction between the two variables did not

(F (1,4) = 2.443, P = 0.0536; Fig. 4.3). Male and female HWO fed flies weighed

significantly different amounts only on Days 10 (P < 0.01) and 15 (P < 0.05), according

to Bonferroni post-test (Fig. 4.3). The weight of male HWO flies increased between Day

0 and 5, but for the remaining time points did not vary significantly (Supp Table A2.5,

Fig. 4.3). The weight of HWO females increased significantly between Day 0 and 5, then again between Day 5 and 10, after which the variation in weight was not significant

(Supp Table A2.5, Fig. 4.3).

Abdominal Area

Mean abdominal area, measured down the ventral midline and across the third segment, was compared between males and females fed one of two diets—honey-water only or honey-water supplemented with milk protein—according to age post-eclosion, by two-way ANOVA.

Only sex (F (1, 4) = 92.40, P < 0.0001) and day post-eclosion (F (1, 4) = 4.578, P

= 0.0021), significantly contributed to the variation in abdominal size of honey-water and protein fed blow flies. The interaction between sex and age did not contribute significantly to the variation in abdominal size of honey-water and protein fed flies (F

(1,4) = 1.851, P = 0.1264; Fig. 4.2; Supp. Table A2.1A). According to Bonferroni post- test, male and female HWP abdominal areas were significantly different at all time points

(Supp. Table A2.1B; Fig. 4.2). Male abdominal area had the lowest measurement on Day

0, peaked at Day 5 post-eclosion, and then over the course of 15 days, dropped back

51 down to the original measurement (Fig. 4.2). While the HWP male abdominal area

followed a Gaussian distribution, the abdominal area of honey-water and protein fed

female blow flies was a logistic, or S-type, curve, starting at the lowest measurement on

Day 0, peaking at Day 5 and remaining constant for the remaining time points (Fig 4.2).

Age post-eclosion did not significantly predict the abdominal area of either males and

females fed a diet of honey-water and protein according to linear regression modeling

(Supp. Table A2.3).

Only sex (F (1,4) = 11.91, P = 0.0009) contributed significantly to the variation in

abdominal size of honey-water only fed male and female L. sericata blow flies (Supp.

Table A2.2). There were no significant differences, according to Bonferroni post-tests

between male and female abdominal size when fed a diet of honey-water only, at any of

the time points, except on Day 15 (p < 0.05), where the female abdominal area grew to be

larger than the male (Fig. 4.3).

Wing Area

Wing area, measured from the origin of the radial vein to the tip of the R2 vein

and across the largest portion of the wing to the second branch of the cubitus vein, was

compared between males and females fed one of two diets—honey-water only or honey-

water supplemented with milk protein—according to age post-eclosion, by two-way

ANOVA.

Only sex contributed significantly to the variation in wing area for honey-water and protein fed flies (F (1,4) = 22.26, P < 0.0001; Fig. 4.2). Day post-eclosion and the interaction between the two variables did not significantly contribute to the variation in

52 wing area for HWO fed flies (Supp. Table A2.2A). According to Bonferroni post-test,

the only significant differences between the wing are of males and females fed HWP, occurred on Days 15 and 20 (P < 0.05 for both) post-eclosion (Supp. Table A2.1B; Fig.

4.2). The wing area of males fed HWP remained relatively constant throughout the entire

testing period, but the female HWO wing are began to gradually increase on Day 15,

peaking at Day 20 post-eclosion. For females fed a diet of honey-water and protein,

according to linear regression models, age post-eclosion is a significant predictor of wing

area (b = (1/26.28), F (1,3) = 23.47, R2 = 0.8867, P = 0.0168; Supp Table A2.3; Fig.4.4),

but not for males fed the same diet.

Sex (F (1,4) = 15.73, P = 0.0002), day post-eclosion (F (1,4) = 3.197, P = 0.0173),

and their interaction (F (1,4) = 3.179), P = 0.178) significantly contributed to the

variation in wing area for honey-water only fed male and female flies (Fig. 4.3). But

according to Bonferroni post-test, the only significant difference between male and

female wing area occurred on Day 20 post-eclosion (P < 0.05; Supp. Table A2.2B, Fig

4.3). While the wing area of HWO males remained relatively unchanged across all time

points, female wing area only peaked slightly on Day 20 post-eclosion.

Ovary Area and Stage

Ovaries, and ovarioles were dissected and measured according to Adams and

Mulla (1967), and Huntington et al. (2007), and staged according to Table 3.1, where 1 is the previtellogenic germarium, 2 is the beginning of vitellogenesis, and 3 is the ovary containing mature eggs.

53 The age post-eclosion and ovary area of female flies fed honey-water and protein

co-vary significantly according to a Pearson correlation (r = 0,9812, P < 0.0001). The

relationship between these two variables is clearly linear. As well, according to linear regression model, age post-eclosion of female flies fed honey-water and protein is a significant predictor of mean ovary area (b = (1/3.812), F (1,6) = 154.7, P < 0.0001; Fig.

4.6).

Female Lucilia sericata blow flies fed a diet of honey water supplemented with

protein show marked increase in ovarian size and ovary stage beginning after Day 3, after

which it is between 24-48 hours before ovaries become fully mature.

Discussion Adult blow flies are know to associate with decomposing organic material both for the purposes of feeding and oviposition. Current practices of post-mortem interval

(PMI) estimation in forensic investigations, however, rely almost exclusively on information gathered from the larval stages. By applying the morphometric analysis in this chapter, data acquired from adults flies associated with carrion could also potentially be used to aid in PMI approximation, as well as give information as to the fitness of blow flies associating with the carrion.

Morphometric Analysis

Morphometric analysis of the abdominal area, wing area, and the gross weight of adults flies was recorded for both male and female Lucilia sericata blow flies at days 5,

10, 15, and 20 post-eclosion. These data were compared between males and females fed

54 the same diet—either honey-water ad libitum (HWO), or honey-water ad lib plus protein

(HWP).

Surprisingly, for both sexes, flies fed a diet of HWO weighed slightly more that those fed a diet supplemented with protein (Fig. 4.2A). The increase in weight follows the same pattern, however, for both diets, with females weighing significantly more than males after post-eclosion Day 5 (Fig. 4.2A, 4.3A). Honey has been established previous research chapter as being of great benefit in the breeding and rearing of laboratory

cultures of Lucilia sericata when part of a simple diet including a broad spectrum source

of protein (Blystone and Hansen, 2014). This is in large part due to the fact that honey is

naturally supplemented with histamines and amino acids, which can aid in filling the

female physiological need for protein (Ball, 2007). It is hypiothesized that without the

presence of a broad spectrum protein source, females will consume more of the available

honey water in order to provide the necessary amino acids for vitellogenesis and sexual

maturation, thus significantly adding to the noted weight increase. This increased consumption of honey also contributes to delayed ovary development (Fig 4.6).

Males fed honey water only also have slightly greater gross weights than those fed a diet supplemented with protein. While males generally do not need a large amount of protein from multiple feedings to fulfill their small physiological need, males do require a protein meal just after eclosing to complete sexual maturation (Roberts, 1974).

It appears that male L. sericata blow flies may have a proclivity for honey as their

weights are significantly higher at all time points when fed a diet consisting only of

honey water ad libitum. That both sexes weigh less when fed a diet supplemented with a

broad spectrum protein suggests that in the presence of a medium that offers complete

55 nutrition, adult L. sericata flies will generally consume less. As well, consuming a broad spectrum of nutrients, seems to contributes to the overall fitness of the organism, in addition to fulfilling physiological need.

It was hypothesized that due to ovarian development, the mean abdominal area of

L. sericata female flies fed a diet supplemented with protein would be greater than the male abdominal area at all time points, and significantly larger than the abdominal area of female flies fed honey-water only. Regardless of diet, female abdominal area was consistently larger than males fed the same diet (Fig 4.2B, 4.3B). Flies of both sexes fed a diet of supplemented with protein, had smaller abdominal areas than those flies fed honey-water only at all time points. For flies fed a protein-supplemented diet, however, the percent change in abdominal area is greater over the time course (Fig 4.2B, 4.3B). It is no surprise that the honey-water only male abdominal area has a low variability over the 20 days of analysis. Given the significant increase in weight between post-eclosion day zero and day five for HWO-fed males, the lack of significant increase in abdominal or wing area is puzzling.

Honey-water and protein fed males experience a significant increase in abdominal which peaks at day 5 post-eclosion, and then decreases to almost to baseline measurements by day 20 (Fig 4.2B, 4.3B). This increase in abdominal size follows the mean weight of HWP fed males, as weight at post-eclosion day 5 is also the largest of the series. That these two metrics of HWP fed male flies follow the same pattern, while

HWO fed males do not, suggests that males are better utilizing the nutrients received from the protein meal for physiological purposes. Knowing how small the need of males is for protein immediately following eclosion, it is speculated given this growth data that

56 males acquire their protein meal within the first five days post-eclosion after which any

physiologic need is met.

Females fed a diet of honey water and protein experience a larger initial

abdominal area growth than do those fed honey water only; and increase which is

supposed to be due in large part to ovarian development (Fig 4.2B, 4.3B). That there is

no significant difference between the abdominal area of flies of either sex when fed

honey water only, also suggests that the female blow fly abdominal area increase is due

to ovary development. Measurement of ovary areas corresponding to the same time

points confirm this. Between eclosion and Day 5, female ovaries, reared on a protein diet,

grow from the pre-vitellogenic germarium, to vitellogenic on Day 3, with mature ovaries

present by day 4 and 5 (Fig. 4.6; Table 6.1).

Age post-eclosion of females fed honey water and protein significantly predicted

ovary area, but surprisingly, it did not predict abdomen area (Fig. 4.4). The ovaries

became fully mature by day five in females fed honey water and protein, and ovaries

became vitellogenic by Day 8 in females fed only honey-water (Fig. 4.4, 4.5). This

pattern which parallels the increase in growth of the female abdominal area. It is

suggested that since protein was present from the time of eclosion in the cages of the

females used for these studies that not only is there a relationship between age-post

eclosion and ovary area, but also between exposure to protein and ovary area. If this is

the case, then the ovary area of female flies associated with carrion may also follow the

same linear pattern as Mohr, et al., recently suggested (2012).

Wing area for all flies fed either diet remained relatively constant across all time points (Fig. 4.2C, 4.3C). Significant differences in wing area between males and females

57 fed either diet only occur toward the latter portion of the time course, with HWO females

peaking at day 20, and HWP females peaking at day 15. Flies younger than 15 days, however, have relatively constant wing area, regardless of diet (Fig. 4.2C, 4.3C).

Growth of wings later in this time course suggests that the increase in area follows the

abdominal area and weight increase of females fed either diet (Fig. 4.4). The area of

male wings remained unchanged for either diet across all of the time points, while female

wings increase over the twenty days of observation, regardless of diet. This suggests that

albeit slightly, wing area increases naturally as female association with a nutritive

medium increases, implying females with better access to nutrients in the larval stages

will have better fitness as adults, and potentially better access to nutrient as an adult.

Both male and female flies frequent carrion for the purposes of feeding, and for

females, oviposition events. Females, however, are almost 25 times more likely to land

on the carrion (personal communication, Jennifer Pechal). Currently, the information

provided by adult flies associating with the carrion is being underutilized by forensic

entomologists. Applying the relationship between ovary area or wing area and time of

protein exposure to female flies found on or around a decomposing medium can provide

information that can strengthen the estimation of the post-mortem interval.

58 FIGURE 4.1: DIAGRAM OF WING AREA MEASUREMENT

Figure 4.1 Diagram taken from Whitworth (2006) and Rognes (1991) to show the R4+5 vein extension and the R1 vein insertion, which were used as points for measurement of wing area.

59 FIGURE 4.2: MORPHOMETRIC COMPARRISONS OF MALE AND FEMALE

FLIES FED HONEY-WATEER AND PROTEIN

Figure 4.2 Male (light grey) and female (dark grey) mean weight (A), abdominal area

(B), and wing area(C) of flies fed honey-water and protein, compared by two-way

ANOVA according to sex and age post-eclosion. Stars indicate significant differences according to a Bonferonni post-test after two-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

60 FIGURE 4.3: MORPHOMETRIC COMPARRISONS OF MALE AND FEMALE

FLIES FED HONEY-WATER ONLY

Figure 4.3 Male (light grey) and female (dark grey) mean weight (A), abdominal area

(B), and wing area(C) of flies fed honey-water and protein, compared by two-way

ANOVA according to sex and age post-eclosion. Stars indicate significant differences according to a Bonferonni post-test after two-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

61 FIGURE 4.4: LINEAR REGRESSION MODELS OF WING AND ABDOMINAL

AREA TO AGE OF FLIES FED HONEY-WATER AND PROTEIN

Figure 4.4 Linear Regression models of female (A) and male (B) wing area (open circles) and abdominal area (open squares) and age post-ecloosion. All models were not significant, excepting female wing area (F (1,3) = 23.47, P = 0.0168).

62 FIGURE 4.5: LINEAR REGRESSION MODEL OF WEIGHT MEASUREMENTS

OF MALE AND FEMALE FLIES FED HONEY-WATER AND PROTEIN

Figure 4.5 Linear regression models of the mean weight measurements according to day post-eclosion of female (A) and males (B) flies fed a diet of honey-water and protein.

Only the female model could significantly predict weight according to day (F (1,3) =

13.93, P = 0.0335).

63 FIGURE 4.6: LINEAR REGRESSION MODEL OF OVARY AREA

AND DAY POST-ECLOSION

Figure 4.6 Linear regression model of ovary areea of females fed a diet of honey-water and protein to the age post-eclosion of the fly. (R2 = 0.9627; P < 0.0001)

64 CHAPTER 5

SCANNING ELECTRON MICROGRAPHS OF THE SENSING ORGANS OF

LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Abstract

Using a scanning electron microscope, examination of the external morphology of both male and female Lucilia sericata (Diptera: Calliphoridae) adult blow flies was undertaken. Studies primarily focused on the major sensing organs—the proboscis, the antenna, and the tarsal pads. Previously, the only Calliphorids detailed using scanning electron microscopy have been Chrysomya pinguis (Walker) and Phormia regina

(Meigen), thus given the recent increase both behavioral and basic research surrounding

L. sericata, examination and documentation of these structures is significant.

Introduction

Lucilia sericata is a blow fly of significance in a number of fields which include forensic, medical, and verterinary entomology (Tomberlin, 2011a, b). As a primary colonizer of decomposing remains, research surrounding this blow fly has recentlyfocused on uncovering its genome as well as understanding its resource-centered behavior (Picard, 2012; Sze, 2012; Tarone, 2013). Key to comprehending the sensing and feeding behaviors of L. sericata, is having a clear picture of its sensing organs.

65 Currently, information regarding the structure of these organs in L. sericata, or other related species of Calliphorids, is relatively scarce.

Sukontason, et al. (2007, 2004) and Shanbhag, et al. (1999), have beautifully shown and described both the macro- and micro-structure of the Chyrsomya pinguis and

D. melanogaster sensing organs respectively, including the various types of sensilla and its corresponding composition, which proved large similarities between the two families.

The olfactory sensing organs, or the antenna, have been extensively studied in a number of species of fly. Three macro structures make up the construction of the antenna: the basal conically-shaped scape, the connecting pedicle, and the sensilla-lined flagellum or funiculus, from which the feather-like aristae originate. While not primarily know for its role in gustatory sensing, the segmented tarsi, or legs, boast both chemo- and mechanosensory sensilla (Amrein and Thorne, 2005). Comparatively little is known about the proboscis, beyond the basic structures and the two types of sensilla that cover the oral discs.

The focus of this study is to thoroughly describe and classify the sensing structures of the antenna, the arista, the proboscis, and the tarsi, using previously published definitions and descriptions, as well as micrographs of other Calliphorids and

Drosophilids.

Materials and Methods

Separation of Pupa and Caging of Flies

After the larval cohort pupated, each of 800 pupa were separately placed in 1 oz. cups, capped with breathable/air permeable lids, and placed in a Powers Scientific

66 Incubator (Dot Scientific, Burton, MI), which was maintained at a 40 +/- 2% humidity,

28 C, with a 12hr light-dark cycle. Adult flies were sexed and placed separately

according to sex into Bug Dorms (0.028 m3 volume; BioQuip, Rancho Dominguez, CA), with 12-20 females or 12-20 males placed in each Bug Dorm. Each cage of flies emerged within 12 hours of each other, from the same cohort of pupae. Newly eclosed flies were fed the respective diets within 2 hours of being separated and caged. One cage of each sex of flies was designated for each of the two diets. All cages were then kept in a portable fly enclosure with temperature maintained at 23 +/-3  C, 35 +/- 3% humidity, on a 12:12 hour light:dark cycle. Temperature, humidity, and light cycle culture parameters were monitored using a portable data logger (HOBO data logger, Onset,

Bourne, MA) over the course of the experiments.

Scanning Electron Microscopy

Fly specimens were prepared for SEM according to Bray, et al. (1993). Briefly, samples were passed through a series of acetone washes (30%, 50%, 70%, 90%, and

100%) with the specimen remaining in the specific wash for no less than 24 hours to desiccate the specimen. After the acetone washes, the sample was placed in a 1:1 acetone: HMDS (Hexa Methyl DiSilazane) (EMS cat# 16700) solution overnight, before a 100% HMDS wash for 24 hrs. The specimen in HMDS was then allowed to dry in the hood completely before mounting with carbon tape (cat # 77835) to a sample stub (cat #

75944) and sputter coating with gold at 45 milliamps. Images were taken on a Hitachi

4800 scanning electron microscope.

67 Results and Discussion An exploratory study of the microstructure of the sensing structures of was

undertaken, and with a few exceptions that will be discussed below, findings were

consistent with the descriptions of sensing organs detailed in Sukontason, et al. (2004).

Sensilla are classified according the terminology found in Zacharuk (1985).

Antennal Structures

Scanning electron micrograph of the male (Fig. 5.1) and female (Fig. 5.2)

antennal areas including the omatidia on either side of the aristate antenna. The scape,

pedicel and flagellum, or funiculus, with accompanying arista, are the three main parts of

the antenna (Fig. 5.7A, B). All three sections contain sensilla, the main sensing organs,

which are concentrated on the flagellum. Microtrichia, tiny non-innervated hair-like

projections, also cover all three sections of the antenna.

The scape is located most proximal to the head, and has six longer, more rigid

projections of trichoid sensilla from the dorsal side, called scape bristles (Fig 5.1B).

These projections are long, pointed, and have grooves starting from the base of the

sensilla that laterally rotate and diminish as the organ comes to a very distinct, sharp

point.

The pedicel is characterized by a longitudinal suture, called the antenna seam that traverses the dorsal side of the structure. It is covered by microtrichia as well, and has three main types of sensilla: the shorter, blunted cylindrical coeloconic sensilla; the short but pointed styloconic sensilla,; and the long trichoid sensilla that forms an organ we are

calling the pedicel bristle (Fig. 5.1B, C, Fig. 5.4). Associated with the styloconic sensilla

68 are the bulbouse seta, which true to its name have round projections at toward the end of

the otherwise pointed structure, and are housed in a semi-circle around the main part of

the associated sensilla (Fig 5.1C, 5.4B). The more blunted lost seta are associated with

the coeloconic sensilla in the same manner as the bulbous seta are associate with the

styloconic (Fig 5.1C, 5.4B). The insertion of the funiculus into the pedicel appears

almost seamless as the antennal sensilla are found on all sides of the third antennal

segment (Fig. 5.1C). These sensilla-setae arrangements are specific to the pedicel.

Figure 5.2 shows a tangential view (A) of the whole female head showing the

structure of the antenna, again including the scape bristle, scape, antennal seam, pedicle, arista, flagellum or funiculus, and the sensory pits. Similar to the pedicel, there are three

main types of sensilla associated with the flagellum, Small basiconic sensilla, large

basiconic sensilla, and the coeloconic, with microtrichia intervening and covering the entirety of the main antennal segment (Fig. 5.1C, D). The slightly curve large basiconic

sensilla, are wide at the point of insertion into the funiculus, gradually tapering out to a

curved point. These sensilla have a lighter texture, almost appearing to have a scale

covered exterior, much different than the grooved exterior of the trichoid sensilla (Fig.

5.4C, D). Small basiconic sensilla have the same basic curved structure as the large

basiconic sensilla, but are shorter and thinner, with the same almost scaled appearance

(Fig. 5.4C, D). Coeloconic sensilla are much shorter and wider than the other types of

flagellar sensilla, with slight curve in the center, and a blunted tip (Fig. 5.4C, D).

Increasing magnification of the feather-like arista can be seen in Figure 5.3. The

attachment of the aristal appendage to the flagellum, however, is very distinct, rising

from a socket-like depression (Fig. 5.3D, E). The base of the arista appears to be almost

69 segmented, as the arista protrudes step-wise from the flagellar socket (Fig. 5.3D, E). The aristal projections, unlike sensilla, do not have a technical point of insertion, as they

appear to simply be branches of the larger structure (Fig. 5.3B). Both the main trunk of

the arista and the branches have soft ridges, that appear almost wave-like, that move from

the base of the structure to the tip (Fig 5.3A, B, C).

Similar to previous findings in Microplitis croceipes (Hymenoptera:Braconidae),

Chrysomya megacephala, Chrysomya rufifaces, and Lucilia cuprina, there were no

morphological or quantitiative differences between the Lucilia sericata male and female

antennal structures or sensilla (Ochieng et al. 2000; Sukontason 2004). Findings were

quite similar to, and confirm the hypotheses of, Sukontason et al. with regard to

Calliphoridae and Muscidae (2004). An exception to these findings was the difficulty

associated with discerning the sensory pits in Lucilia sericata. While two on the left

antennal flagellum were located, according to Sukontason who found numerous sensory

pits on the antenna of Lucilia cuprina easily, there should be many more, and the number should be sexually dimorphic, with females having a larger frequency (2004; Amrein

2007). L. sericata as a decomposition volatile odor-profile seeking Dipteran, should have

roughly the same number of sensory pits as other Calliphorids: Phormia regina females

have 11-16 sensory pits, while males have only 9-11 (Dethier 1971). Sadly, more

information on the sensory pits in L. sericata could not be gleaned from this study as

higher magnification micrographs had high background and were not focused properly.

Tarsi

Figure 5.5 shows a scanning electron micrograph of the female foreleg, with an

70 overview (A, B) of the tibia, tarsal segments and pad. The tarsal segments protrude from

the tibia in decreasing sizes of magnitude, ending with the smallest tarsal segment, the

tarsal pad. Similar to the other sensing appendages, the forelegs are covered with the uninnervated hair-like microtrichia and chemosensing sensillar structures. Two to four

especially long grooved and pointed trichoid sensilla protrude from the base of each

tarsal segment (Fig. 5.5A, B). Shorter, sharp contact chemosensilla line the segments

(Fig. 5.5D). The tarsal pad has five smooth hooks that originate in the base of the pad

and extend out and around the dorsal side of the structure. The tarsal pad is as densely

covered as the antenna with sensillar hairs and structures (5.5C)

It is not surprising that the trichoid sensilla compose most of the sensing organs

on the tarsal segments, as they have been suggest to function as contact chemoreceptors

in the centipede Geopphilus longicornus and as mechanoreceptors in both the human bot

fly, Dermatobia hominus, and the ground beetle, Benbidon poperans (Eisenbeis and

Wichard 1987; Fernandes et al. 2004; Merivee et al. 2002). The tarsi are purposed for

both mechano- and chemo-reception, and are not only a vital part of orientation, but of

the taste complex as well (Amrein and Thorne, 2005).

Mouthparts

Figure 5.6 shows the Lucilia sericata mouthparts in detail. A broad view of the

fly head with a fully extended proboscis is seen in figure 5.6A. The major mouthparts are

easily distinguished: the sensilla-lined maxillary palps extending from the conical

membranous rostrum; the smooth separated parts of the labrum; the sensilla and

microtricha-lined haustellum; the smooth labellum; and the mouth opening (mo) on the

distal portion of the oral disk. The rostrum is lined with non-sensing or -innervated small

71 setae on the outer portion, and large setae toward the midline (Fig. 5.6B). The maxillary

palps located on the proximal portion of the rostrum, are lined with gustatory sensilla and microtrichia (Fig. 5.6B). A side-view of the proboscis shows the connection between the

smooth outer shell of the haustellum (hs), and the more fibrous inner labrum, through

which the labrum-epipharynz and labrum-hypopharyx progress (5.6C). The spongy

appearance of the underside of the oral disc is created by the smooth undulations of the

pseudotrachea (Fig 5.6D). The external portion of the labellum appears to be a hard outer shell for the soft oral disc it contains. The labellum is lined with microtichia and three types of sensilla: the large, pointed, and grooved trichoid sensilla through which most of

the sensing occurs; the large, pointed and curved basiconic sensilla; and the short and

stout small basiconic sensilla (5.6D).

Overall, scanning electron micrograph findings are similar to those previous

published, but unique in that they uniquely describe the sensing organs of Lucilia sericata.

72 FIGURE 5.1: SEM OF THE MALE ANTENNA

73 Figure 5.1 (continued) Scanning electron micrograph of the male antenna structures. An overview (A) shows all of the major

features of the male antennal area, including the omatidia (o), scape (s), antennal seam (as), pedicle (p), arista (a), flagellum or

funiculus (f), and the sensory pits (arrow heads). A closer look (B) at the scape (s) and pedicel (p), show a clear view of the ridged and uninnervated scape bristle (st), the microtrichodia (mt) which are also uninnervated, the short, pointed styloconic (sc) sensilla, the

more blunted coeloconic sensilla (cs), and the long trichoid sensilla (tc) that forms an organ we are calling the pedicel bristle. C shows

the seam between the pedicel (p) and the flagellum or funiculus (f), as well as the aristal insertion (ai), the arista (a), and the

microstructure of the pedicel. Associated with the styloconic sensilla (sc), as seen in C, are the bulbouse seta (bs); lost seta (ls) are

associated with the coeloconic sensilla (cc), and the trichoid sensilla (ts) are characterized by a lack of either type of seta. The aristal hairs (ah) branching from the arista (a) are seen in D, alongside the flagellum (f).

74 FIGURE 5.2: SEM OF THE FEMALE ANTENNA

75 Figure 5.2 (continued) Scanning electron microscopy images of the female antenna, each showing progressive magnification. A tangential view (A) of the whole female head showing the ultrastructure of the antenna, including the scape bristle (st), scape (s), antennal seam (as), pedicle (p), arista (a), flagellum or funiculus (f), and the sensory pits (sp). B shows a higher magnification of the flagellum and the arista. Small basiconic sensilla (sb), large basiconic sensilla (lb), and microtrichia (mt) are shown at increasing magnification (C, D).

76 FIGURE 5.3: SEM OF FEMALE ARISTA

77 Figure 5.3 (continued) Scanning electron micrograph that shows increasing magnifications of the female arista: the ultrastructure of the middle section of the arista (A), the microstructure of the attachment of one of the aristal hairs (B), and a closer look at the attachment of the aristal hairs showing ridged nanostructure (C). The insertion of the arista (D) shows the heavy microstructure of the flagellum in comparison the smooth structure of the arista (E).

78 FIGURE 5.4: SEM OF FEMALE PEDICEL

79 Figure 5.4 (continued) Scanning electron micrograph of the L. sericata female pedicel. Vertical overview of the pedicel (A) showing the small hair-like microtrichodia (mt), the small sensory hair-lined sensory pit (sp), a grouping od peg-like styloconic sensilla (sc), the long angular trichodia sensilla (ts), the ridged scape bristles (st), the scape (s), and an aristal projection (a). A grouping (B) of curved large basiconic (lb) sensilla, small basiconic (sb) sensilla, the ridged styloconic sensilla (ss), and a shorter, blunted coeloconic sensilla (cs). A closer view (C) of the large basiconic (lb) and small basiconic (sb) sensillar structures on the flagellum. Blunted coeloconic sensilla (cs) and a small basiconic (sb) sensilla also on the flagellum (D).

80 FIGURE 5.5: SEM OF FEMALE TARSI

81 Figure 5.5 (continued) Scanning electron micrograph of the female tarsi, showing an overview (A, B) of the tarsal segments (tm) and pad (tp). A closer view (C) of the nanostructure of the tarsal pad, and of the tarsal segments (D), showing the pointed, grooved contact chemosensilla (ccs) among the smaller sensillar hairs.

82 FIGURE 5.6: SEM OF THE BLOW FLY MOUTHPARTS

83 Figure 5.6 (continued) Scanning electron microscopy of the Lucilia sericata mouthparts. A shows a broad view of the flye head

including the major mouthparts: the maxillary palps (mp), the smooth separated parts of the labrum (lb), the sensilla and microtricha- lined haustellum (hs), the conical membranous rostrum (rt), the smooth labellum (ll), and the mouth opening (mo) on the distal portion of the oral disk (od). The rostrum (rt) is lined with both small setae (ss) on the more outer portion, and large setae (ls) toward the midline (B). The maxillary palps (mp), located on the proximal portion of the rostrum (rt), are lined with gustatory sensilla (gs) and microtrichia (mt) (B). A side-view of the proboscis (C) shows the connection between the haustellum (hs), and the labrum (lb) through which the labrum-epipharynz and labrum-hypopharyx progress. The pseudotrachea (pt) create the spongy appearance of the distal end of the oral disk (od) (D). The external portion of the labellum (ll) is lined with microtichia (mt) and three types of sensilla, the large trichoid sensilla (lt), the large basiconic sensilla (lb), and the small basiconic (sb) sensilla (D).

84 CHAPTER 6

THE EFFECT OF AGE, SEX, AND DIET ON THE GUSTATORY SENSING

CAPABILITIES OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Submitted to the Journal of Insect Behavior

Introduction

The blow fly, Lucilia sericata, is of primary forensic, medical, and veterinary importance as an initial colonizer of decaying organic material. As well, L. sericata has been the focus of recent behavioral studies, due to its amazing and natural ability to sense and respond to decomposition (Tomberlin, et al., 2012; Frederickx, et al., 2012). While indigenous in moist, warm climates, such as the islands of the South Pacific, it has spread and colonized pandemically and is now found from the northern temperate zones of

Northern Europe and North America, to the warm, humid climes of South America and the Pacific Islands (Byrd and Allen, 2001; Greenberg, 1971). Given the large body of recent research on this species, specifically its previously unknown genetic make-up and food-orienting behaviors (Picard, et al., 2013; Sze, et al., 2012), it is easy to see that this organism very well could emerge as a model organism for Calliphorids.

While differences do exist, as a Dipteran, this particular organism shares a number of traits and characteristics with the order’s model organism, Drosophila

85 melanogaster, which include the structure and function of sensing organs (Amrein and

Thorne, 2005).

Blow flies are just one of many Dipterans whose tarsi are lined with contact chemosensilla, a classification of sensing organ which allows the organism to essentially

“taste” a substance before extending its proboscis for the purpose of feeding (Amrein and

Thorne, 2005; Geisert and Altner, 1974). This behavior is referred to as the proboscis extension reflex, or PER, and facilitates an understanding of the association between an insect’s need for nutrients/nutrition and an externally exhibited behavior (Gelperin, 1966,

1972; Getting, 1971; Getting and Steinhardt, 1972; Dethier, 1954).

The proboscis extension reflex assay has been used in a variety of species to determine taste preference (Ebbs and Amrein, 2007; Slone et al, 2007). When this reflex is studied in the laboratory setting, the fly is immobilized in a prone position to allow the treatment to contact the tarsi, after which the proboscis may extend in response to the challenge (Amakawa, 2001; Shiraiwa, 2007). This reflex, while utilized extensively in experiments using Drosophila, has only been used in a few studies on blow flies

(Amakawa, 2001; Tully and Hirsch, 1983). Dethier, who is known for his comprehensive work on blow fly physiology, sensing, and diet, discovered a connection between the reception of sensory information from the chemosensilla on the tarsi and the motor reflex elicited in the extensor muscle of the proboscis (Gelperin, 1966; Getting,

1971; Getting and Steinhardt, 1972; Amakawa, 2001). Getting and colleagues (1971) studied the effect of sugar on the proboscis response through a combination of observational and electrophysiological experiments, but did not carry their work one step further to look at the influence of nutrient consumption (diet) on this reflex.

86 Amakawa, through the process of injecting Phormia regina (another forensically important blow fly) with a sucrose solution, found that a raised hemolymph sugar level significantly reduced the gustatory responsive to sugars (2001). From these data, from un-quantified observation of blow flies either ovipositing or feeding on a complex protein source, and from recent data that suggests the importance of a nutrient enriched diet for L. sericata (Blystone and Hansen, 2014), we hypothesize that food consumption could affect the gustatory response of the blow fly.

Typically the PER of both blow flies and fruit flies has only been tested with sugar solutions, despite a Calliphorid’s marked attraction to decomposing material as a food and oviposition source (Huntington and Higley, 2010). A large body of literature also exists that points to protein as a physiological requirement for female blow flies, since without protein in the diet females will be unable to complete vitellogenesis, achieve sexual maturity, or produce the sex pheromones necessary for mating (Browne,

1993; Mullins and Gerry, 2006; Qin, 1995). Until Toshima and Tanimura (2012) showed that the Drosophila response to amino acids was dependent on the fly’s nutritional state, many leading researchers in the field believed this response to be an impossibility.

Early experimentation revealed that blow flies do exhibit a PER to amino acids, but in a sexually divergent manner, which was further examined as the age of the flies increased. The amino acids tested were narrowed to the six most closely related to volatile organic compounds released early in the decay process: cadaverine, the most widely decomposition-associated volatile, is produced by the decarboxylation of lysine; putrescine and spermine, also known to be released by carrion, are closely related to the breakdown of methionine; tryptophan shares the same aromatic ring as indole which is

87 used in blow fly-specific traps; tyrosine and phenylalanine were include for their

aromatic nature (Statheropoulous, et al., 2005; Vass, et al., 2008; Vass, 2012).

Additionally the flies were fed two different diets and then tested against the same

amino acids, under the same conditions. The results were fascinating, with only females

responding significantly to the challenged amino acids, and the addition of a complex

protein source to the diet producing a marked decrease in even the female’s response.

This decrease in response follows closely the ovary development and sexual maturation

of females, indicating that achieving sexual maturity results in a decreased need for

protein in the diet. These findings strongly suggest a link between the physiology as

related to sexual development, and feeding behavior of the Lucilia sericata females, which could help elucidate the time line of adult blow fly association with carrion.

Materials and Methods

Flies

Lucilia sericata pupae were received as a gift from Dr. Aaron Tarone’s stock of

fly eggs originally removed from a carcass in southern California in 2006. They were

reared and bred using honey-water and liver, before being subjected to experimentation.

Separation of Pupae and Caging of Flies

After the larvae pupated, each of 400 pupae were separately placed in 1 oz cups,

capped, and placed in an incubator (Powers Scientific, Dot Scientific, Burton, MI) which

was kept at 40 +/- 2% humidity, 28 C, on a 12hr light:dark cycle. Within 12 hours of

88 eclosion, adult flies were classified according to sex and placed into cages (Bug Dorms,

0.028 m3 volume; BioQuip, Rancho Dominguez, CA) with 40-50 males or females

housed in each cage.

Fly Diet

Immediately upon caging, one cage of all males and one cage of all female flies

were fed ad libitum honey-water (HWO) according to Blystone and Hansen (in review).

Separate cages of all males or all females were fed both ad libitum honey-water and less than 5 grams of liver from the moment of caging (HWL). Water was also provided ad libitum. Honey-water and liver treatments were replaced every other day to allow the flies access to fresh food.

Amino Acid Solutions

All twenty essential amino acids were purchased from Sigma (Sigma-Aldrich

Corp. St. Louis, MO, USA) and used to create 100mM solutions for testing. 100 mM solutions were made and aliquoted into epiTubes (Eppendorf, Hauppauge, NY) and frozen at -20 C until the day of testing to prevent amino acid degradation. All amino acids were solubilized fairly easily, with the exception of Phenylalanine, Tyrosine, and

Tryptophan, which after mild application of heat went into solution. 40-60 minutes before testing amino acid solution aliquots were removed from the freezer and allowed to thaw at room temperature.

89 Behavioral Assays

Proboscis extension reflex (PER) assays were performed according to Wang, et al.

(2004), Shiraiwa and Carlson (2007), and Slone, et al. (2007). Briefly, (with the

exception of Day 1 flies which were tested within 12 hours of emergence and never

caged) at 24 hours prior to testing, 5 male and 5 female flies were removed from cages

and placed in one ounce cups with a water-saturated strip of paper towel, capped, and

placed aside. One hour before testing, cupped flies were anesthetized on ice for 7-10

minutes, then immobilized in a prone position by the taping the wings to a glass

microscope slide, so that the tarsi were exposed to the experimenter. After a 15-20

minute period during which the flies came out of anesthesia and became accustomed to

their new orientation, the flies were then given water dispensed from a micro-pipette tip

until satiated. The flies were then challenged with 100 mM amino acid or sugar solutions

applied to the tarsi in droplet form at the end of a micro-pipette tip. Each fly was challenged with a treatment for three seconds. The largest response that occurred during the challenge was recorded. After each challenge, water was applied to the tarsi to confirm a non-response. If the fly responded to the challenge of water, the fly would be allowed to drink until satiated (extension ceased), and then challenge with the treatment solution would begin again. Twenty to thirty seconds were allowed to expire between

each of the three challenges and the subsequent water test, with another twenty to thrity

second interval between the water and the following challenge. Responses were graded

on a 1-7 scale, as described in Figure 6.1. Amino acid solutions were administered in a

blind manner to eliminate any potential for experimenter bias.

90 Ovary Measurement and Staging

Three to five females were removed from the HWL fed cage each day for 10 days, and preserved in 70% ethanol. Ovaries and ovarioles were dissected and measured according to Adams and Hint (1967), and Huntington (2010). Briefly, whole ovary area was measured using an Olympus D-40 microscope (Olympus America, Center Valley,

PA, USA), a scope-mounted camera (Canon, Melville, NY), and Image J (NIH, free access) to take whole ovary area measurements. The ovaries were then staged according to Table 6.1, where 1 is the previtellogenic germarium, 2 is the beginning of vitellogenesis, and 3 is the ovary containing mature eggs (Adams and Hintz, 1967;

Huntington, et al., 2010).

Statistical Analysis

The three responses recorded for each solution test per individual fly for each challenge were recorded and averaged; all male or female averaged responses for each testing period were collated and subjected to statistical analysis. Two-way ANOVA was used to determine whether the differences in response of the sexes was statistically significant, as well as if interaction exists between the sex and treatment, and the day and treatment. Bonferroni post-tests were then performed to establish statistical significance of any differences. Correlation and linear regression models were used to test the linearity of the relationship between ovary size and day post-eclosion. GraphPad Prism version 5.0 (GraphPad Software, San Diego CA, USA, www.graphpad.com) was used to conduct all statistical analyses.

91 Results

Proboscis Extension Reflex Assay

The proboscis extension reflex (PER) of Lucilia sericata was graded on a scale of

1 to 7, with a rating of 7 assigned to full proboscis extension, and 0 assigned to no

response (Fig. 6.1).

PER of Honey-Water-Only (HWO)-fed Flies to Amino Acids and Sugars

Initial proboscis extension reflex assays (PER) were performed on flies of age 5-7

days post-eclosion, which revealed that unlike Drosophila melanogaster (Amrein and

Thorne, 2005), L. sericata does respond when challenged with 100 mM amino acid

solutions (Fig. 6.2). Not only did they respond, there was a marked sexual dimorphism in

the response, with only females exhibiting any PER. This initial data set was used to

select the appropriate age range span for the next set of experiments.

PER was then performed on male and female flies fed only honey-water (HWO),

aged 1, 5, 7, and 9 days post-eclosion. Flies were offered solutions of six decomposition-

related amino acids (100 mM each; tryptophan , tyrosine, proline, methionine,

phenylalanine, and lysine) and two sugar sources (100 mM; sucrose and honey-water),

with sex and age differential responses exhibited to the amino acids of interest (Fig. 6.3).

Days 5 and 7 are clearly important time points for females to acquire a protein source,

because on these two days the female flies exhibit not only preference, but also the

greatest difference in PER response, while the male PER response was consistently low

or non-existent (Fig. 6.3). The Day 1 female PER to the six amino acids shows no

difference from the low Day 1 male response, excepting Tyrosine and Proline. The male

92 PER remains consistently low for all time points. At Days 5 and 7, however, the female

response to Trp (pDay5<0.01, pDay7<0.001), Pro (pDay5, 7<0.001), and Met (pDay5, 7<0.001)

was significantly larger than the male response (Fig. 6.3) according to a two-way

ANOVA with Bonferroni post-test (Supp. Table A3.1). The female response to Lys

(p<0.001) and Phe (p<0.001), according to a two-way ANOVA with Bonferroni post-test,

was significantly larger than the male response only on Day 7. The treatment explained a

significant source of the variation according to two-way ANOVA on all days between

male and female flies fed a diet of HWO (Day 1: F(1,7)= 3.875, MS = 130.9, p < 0.0001;

Day 5: F(1, 7) = 84.99, MS = 91.75, p < 0.0001; Day 7: F(1, 7) = 87.22, MS = 29.97, p <

0.0001; Day 9: F(1, 7) = 28.04, MS = 24.42, p < 0.0001; Supp. Table A3.1).

There was no statistically significant difference between the male and female PER

response to HWO on all days, with the Day 1 PER pushing the limits of the grading scale

for both sexes, plateauing through Day 7, then sharply and significantly decreasing on

Day 9 (Fig. 6.3; Supp. Table A3.1). Significant differences do exist between the male

and female PER response to sucrose, however, for flies fed only a diet of HWO, on Days

1 (p<0.001), Day 5 (p<0.001), and Day 9 (p<0.001), with the female responding

significantly lower than the male response.

Alteration in Diet Changes the PER to Amino Acids and Sugars

Male and female flies fed a diet of honey-water and bovine liver ad libitum

(HWL) showed no difference over the course of seven days (aged 1-7 days post-eclosion) in proboscis extension reflex (PER) to 100 mM solutions of the tested amino acid treatments, which included Lys, Trp, Tyr, Phe, Met, and Pro, or the two sugar solutions

(Fig. 6.4). Day 9 data are omitted, since for all three trials all the males died prior to, or

93 during PER assays. Treatment accounts for close to 100% of the variation in response,

given the large amount of variation between the PER to the sugar sources and amino acid

solutions (Day 1: F(1, 7) = 157.1, MS = 67.54, p < 0.0001; Day 5: F(1, 7) = 77.71, MS =

69.51, p < 0.0001; Day 9: F(1, 7) = 74.66, MS = 53.31, p < 0.0001); Supp Table A3.2).

When the PER of males fed HWO is compared to those fed HWL, again the only

significant difference in response is associated with the sugar source, while the response

to the amino acid treatments remains almost nonexistent across both diets (Fig. 6.3, 6.4).

The female PER to tested amino acids, was considerably altered by the addition of

protein to the diet (Fig. 6.5). Females fed HWO had a significantly larger PER than those

fed honey-water and liver, with the interaction between the diet and amino acid

(treatment) accounting for over 60% of the variation in response according to two-way

ANOVA (Day 1: F(1,7)= 101.9, MS = 79.35, p < 0.0001; Day 5: F(1, 7) = 40.99, MS =

56.29 p < 0.0001; Day 7: F(1, 7) = 108.0, MS = 39.95, p < 0.0001; Day 9: F(1, 7) = 28.32,

MS = 13.25, p < 0.0001; Supp. Table A3.3). At days 1 and 9, PER of female flies fed a protein-supplemented diet (HWL)was not significantly different from those with a HWO diet when challenged with amino acid solutions (Fig. 6.5). The PER to the sugar solutions, however, was significantly different on days 7 and 9, with the PER of the HWL fed flies

being higher than those fed the protein-supplemented diet (HW: pDay 7 < 0.01, pDay 9 <

0.05; Suc: pDay 7 < 0.001, pDay 9 < 0.001; Supp. Table 3). Responses to the sugar sources varied, with those fed the HWL diet having a lower response to honey-water, but a higher response to sucrose (Figs. 6.4, 6.5). On Day 5, female flies fed a diet of HWO, responded significantly higher to all amino acid treatments, except for Phe and Lys (pall < 0.05 ),

than did female flies fed a HWL diet. The response of HWO-fed female flies to all

94 amino acid solutions, except Tyr, was significantly higher than the response of flies fed

HWO on Day 7 (pall < 0.001) (Supp. Table A3.3).

Ovary Area and Stage

Ovaries and ovarioles were dissected and measured according to Adams and

Hintz (1967) and Huntington and Higley (2010), and staged as described in Table 1,

where Stage 1 is the pre-vitellogenic germarium, Stage 2 is the beginning of

vitellogenesis, and Stage 3 is the ovary containing mature eggs (Table 6.1).

The age post-eclosion and ovary area (using whole area measurements taken in

Image J) of female flies fed HWL co-vary significantly using a Pearson correlation (r =

0.9812, P < 0.0001). The relationship between these two variables is clearly linear.

Using a linear regression model, the age post-eclosion of female flies fed HWL is a

significant predictor of mean ovary area (R2 = 0.9627, b = (1/3.812), F (1,6) = 154.7, P <

0.0001) (Fig. 6.6).

Female L. sericata blow flies fed a diet of HWL show a marked increase in ovarian size and ovary stage after Day 3 post-eclosion, with a 24-48 hour interval before ovaries become fully mature (Fig. 6.7).

Adult Age and Feeding Behavior

Figure 8 shows a graphical illustration of what we, given the data from this study, believe to be the time line in both the male and female adult life cycle, taking into account feeding behavior as assessed by exhibition of a gustatory response, and physiological development of female ovaries.

Discussion

To help answer the question as to whether or not the female Lucilia sericata flies

have a greater PER response to amino acids than to the males due to their increased need

for protein, the diet was altered, adding an ad libitum source of complex protein. It was

projected that the female PER to amino acids would decrease with the alteration in diet.

Difference in Response to Amino Acids and Sugars

When challenged with essential amino acid solutions, Lucilia sericata exhibits a

proboscis extension reflex (PER), indicating that the organism is attracted to, or

physiologically requires the treatment. Given the fact that blow flies are known

colonizers and consumers of decaying remains, we expected a PER response from both

sexes for the six tested amino acid treatments. While differences in response to the

amino acids were predicted, divergence in the PER to the two sugar solutions was

surprising given that sugar is a common dietary requirement. Of particular interest,

though, is the sexually dimorphic response to the tested amino acids, with the females

exhibiting significantly larger responses than the males on Days 5 and 7 post-eclosion.

Females require a complex protein source to complete vitellogenesis, achieve sexual maturity, and to produce pheromones necessary to attract a mate (Browne, 1993; Mullins and Gerry, 2006; Qin, 1995). It is thus anticipated that the females would respond when challenged with the selected decomposition-related amino acid solutions.

Blow fly colonies reared under laboratory conditions are typically introduced to a complex protein source between Days 7 and 14 post-eclosion; however, our results

96 suggest that the female interest in protein can begin as early as Day 5. The magnitude of the female PER responses between Day 1 and 5 suggest that during those few days they undergo a physiological change in preparation for vitellogenesis, as evidenced by ovary staging results that show females with fully developed ovarioles by Day 5 when fed protein (liver) from Day 1 post-eclosion (Fig. 6.3, 6.6, 6.7). That the attraction to amino acid solutions declines around Day 9 implies that for females, Days 5-9 are of primary importance for protein acquisition (Fig. 6.3).

Knowing that the volatiles attractive to blow flies are by-products of decomposition, the amino acids tested in this study were selected based on relationship to

VOCs released by decomposition: cadaverine is produced by the decarboxylation of lysine; putrescine and spermine are closely related to the breakdown of methionine; tryptophan and indole share the same aromatic ring structure (Statheropoulous, et al.,

2005; Vass, et al., 2008; Vass, 2012). Not surprising, females gave the largest PER to these three amino acid solutions, while the males were markedly non-responsive (Fig.

6.3). That the males do not significantly respond to these or any other amino acids suggests that males may not have a physiological need for consumption of a complex protein source during the early stage of their life cycle (Fig. 6.3, 6.4). Previous research on Phormia regina has revealed that male blow flies require small amounts of protein within a few hours post-eclosion for somatic growth (Roberts and Kitching, 1974); males of Lucilia cuprina, a closely related sister species to L. sericata, however, did not exhibit this same requirement (Roberts and Kitching, 1974). Knowing that both male and female

L. sericata can detect decomposition-related VOCs, as evidenced by the fact that both sexes can be found near carrion and according to electroantennogram studies (Frederickx,

97 et al., 2012), but that the males typically do not exhibit a PER to the tested amino acids

implies that the males are only attracted to decaying remains for the purposes of mating, i.e. finding the females. Further experimentation in this area is necessary to more fully understand this sexual divergence in behavior. Olfaction and gustation are closely linked in Dipterans (Amrein and Thorne, 2005), thus it is reasonable to hypothesize that this sexually dimorphic gustatory response could also translate into differences in the olfactory response of these flies to decomposition-related volatiles.

Alteration in Diet Changes the PER to Amino Acids and Sugars

Given that flies reared on a diet without protein exhibit a PER to amino acids, we then asked if adding that component to the diet would alter the behavioral response. It stands to reason that if L. sericata females are exhibiting a PER due to a physiological need to consume the treatment/protein, then once that need is fulfilled, the behavior should cease. This hypothesis was confirmed: after the diet was supplemented with

bovine liver as a source of complex protein, the female PER was reduced to that of the

typical male response (i.e. negligible), with there being no significant difference on post- eclosion Days 1, 5, or 7 between the two sexes to the decomposition-related amino acids

(Fig. 6.4). When females fed a diet supplemented with protein (HWP) were compared to females fed honey-water only (HWO), again Days 5 and 7 emerge as key time points (Fig.

6.5). When compared to the ovary staging of females given the same diets, these results confirm that as the ovaries undergo vitellogenesis, the PER to amino acids decreases, suggesting a link between egg development and food-seeking behavior (Figs. 6.5, 6.6, 6.7,

6.8). These data confirm that L. sericata females do require a dietary protein source for physiological reasons, but suggests that once that need is met, whether in the lab from a

98 supplement of bovine liver, or in the field from a decomposing organism, visiting such a

protein source is simply for the purposes of either mating or oviposition behavior, but not

for feeding (Fig. 6.5, 6.7, 6.8). This idea is supported by our data showing that males,

when fed the two diets described here, exhibited no significant change in PER (Fig. 6.3,

6.4). In addition, no previous research has shown that males have physiological requirement for a diet supplemented with a complex protein source for long periods of time.

The male flies in this study showed consistently low or non-existent PER to any amino acids, but can and do readily respond to sugar sources, regardless of diet (Fig. 6.3,

6.4). This difference in response to sugar solutions sheds new light on the question of whether reflexive and sensing behavior is first initiated at the level of the odorant binding protein or at the higher level of the antennal lobe ganglion. The males had the highest

(relatively speaking) responses to serine, arginine, and asparagine (Fig. 6.2), suggesting that the ability to exhibit a PER to amino acids is present, but that when those signals are processed in the ganglion, and a physiologic need is not present, the PER is diminished.

While these questions are beyond the scope of this paper, this information clearly describes the need for further work in this area.

Ovary Stage and Ovary Area

Females fed a diet of HWL experience a larger initial abdominal area expansion than do those fed HWO, an increase which is purported to be due in large part to ovarian development (Fig. 6.6, 6.7). That there is no significant difference between the abdominal area of flies of either sex when fed HWO, also suggesting that abdominal area increase is due to ovary development (data not shown). Ovary areas corresponding to the

99 same time points confirm that between eclosion and Day 5, ovaries of females fed protein

grow from the pre-vitellogenic germarium, to vitellogenic on Day 3, with mature ovaries

present by Days 4 and 5 (Table 6.1; Figs. 6.6, 6.7).

Age post-eclosion of females fed HWL was a significant predictor of ovary area.

The ovaries became fully mature by Day 5 in females fed HWL, and ovaries became

vitellogenic by Day 8 in females fed HWO (Figs. 6.6, 6.7). This pattern precedes the

PER of female flies to decomposition amino acids by just a few days for both diets: the

PER of flies fed HWO to amino acids declines by Day 9, with the ovaries increasing in

stage from pre-vitelloginic to vitellogenic by Day 9; the PER of female flies fed HWL to

amino acids is significantly decreased from Day 1, with the ovaries reaching full

maturation by Day 3 (Figs. 6.5, 6.7). We suggest that since protein was present from the time of eclosion in the cages of the females used for these studies there is not only a

relationship between age-post eclosion and ovary area, but also between exposure to

protein and ovary area (Fig. 6.8). If this is the case, then the ovary area of female flies

associated with carrion in the field may follow a similar pattern.

These findings indicate that the diet, age, and sex of an adult blow fly all affect

gustatory sensing behaviors (Fig. 6.8). When fed a diet supplemented with protein from

post-eclosion Day 1, female blow flies will achieve fully developed ovaries by Day 5,

while simultaneously decreasing the proboscis response to decomposition-related amino

acids. This suggests that female blow flies, in the absence of a protein meal, will alter

their behavior to actively respond to odors associated with a protein source; however, as

the ovaries mature, the interest in protein-associated nutrients declines as does the

physiological need. Understanding the sexual dimorphism in proboscis extension reflex

100 to amino acids is not only an important finding that will affect the fields of forensic, medical, and veterinary entomology, but also allows insight into Dipteran sensing.

101

FIGURE 6.1: PROBOSCIS EXTENSION STAGES

Figure 6.1 Scanning electron micrograph of the ffully extended Lucilia sericata proboscis, showing the scale used in this study to stage the proboscis extension reflex; “0” is a non- response, with no extension, and “7” is a maximal response, with full extension of the proboscis and labrum.

102 FIGURE 6.2: DAY 5-7 PROBOSCIS EXTENSION REFLEX TO AMINO ACIDS AND SUGARS

Figure 6.2 Mean (±SE) Proboscis Extension Reflex to 100M solutions of all twenty essential amino acids (arginine, proline, methionine, leucine, serine, glutamic acid, asparagine, glutamine, alanine, valine, tyrosine, tryptophan, isoleucine, aspartic acid, threonine, glycine, cysteine, histidine, phenylalanine, and lysine) and four sugar sources

(honey-water, and  of glucose, fructose and sucrose) of both males and females aged five to seven days post-eclosion.

103 FIGURE 6.3: MEAN PROBOSCIS EXTENSION OF HWO fed FLIES

Figure 6.3 Male (circles and dotted lines) plotted with female (squares and solid lines) mean (±SE) proboscis extension reflex across days 1, 5, 7, and 9 to honey-water (A), sucrose

(B), tryptophan (C), tyrosine (D), proline (E), methionine (F), phenylalanine (G), and lysine

(H). Stars indicate statistical significance between rresponses of the sexes to amino acids according to day, established by Bonferroni post-tessts after two-way ANOVA .

104 FIGURE 6.4: MEAN PROBOSCIS EXTTENSION OF HWL fed FLIES

Figure 6.4 The mean (±SE) proboscis extension reflex across days 1, 5, 7, and 9 of males

(filled circles, solid lines) plotted with females fed a protein-supplemented diet (open circles, dotted lines) to honey-water (A), sucrose (B), tryptophan (C), tyrosine (D), proline (E), methionine (F), phenylalanine (G), and lysine (H). Stars indicaatte statistical significance according to Bonferroni post-tests after two-way ANOVA.

105 FIGURE 6.5: COMPARISON OF HWO- AND HWL-fed FEMALE PER

Figure 6.5 The mean (±SE) proboscis extension reflex across days 1, 5, 7, and 9 of females fed honey-water only (filled circles, solid lines) plotted with female fed a protein- supplemented diet (open circles, dotted lines) to honey-water (A), sucrose (B), tryptophan

(C), tyrosine (D), proline (E), methionine (F), phenynylalanine (G), and lysine (H). Stars indicate statistical significance according to Bonferroni post-tests after two-way ANOVA.

106

FIGURE 6.6: LINEAR REGRESSION MODEL OF OVARY AREA

AND DAY POST-ECLOSION

Figure 6.6 Linear regression model of ovary areea of females fed a diet of honey-water and protein to the age post-eclosion of the fly. (R2 = 0.9627; P < 0.0001)

107

FIGURE 6.7: COMPARISON OF OVARY STAGES OF FEMALE FLIES FED DIFFERENT DIETS

Figure 6.7 Comparison of the ovary stage according to Adams and Mulla (1967), and

Huntington (2007) (where 1 is the previtellogenic germarium, 2 is the beginning of vitellogenesis, and 3 is the ovary with mature eggs) using ovary areas from this study, of honey-water and liver (HWL) fed female flies (dashed line) and honey-water only

(HWO) fed female flies (solid line).

108

FIGURE 6.8 PROPOSED LUCILIA SERICATA ADULT TIME LINE

Pre-Vitellogenic

Vitellogenic

Mature Ovary Status

Oviposition

Likely to Occur

Female Response to Gustatory Cues

Male Responses Sensing Gustatory Gustatory Cues

Occurrence of Feeding Behavior

Day/Age Post-Eclosion 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 6.8 Proposed time line of Lucilia sericata adult feeding behavior, sensing responses, and ovarian status at 27C.

109

TABLE 6.1: OVARY AND OVARIOLE STAGES

Ovarian Stage Ovary Area Ovariole Stage Ovariole Shape Ovariole Size Stage 1: Previtellogenic Germarium < 2.00 μm2 Stage 1 Pear-shaped 50 x 27 μm Stage 2 Spherical 50 x 38 μm Stage 3 Elongated 100 μm Stage 2: Beginning of Vitellogenesis 2.01 – 2.49 μm2 Stage 4 Elongated 171 μm Stage 5 Elongated 254 μm Stage 6 Elongated 409 μm Stage 7 Elongated 544 μm Stage 3: Ovaries with Mature Eggs > 2.50 μm2 Stage 8 Elongated 705 μm Stage 9 Elongated 772 μm Stage 10 Elongated 910 μm

Table 6.1 Table of ovary and ovariole stages used to classify ovaries, based on ovary areas found in this work, as well as Adams and Mulla (1967), and Huntington (2007).

110 CHAPTER 7

THE EFFECT OF AGE, SEX, AND DIET ON THE OLFACTORY SENSING

CAPABILITIES OF LUCILIA SERICATA (DIPTERA: CALLIPHORIDAE)

Best known for its role as a forensically important primary colonizer of decomposing remains, Lucilia sericata larva helps establish a victim’s post-mortem interval (PMI), or time since death, in many criminal investigations. As the organism

progresses through the egg, three larval stages, and a pupal form, investigators can

estimate the time since the adults oviposited eggs in the victim, and thus the period of

elemental exposure the victim has experienced. Lucilia sericata, a keen biosensor for the

volatile suite emanating from carrion, arrives at the body within minutes. It has been assumed that the first flies to the carrion arrive with the sole purpose of oviposition, but this assumption has recently been challenged by discoveries of the feeding behavior of L.

sericata (Blystone and Hansen, 2014) and Cochlimya homnivorax (Chaudhury, et al.

2010).

Before a blow fly can locate and feed on a decomposing medium, it first must discern the carrion-specific volatile suite from other environmental scents. Nine major

111 classes of volatiles are released during the various stages of decay by carrion: acids,

esters, ketones, aldehydes, alcohols, nitrogenous, halogen and sulfurous compounds, as well as cyclic hydrocarbons (Dekeirsschieter, et al., 2009; Statheropoulos, et al., 2005;

2011). Sulfur-rich compounds, associated with all stages of decomposing carrion and

thought to be generated and released by enterobacteria, initiate the activation of attraction

behaviors such as, orientation to, flight toward, and landing on the carrion (Ashworth, et

al., 1994; Kasper, et al., 2012; Dekeirsschieter, et al., 2009; Frederickx, et al., 2012a, b).

Ammonia-rich compounds, released during active decay upon breakdown of protein and

amino acids, are more attractive to gravid females, and act as an oviposition cue

(Ashworth, et al., 1994; Kasper, et al., 2010; Dekeirsschieter, et al., 2009).

This association between blow flies and volatiles as behavioral cues was used in a

legal context in the 2008 Casey Anthony trial during which an alleged cadaveric volatile

suite was collected and submitted into evidence alongside forensic entomological

specimens. A flurry of research has recently focused on this connection between

colonizers and the “smell of death” (Kasper, et al., 2010). Frederickx, et al. (2012b)

confirmed that a number of these decomposition-related volatiles are attractive to and can

elicit neuronal responses in blow flies, specifically Lucilia sericata, as assessed by

electroantennography (EAG).

The electroantennogram (EAG) is a bioassay used to determine and measure

volatile detection of the insect olfactory system, specifically the antenna, and has been

extensively used in various branches of the field of entomology (Kendra, et al., 2005;

2009; Syed, et al., 2010; Frederickx, et al., 2012). Experimentation designed to quantify

the antennal response of diverse insects to a number of odorants and volatiles began

112 around 1953 as a measurement of gross ion concentration changes within a neuron.

Schneider (1957) was the first to record voltage fluctuations in the insect antennal structure after exposure to pheromones, and discovered what remains today to be the main principle of EAG: “measured voltage fluctuation is caused by an electrical depolarisations [sic] of many olfactory neurons in the insects’ antenna.”

To measure these voltage fluctuations, an electrode is placed at both the tip and base of the antenna as a volatile is pushed across the cuticle of the olfactory apparatus by a vapor delivery system. These electrodes are then connected to an amplifier that magnifies the incoming signal, reduces the signal to noise ratio, and then transforms the signal to readable output by specialized computer software. After stimulation, the amplitude of the depolarization signal is measured in millivolts (mV) and compared against both a positive control, (a volatile known to elicit a full depolarization of the olfactory nerves), and a negative control (ambient air).

Knowing that both feeding and ovipositing behaviors are closely connected with aromatic cues received and translated by the olfactory system, further examination of the blow fly response was undertaken using electroantennography. In this study we assess how culture diet affects the EAG response of both males and females across daily time points over fourteen days. Our previous research suggests that a diet supplemented with protein fulfills a physiological need and therefore reduces the gustatory response of female flies to decomposition-related amino acid. Quite a bit is known about the gustatory and olfactory systems in those of the Dipteran model organism, the fruit fly,

Drosophila melanogaster. We anticipate that the D. melanogaster and L. sericata gustatory and olfactory systems are similar in both structure and function (Jacquin-Joly

113 and Merlin, 2004). We hypothesized that feeding the blow flies with protein prior to a

challenge with volatiles and using the EAG to determine depolarization response, should

result in EAG responses lower than those fed only a sugar-based diet. While the

responses were varied, we did find this maxim to hold true at certain time points for adult

female L. sericata flies. There was, however, a large divergence in the way males

responded to olfactory stimuli. Using this information, the goal of this study was to

better understand how the olfactory response of both male and female L. sericata blow

flies is affected by ordinary factors such as diet, age, and sex.

Materials and Methods Flies

Lucilia sericata pupa were received as a gift from Aaron Tarone’s stock of fly eggs originally removed from a carcass in southern California in 2006, used in Sze, 2012 and Tarone, et al., 2011, and were raised and bred using honey-water and liver, before being subjected to experimentation.

Separation of Pupa and Caging of Flies

After the larvae pupated, each of 400 pupa were separately placed in 1 oz cups, capped and placed in an incubator (Powers Scientific), which was kept at 40 +/- 2% humidity, 28 C, with a 12hr light:dark cycle. Within 12 hours of emergence, adult flies were classified according to sex and placed into cages (Bug Dorms) with 40-50 males or females in each cage.

114

Fly Diet

Immediately upon caging, one cage of all males and one cage of all female flies

were fed ad libitum honey-water according to Blystone and Hansen (2014). Separate

cages of all males or all females were fed both ad libitum honey-water and approximately

5 grams of liver from the moment of caging. Water was also provided ad libitum.

Honey-water and liver were replaced every other day to allow the flies access to fresh food.

Volatiles

Volatiles were acquired in either liquid or powdered form from Sigma-Aldrich

(Putrescine, P5780; Cadaverine, 33211, Butanoic Acid, B103500, Dimethyl Disulfide,

40221; Phenol, P1037). Compounds were then diluted initially in a weight by volume

(w/v) manner to create a 1:10 dilution, from which decadic dilutions were made according to Syed, et al. (2010). Hydrophilic compounds were diluted using Type I lab water (18 ohms), and hydrophobic compounds were diluted using pure hexane, as an inert liquid. 200μl aliquots were absorbed onto a 5cm by 2cm rectangular strip of filter paper (Fisher Scientific), which was then placed in a headspace vial (Fisher Scientific).

Volatiles were allowed to equilibrate and accumulate in the headspace vials for 30 to 45 minutes at room temperature before testing began. Concentrations were chosen according to Frederickx, et al. (2012).

115

Electroantennography

Electroantennogram responses were recorded with the Syntech® IDAC-4 system

and accompanying EAGPro software (Hilversum, Netherlands) according to methods

previously described by Kendra, et al. (2005; 2009). Briefly, freshly dissected whole

head preparations were mounted using conductive gel (Spectra 360; Parker Laboratories,

Fairfield, NJ, USA) between glass micropipette electrodes filled with 0.1N KCL, and placed under purified, humidified continuous air flow. 200μl aliquots were drawn from the headspace vials using gas-tight syringes (Hamilton Co., Reno, Nevada, USA), and injected into the flowing airstream, which was presented at a distance of 1cm from the antenna. To prevent antennal adaptation, stimuli were separated by 2 minute intervals of purified air. EAG responses were recorded in volts (maximum peak height of depolarization) using the EAG Pro software supplied with the Syntec ® system, with any response to the solvent (pure water or hexane) subtracted from the stimulus response.

Each volatile (stimulus) was presented to each of three to five flies a minimum of three times per samoling point. Male and female flies from each of the two diets were tested every other day, starting on post-eclosion Day 2, for a total of 14 days. Responses were averaged according to stimuli and fly, after which means were collated and subjected to statistical analysis.

Statistical Analysis

Maximum depolarizations for each stimulus for each fly were recorded and averaged. Means for all male or female responses according to stimuli were then collated

116 and subjected to statistical analysis. Two-way ANOVAs were run to determine whether the differences in response of the sexes have statistical significance, as well as what interaction exists between the sex and treatment, and the day and treatment. Bonferonni

post-tests were then performed to establish statistical significance of any differences.

GraphPad Prism version 5.0 (GraphPad Software, San Diego CA, USA,

www.graphpad.com) was used to conduct all statistical analyses.

Results According to the previous proboscis extension reflex assays (PER) (Chapter 6), a

diet of honey-water supplemented with protein, and the fly age post-eclosion affect the

behavioral responses of Lucilia sericata. As well, previous research on all of the tested

volatiles has shown to elicit a measurable response via electroantennogram in L. sericata,

with the exception of phenol (Frederickx, et al., 2012). In our studies flies responded to

all of the tested volatiles. Further studies to examine the effect of diet and age on the

olfactory response, as measured by electroantennogram, were conducted.

Female EAG Response According to Diet

Mean electroantennogram responses to five decomposition-related volatiles

(putrescine, cadaverine, butyric acid, dimethyl disulfide, and phenol), measured

according to maximal depolarization (volts), were compared between female blow flies fed one of two diets—honey-water only, or honey-water supplemented with liver—by

two-way ANOVA. There was a significant interaction between diet and age post-

eclosion for all volatiles except for phenol (FPut(1, 4) = 4.21, Pput = 0.0047; FCad(1, 4) =

9.455, PPut< 0.001; FBA(1, 4)=4.008, PBA= 0.0058; FDMDS(1, 4)= 8.992, PDMDS< 0.0001;

FPhenol(1, 4)= 1.028, PPhenol = 0.4027, Supp. Table A4.1). Age post-eclosion of these

117 female flies significantly effected the response for all volatiles tested (FPut(1, 4) = 3.664,

Pput = 0.0101; FCad(1, 4) = 14.39, PPut< 0.0001; FBA(1, 4)=4.631, PBA= 0.0024; FDMDS(1,

4)= 17.65, PDMDS< 0.0001; FPhenol(1, 4)= 6.094, PPhenol = 0.0005, Supp. Table A4.1, Fig

7.2) With the exception of phenol, there were significant differences in the responses of

females fed different diets at post-eclosion Day 4 to all other volatiles, (PPut < 0.05; PCad,

BA, DMDS < 0.001, Supp. Table A4.1, Fig. 7.1), according to Bonferonni post-tests.

Putrescine and cadaverine also elicited significantly different responses between females

fed various diets on Day 10 (PPut < 0.05; PCad < 0.001, Supp. Table A4.1, Fig. 7.1)

according to Bonferonni post-tests. Interestingly, flies fed a diet supplemented with

protein (HWL), exhibited significantly higher responses to all volatiles, except for phenol,

on Day 4, while flies fed a honey-water only diet had significantly higher responses on

Day 10 to putrescine and cadaverine only.

Two-way ANOVA analysis reveals that the response to volatiles among flies fed

honey-water only was significantly affected by the age post eclosion (F(4, 16) =13.45,

P<0.0001), and volatile tested (F(4, 16) = 3.488, P = 0.0096). The interaction between

day and volatile for female flies fed honey-water only was not significant (F(4, 16) =

1.034, P = 0.04255). The interaction between day (age post-eclosion) and volatile for

honey-water and liver fed female flies was, however, significant (F(6, 24) = 1.867, P =

0.0113). For flies fed a diet supplemented with protein, both the effect of day (F(6, 24) =

11.90, P < 0.0001) and volatile (F(4, 24) = 9.749, P < 0.0001) significantly effected

response.

118 Male EAG Response According to Diet

A two-way ANOVA was also conducted to examine the effect of diet and day

(age post-eclosion) on the mean EAG response of male flies fed different diets to each of

five decomposition-related volatiles (putrescine, cadaverine, butyric acid, dimethyl

disulfide, and phenol). There was a significant interaction between diet and age post-

eclosion for all volatiles (FPut(1, 6) = 49.05, Pput < 0.0001; FCad(1, 6) = 111.0, PPut<

0.0001; FBA(1, 6)=5.022, PBA= 0.0002; FDMDS(1, 6)=10.96, PDMDS< 0.0001; FPhenol(1, 6)=

1.028, PPhenol < 0.0001, Supp. Table A4.2). Age post-eclosion of these male flies

significantly affected the response for all volatiles tested, with the exception of butyric

acid (FPut(1, 6) = 56.10, Pput < 0.0001; FCad(1, 6) = 120.7, PCad< 0.0001; FBA(1, 6)=2.020,

PBA= 0.0742; FDMDS(1, 6)= 7.168, PDMDS< 0.0001; FPhenol(1, 6)= 9.569, PPhenol < 0.0001;

Supp. Table A4.1, Fig 7.2). Diet exerted a significant effect on the male response to only

Putrescine (F(1, 6) = 16.23, P < 0.0001), and Cadaverine (F(1, 6) = 30.56, P < 0.0001)

(Supp. Table A4.2).

Unlike the female response, the male response to volatiles when fed different

diets did not follow a uniform pattern. Honey-water only fed males had significantly

higher responses to Putrescine and Cadaverine on Day 6 and 4, respectively (PPut,Cad <

0.001), according to Bonferroni post-tests. This peak in HWO fed male response was quickly followed by a sharp decrease in response to Putrescine and Cadaverine on Day 8 and 6, respectively, with the HWL fed males responding significantly higher (PPut,Cad <

0.001). Conversely, HWL fed male flies responded significantly higher, earlier , with responses peaking on Day 4 and 2 to Butyric Acid (PDay4<0.01), Dimethyl Disulfide

119 (PDay4<0.001), and Phenol (PDay2<0.05, respectively. HWO fed flies responded higher,

later to both Dimethyl Disulfide (PDay10<0.001) and Phenol (PDay6<0.001).

Two-way ANOVA analysis reveals that the EAG response of honey-water only

fed males is significantly effected by the interaction between day and volatile (F(6,24)=

6.641, P<0.0001). Both age post-eclosion (F(6, 24) = 52.20, P<0.0001) and volatile

tested (F(4, 24) = 7.505, P<0.0001) tested significantly affected the EAG response of

HWO fed males. Males fed a diet supplemented with protein also had a significant

interaction between age post-eclosion and volatile tested (F(6, 24) = 2.250, P = 0.0014).

Similar to HWO fed male flies, both age post-eclosion (F(6, 24) = 23.72, P<0.0001) and

volatile (F(4, 24) = 9.655, P<0.0001) tested significantly affected the EAG response of

HWL fed males.

Comparison of Males and Females fed the Same Diet

Among honey-water only fed male and female flies, when compared by two-way

ANOVA analysis according to sex and day, only EAG responses to cadaverine and

phenol had a significant interaction (FCad(1,4) = 14.46, P< 0.0001; FPhenol(1, 4) = 3.410, P

= 0.0163). Within this analysis, the age post-eclosion (day) had a significant effect on the

response to all volatiles, except for Butyric Acid (FPut(1, 4) = 56.105.467, Pput = 0.0009;

FCad(1, 4) = 19.24, PCad< 0.0001; FBA(1, 4)=1.679, PBA= 0.1680; FDMDS(1, 4)= 34.63,

PDMDS< 0.0001; FPhenol(1, 4)= 6.798, PPhenol = 0.0002; Supp. Table A4.4, Fig 7.2).

According to Bonferroni post-tests, male and female flies fed honey-water only had

significantly different responses from one another to both Putrescine (PDay10<0.01) and

Cadaverine (PDay2,4,10<0.001). The male and female responses to Butyric Acid, Dimethyl

120 Disulfide, and Phenol were not significantly different at all time points according to

Bonferroni post-tests.

The EAG response to all five decomposition-related volatiles of honey-water and liver fed male and female flies were analyzed by two-way ANOVA to determine the effect of sex and age-post eclosion. For all five volatiles, the response was significantly

effected by the age post-eclosion of the fly (FPut(1, 6) = 5.773, Pput < 0.0001; FCad(1, 6) =

20.72, PCad< 0.0001; FBA(1, 6)=2.020, PBA= 8.027; FDMDS(1, 6)= 16.86, PDMDS< 0.0001;

FPhenol(1, 6)= 13.23, PPhenol < 0.0001; Supp. Table A4.3, Fig 7.2). Only the responses to

Phenol (F(1,6)=2.304, P = 0.0445) and Cadaverine (F(1, 6)=2.801, P = 0.0167) elicited a

significant interaction between both sex and age. According to Bonferroni post-test, there were no days during which the male and female response to any of the decomposition-related volatiles were significant, with the exception of the response to

Cadaverine and Phenol on Day 2 (PCad,Put<0.01).

Discussion Previous research as described in Chapter 6, strongly suggests that when protein

is added to the diet, the sex, and the age post-eclosion of a Lucilia sericata blow flies influence the gustatory response to decomposition-related amino acids. Gustatory and olfactory sensing are closely related in Dipterans, even to the point of sharing sensilla,

organ structure, neuron circuits, and ganglion (Jacquin-Joly and Merlin, 2004). Given

these similarities, as well as the hyper-relatedness of gustation and olfaction, it was

hypothesized that diet, sex, and age post-eclosion would also effect the olfactory response as measured by electroantennography. Recently published research suggests that

121 decomposition-related volatiles do elicit a measurable response via electroantennogram

in L. sericata (Frederickx, et al., 2012). In the present study, flies responded to all of the

tested volatiles, (Putrescine, Cadaverine, Butyric Acid, Dimethyl Disulfide, and Phenol).

Responses, however, were modulated by the age of the fly post-eclosion, the diet

consumed by the flies, and by the sex of the fly.

Female EAG Response According to Diet

The female response to the decomposition-related volatiles varied by the age of

the fly according to the whether or not the fly was fed a diet supplemented with protein.

Females fed a diet of honey-water only had the largest EAG response to the

decomposition-related volatiles around Day 10, while female flies fed a protein- supplemented diet had the largest response to the volatiles, much earlier at post-eclosion

Day 4. This response is significantly different according to analysis by two-way

ANOVA, after a Bonferroni post-test (Supp. Table A4.5), which suggests that similar to

the PER results, this response is related nutritional requirements. Female flies starved of

protein, and fed only honey-water, physiologically need to find and feed on protein in

order to complete vitellogensis, produce essential sex pheromones, and to achieve sexual

receptivity. That decomposition-related volatiles elicit significantly larger olfactory responses in female flies fed only honey-water at Day 10 suggests that previous to this time point, females are allocating for somatic growth and not necessarily for sexual maturation. While females consistently responded to these volatiles, the Day 10 difference suggests that this is the time point by which females are physiologically required to have acquired a protein meal. Previous research shows that in laboratory settings, flies are typically not given a complex protein meal until 14 days post-eclosion.

122 The PER results presented in Chapter 5 and these results, however, suggest that the requirement for a protein meal affects the behavior of honey-water only fed females as early as Day 5 and Day 10 post-eclosion.

Consistently, female blow flies fed a diet of honey-water supplemented with a broad spectrum protein source (liver) had a significantly larger EAG response to decomposition-related volatiles significantly earlier than those fed a sugar source only

(Figs. 7.1, 7.2). While HWL fed females had the largest EAG responses on Day 10, female flies fed HWO had the largest response on Day 4. This finding is consistent with the PER experiments, during which the peak response to decomposition-related amino acids was on Day 5. That these findings are consistent suggests that without a local source of broad spectrum protein post-eclosion Day 4 or 5 females will alter behavior to locate and feed on carrion.

Male EAG Response According to Diet

Regardless of diet, males continually responded to all of the decomposition- related volatiles as assessed by EAG (Fig. 7.2). For all volatiles tested the interaction between diet and day significantly affected the olfactory response. The response to

Putrescine and Cadaverine, for males fed honey-water only, was significantly higher than the response of those fed HWL (P < 0.001) on post-eclosion Day 6 and 4, respectively.

Interestingly enough, the next time point tested showed completely opposite results, with the HWL fed males flies responding significantly higher on Day 8 and 6 than the HWO fed male flies (P < 0.0001) to Putrescine and Cadaverine, respectively. As these are the two volatiles most closely related to decomposition, it is hypothesized that the initial response of HWO fed males flies correlates to their small need for protein early in their

123 somatic development. Once this need has been met, however, the later response of HWL

fed male flies may signify their interest in mating, as their significantly higher response

to decomposition-related volatiles coincides with the time by which females typically are

looking for an oviposition medium, and thus likely found near carrion. The male

response to Butyric acid, Dimethyl Disulfide, and Phenol, volatiles that have been

associated with decay, but have not been found to be released by a human body, is

opposite to the response elicited by volatiles directly related to and released by a

decomposing body (Dierkesshier, et al., 2009). Male flies fed a diet supplemented with

protein have a significantly higher EAG response to BA, DMDS, and Phenol, much

earlier (Days 4, 4, and 2, respectively) than the peak responses to Putrescine and

Cadaverine. This time difference is significant according to two-way ANOVA and

Bonferroni post-tests, is not shared with gustatory sensing, and suggests that the addition of liver to the male diet either increases the sensitivity of olfactory sensing, or that withholding protein modulates the attraction to carrion.

Comparison of Males and Females fed the Same Diet

The gustatory responses of males and females fed a diet of honey-water only were significantly different from each other, with only females responding to decomposition- related amino acids (Fig 7.2). This disparity in response was so marked that it was hypothesized that the olfactory response would show the same difference in response,

with females responding higher to decomposition-related volatiles. While this was

expected, the results from EAG studies showed the exact opposite—upon comparison of male and female flies fed honey-water only, males had the larger EAG responses to decomposition-related volatiles. Both sexes responded consistently to all five of the

124 tested volatiles (Putrescine, Cadaverine, Butyric Acid, Dimethyl Disulfide, and Phenol),

but with the exception of the larger response of females to Putrescine and Cadaverine on

Day 10, males responded significantly higher on to all volatiles on Day 6. Again we see that Day 9 and 10 are significant within the spectrum of female sensing. This sexually

dimorphism in response on Day 10 suggests that the sensing mechanism of females is

closely tied to their physiological requirements, and is affected by the female need for a

protein meal.

Similar to the results of comparing honey-water and liver fed male and female

gustatory responses, there were no significant differences between the male and female

olfactory response as recorded by EAG to decomposition-related volatiles, with the

exception of the male response on Day 2 post-eclosion to both Cadaverine and Phenol, which was significantly higher than that of the female. The five volatiles tested did consistently elicit responses from both male and female HWL fed blow flies, which is a divergence from the gustatory experiments, during which neither male nor female responded to the decomposition-related amino acids. This difference implies that while the gustatory response is diminished, indicating that feeding behavior after consumption of liver is no longer required, olfactory sensing remains essential and sharp. This difference between olfactory and gustatory sensing suggests that the systems entailed in the proboscis extension reflex may be more closely tied to physiology than the neuronal systems examined through EAG, and thus may rely more heavily on the up and down regulation of the easily manipulated odorant binding protein genes. That the tested decomposition-related volatiles continually elicit responses as measure by EAG, suggests that maintaining a minimal level of olfactory sensitivity and activity may be necessary for

125 both sexes to perform normal resource-seeking behaviors. A basal level of EAG activity in response to the tested volatiles also suggests that the flies can continually “smell” or sense olfactory cues, but the behavioral response relies heavily on the physiological needs of the organism.

126

FIGURE 7.1: TWO-WAY ANOVA OF MEAN EAG RESPONSE OF HWO- AND HWL- fed MALE AND FEMALE FLIES

127 FIGURE 7.1: TWO-WAY ANOVA OF MEAN EAG RESPONSE OF HWO- AND

HWL- fed MALE AND FEMALE FLIES

(Continued)

Figure 7.1 Mean (± SEM) maximal depolarization response to each of 5 five volatiles:

putrescine (A, F), cadaverine (B, G), butyric acid (C, H), Dimethyl Disulfide (D, I), and

Phenol (E, J), as measured via electroantennogram (EAG) of females (circles, solid lines) and

males (squares, dashed lines) fed either a diet of honey-water and liver (A-E) or honey-water only (F-J). Variation analyzed by two-way ANOVA. Significant differences in response determined by BonFerroni post-test. For comparisons of females and males within the same diet, p<0.05 (a), p<0.001 (b), p<0.001 (c). For comparisons of females fed different diets, p<0.05 (d), p<0.001 (e), p<0.001(f). For comparisons of males fed different diets, p<0.05 (g), p<0.001 (h), p<0.001 (i).

128 FIGURE 7.2: TWO-WAY ANOVA OF THE AGE EFFECT OF MEAN EAG RESPONSE OF

HWO- AND HWL- fed MALE AND FEMALE FLIES

129 FIGURE 7.2: TWO-WAY ANOVA OF THE AGE EFFECT OF MEAN EAG

RESPONSE OF HWO- AND HWL- fed MALE AND FEMALE FLIES (Continued)

Figure 7.2 Mean (± SEM) maximal depolarization response to each of 5 five volatiles: putrescine (circle, solid line), cadaverine (square, dashed line), butyric acid (triangle up, dotted line), Dimethyl Disulfide (triangle down, dash and dot line), and Phenol (rhombus, dash, double dot line), as measured via electroantennogram (EAG) of females fed a diet of honey-water only (A) or honey-water and liver (B), and males fed honey-water only (C), or honey-water and liver (D).

130 CHAPTER 8

SUMMARY AND CONCLUSIONS

With lofty dreams and admirable aspirations of designing a biomimetic biosensor in the course of a doctoral program, this research commenced four, almost five years ago.

Many iterations of drafted proposals, insightful ideas, and serendipitous findings later, the research described in this dissertation reflects a refined, honed focus on the effect of diet, sex, and age on the sensing capabilities of the forensically important blow fly, Lucilia sericata.

Initial studies focused on the continuous culture of L. sericata, and the establishment of a simple easy diet that would allow for easy replication of studies as well as comparison across forensic and medical entomology labs. Not only was this project undertaken to benefit the greater body of science, but more importantly and specifically to develop protocols for the blow fly sensing laboratories here at the

University of Dayton (UD). Finding that two highly respected labs, sharing the same culture facility, cultured their flies in two notably different manners, but with very similar and successful results, the question was posed as to whether these diets were affecting

131 various aspects of growth, development, and specific to our lab, the sensing abilities blow

flies. With this design, nine different variations of three different diets were sampled and

tested for life history metrics, the results of which are found in Chapter 3.

Finding that given the proper and most nutritive diet of honey water supplemented

with a broad-spectrum protein source, Lucilia sericata can live well beyond the expected

and published 30-60 days to a whopping 120, was one of the most fascinating results of

the Chapter 3 culture experiments (Fig. 3.1). While extending the life span of the blow fly is interesting, it is a useless metric for laboratory colonies unless the amount of effort

invested into the colony by the investigator has a meaningful return. This return on

investment can be measured in number of oviposition events, number of eggs per oviposition events, and total number of eggs produced throughout the time course of the colony (Figs. 3.2-5). After careful analysis of each of these metrics it was determined that a simple diet of honey-water and broad spectrum protein (bovine liver) is optimal for perpetuation of L. sericata colonies. As this is a diet that is much closer to what flies find naturally in the environment than a complex diet of mixed sugar, agar, milk protein, and yeast, it is expected that results found in the laboratory using this diet will be mimic

nature more closely. Hopefully these findings help the Forensic Entomology community

better respond to the demands of the 2009 National Research Council and the 1993

Daubert v. Merrell Dow Pharmaceuticals decisions’ demands: that the theory or

technique used to gather evidence must be testable; it must be accepted and supported by

the scientific community; the error rate of the methodology must be known; and it must

meet existing professional standards.

132 Post-mortem interval estimation is one of the most important uses of

entomological evidence in criminal investigation, thus one of the main goals of research

in the field is to better inform the ability to make that estimation. As the field moves

more toward a genetic basis for PMI estimation, establishing common practices, a simple

diet, and easy culture standards, that hopefully will be adopted by a number of laboratories, will help decrease the source of variation between published results of different labs.

Even as the field is moving more toward the use of genetics, living entomological evidence is still paramount in criminal cases. Currently, larval stages play a large role in the estimation of a post-mortem interval of a victim. Adult flies, however, are known to associate with the carrion for undetermined amounts of time before oviposition occurs, eggs hatch, and larvae start feeding. Sadly, adult blow flies as a source of entomological evidence is largely untapped in the current field. Understanding this deficit, having established a simple diet, and desiring to investigate the effect, if any, this diet ultimately has on the sensing of this organism, an intensive study of examining the how the fitness of adult blow flies affects resource utilization and location was undertaken. The main focus of this study was the utilization of macrostructures that can be located and easily measured in the field.

The most applicable research from Chapter 4, however, is the linear regression analysis that shows that age post-eclosion of females fed a diet of honey-water and liver, is a significant predictor of ovary area. Protein was present for consumption from the time of eclosion in this study, and ovaries were fully mature within four days (Fig 4.6).

The research detailed in the Chapter 3, however, did not present either sex with protein

133 until day 14 post-eclosion, after which the first oviposition event occurred within 5 days

(Fig. 3.4). Together, these data suggest that within four days of exposure to protein,

ovaries are fully mature, and within five days oviposition can and does occur. This

suggests that an adult female fly can associate with a decomposing medium for up to five

days before oviposition. If future research of colleagues can also suggest that this hypothesis holds true for other carrion-seeking species of blow fly, this information could greatly inform metrics used to asses entomological evidence in criminal investigations.

Beautiful scanning electron microscopy accompanies the morphometric analysis of

Chapter 4, showing the microstructure of the sensing organs of Lucilia sericata for the

first time in published history.

Before a blow fly can even begin colonize a carcass, there is the problem of

sensing the nutritive medium from which “pleasing” aromas emanate. With the

suggestion that diet can affect the macrostructure of the adult fly, specifically the wings

and ovaries, it was hypothesized that diet could also affect the fly’s ability to sense the

medium from which it receives this meal. Utilizing the proboscis extension reflex in

which gustatory receptors on the tarsi are stimulated, thus triggering the behavioral

response of extending the proboscis in a feeding movement, this hypothesis was tested on

Lucilia sericata. Not only was the normally high female response to decomposition-

related amino acids significantly decreased by the addition of protein to the diet, it was

decreased to that of the almost non-existent response elicited by the males fed either diet

(Figs. 6.3, 6.4, 6.5). Also interesting, is that for almost all of the amino acids, the PER

response of protein-starved females peaked at Day 5 post-eclosion.

134 Electroantenogram assessment of olfactory response to decomposition-related volatiles also suggested Day 5 post-eclosion was important in the life-cycle of adult female L. sericata blow flies. The female response to some volatiles was affect by both diet and age, but the trend remained that they would consistently respond to the volatiles after post-eclosion Day 3 or 4 regardless of diet. While male flies did have olfactory response to decomposition volatiles, which was much different from their lack of gustatory response to the tastants, their response tended to remain low, but peaked around post-eclosion Day 8.

Piecing the olfactory and gustatory data together with information gleaned from

Chapter 4 and 5, suggests a number of theories about the early L. sericata life cycle (Fig.

8.1). Together these data suggest that 5 day old adult females begin to seek a protein source for the purposes of feeding. Within 4 days of discovering and feeding on a broad spectrum protein source, such as carrion, the ovaries are fully developed, sexual maturation occurs, and female becomes sexually receptive. Fertilization can happen almost immediately, or can take a few days as females may store sperm in the spermatheca. Five days after feeding begins, the L. sericata female can oviposit. It is around this same time, between 8 to 10 days post-eclosion, that L. sericata males have the largest olfactory response to decomposition-related volatiles. This suggests that males instinctively “know” when females will be sexually mature and receptive, based on their age and days feeding, and are programmed to seek out the source of such decomposition volatiles at this time. It is implied that this olfactory response is for the purposes of mating, seeing as though protein feeding happens, if at all, in small amounts

135 very near to eclosion. After which the male adult does not exhibit attraction to a protein source, based on the lack of gustatory response to decomposition-related amino acids.

If the proposed time line of the first 8 to 10 days of the adult blow fly life cycle is correct, the implications for medical and forensic entomology, as well as for insight into

dipteran sensing as a whole, are broad.

Flies continue to mate throughout their seemingly long lifespan, and can travel

great distances to colonize carrion. This research, despite its focus on the first five to

fourteen days of L. sericata’s life, suggests that female flies can associate with a

decomposing medium for up to four days before oviposition can occur. After

ovisposition occurs, the larvae feed, and pupation occurs, it can be between five to eight

days before those flies also emerge depending on environmental conditions. For medical

entomology, that this life cycle can turn over so quickly is helpful for producing large

robust colonies quickly. For forensic entomology, the estimation of a post-mortem

interval could be greatly aided by these details of the L. serciata adult life cycle,

specifically, if the body is in an enclosed space for weeks after death.

Informing the suggestion that the adult life cycle can be affected by the addition

of protein, is the understanding that the physiological state, specifically of females, can

also be impacted by a diet supplemented with protein. The research of Dethier, Brown,

and Hobson all point the knowledge that for adult anautogenous Lucilia sericata females,

protein is required to alter the physiological state. The addition of protein does alter the

stage of the female ovaries, thus moving her from immature juvenile status to that of a

mature adult (Fig 4.6, 4.6). This suggests that the diet consumed by an adult blow fly,

specifically the addition of protein to the diet, can affect the physiology of the fly. This

136 protein supplementation, in turn affects the gustatory and olfactory sensing abilities of

both male and female flies (Figs. 6.2-5, 7.1-2) and the respective responses to

decomposition-related tastants or volatiles.

Armed with the understanding that specific volatiles are emitted at different decay stages, and that insect succession closely follows these stages, the results of these sensing studies suggest that there could possibly be species-specific succession on the carrion

based on physiological need. Both newly eclosed Lucilia sericata male and female flies

show little to no interest in exhibiting either a gustatory response to amino acids, or an

olfactory response to putrefactive volatiles. By day 4 or five post-eclosion, however, this

attraction has changed dramatically for females, as both amino acids and VOCs elicit

sensing responses. Once the four to five day old adult females have fed on the carrion,

their physiological status changes. These mature females now respond differently to

these same tastants and volatiles; a response still occurs, but is significantly different

from that of pre-vitellogenic or vitellogenic flies (Figs. 4.5-6, 5.2-4, 6.1-3). If a female

needs protein to complete vitellogenesis, then the fly should exhibit attraction to specific

volatile cues. A gravid female, however can be attracted to a separate volatile cues, that

are associated with a specific stage of decay

A fly will cease to exhibit a proboscis extension reflex (PER) gustatory response

when the fly is sated; a mechanism which is under neural control of the cephalic ganglion

(Dethier, 1962). Satiation may be determined by the supra- and sub-esophageal ganglion,

but it appears, given the data presented in Chapters 6, response and attraction to a specific

nutrient at a certain age is determined by physiology. As the female feeds on protein, a

behavior governed by physiological need and evolutionary drive, ovarian status

137 progresses from pre-vitellogenic to mature. This physiological development then occurs at the same time that a decrease in the olfactory response to decomposition-related VOCs occurs. Similar to gustatory behavior, in which satiation is governed by the insect “brain,” the olfactory system is also known to have a high component of neuronal control and involvement. Given the evidence presented in Chapters 6 and 7, it can be supposed that neuronal control of behaviors can be affected by physiological needs and status.

That physiology may play a role in the regulation of neutrally-controlled processes, suggests that gustatory sensing could occur at the organ level, starting with discrimination in the sensilla. If the tastant provides for a physiological need, then it is possible that sensing will govern behavior. If it is not physiologically relevant at the time the fly is challenged by the tastant, then it will elicit no response. Females support this supposition, as when sated with protein, will not exhibit a gustatory response to amino acids related bodily decay (Figs 6.3-6). This points toward local control of the response, due to close physiological (chemical) association (Fig. 7.2).

Gustatory and olfactory sensing are known to be closely related, sharing similar organization and response structure, as well as often the same sensillar structures. Unlike the gustatory sensing data of Chapter 5, the olfactory sensing data in Chapter 6, however, does not necessarily point toward local control of the response. A prime example of this difference in response is Lucilia sericata’s response to water: if the fly is “thirsty”, it will have a gustatory PER response will be elicited by a challenge with water; however, no olfactory response to water was ever elicited by humidified air. Hexane, humidified air, and “water” did not elicit responses as measured by EAG, similar to the way in which various amino acids did not elicit a gustatory response in male flies. That a response was

138 recorded through the use of an electroantennogram to all of the volatiles tested in Chapter

6 for both males as females throughout the time course of testing, suggests that all of the decomposition-related VOC’s are bound by odorant binding proteins, and thus can trigger depolarization of gustatory receptor neurons. This implies that while discrimination of gustatory input may occur at the local level, discrimination of olfactory cues is more likely to occur at the neural level within the antennal lobe of the cephalic ganglion.

Examining the data discovered in the pursuit of a better understanding of the olfactory and gustatory sensing systems of Lucilia sericata, a number of important theories and applications have been proposed that will impact forensic, medical, and classical entomology. Applying a common diet consisting of honey-water and a broad- spectrum protein source, such as bovine liver will allow for continuous and replicable culture of laboratory colonies of Lucilia sericata in conditions similar to those found in the natural environment. Details of the post-eclosion adult female and male time line as affected by the consumption of protein should help inform post-mortem interval estimation. Combining data from the diet and morphometric studies with the olfactory and gustatory sensing findings allows insight into where Calliphorid sensing occurs a the local level of the sensilla or at a higher processing level in the antennal lobe of the cephalic ganglion.

139 FIGURE 8.1 PROPOSED LUCILIA SERICATA ADULT TIME LINE

Pre-Vitellogenic

Vitellogenic

Status

Ovarian Mature

Oviposition

Likely to Occur

Female Response to Olfactory Cues

Female Response to Gustatory Cues

Male Responses Sensing Gustatory Cues

Male Responses To Olfactory Cues

Occurrence of Feeding Behavior

Day/Age Post- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Eclosion

Figure 8.1 Proposed time line of Lucilia sericata adult feeding behavior, sensing responses, and ovarian status.

140 FIGURE 8.2 PROPOSED FRAMEWORK FOR LUCILIA SERICATA SENSING

Figure 8.2 Proposed synthesis of the interaction between Lucilia sericata’s physiological needs, diet, behavior, and neuronall control of the included systems combining research gathered from all four of the dissertation research chapters.

141 CHAPTER 9 PROPOSED POST-DOCTORAL RESEARCH

Introduction and Background

Almost the minute an organism’s vital functions cease—the beating of the heart, the breathing of the lungs, the function of the brain—abiotic post-mortem phenomena such as the loss of consciousness, muscle tone, and circulation can be observed, as well

the transformation and creation of putrefaction in the corpse release putrid volatile

organic compounds (VOCs) (Dekeirsschieter, et al., 2009; Hensage, et al., 1995;

Rodriguez, 1997; Statheropoulous, et al., 2005). Even from a distance of 20 km

Calliphorid species can sense these odors and fly to the source with the purpose of

finding viable food, as well as laying eggs within the carcass (Browne, 1987;

Campobasso, et al., 2001).

Arthropod colonization of carcasses has been studied for just under 250 years;

Carl von Linne in 1767 was the first to characterize the succession of insects that

contribute to the decomposition of carrion (Bass, 1997; Campobasso, et al., 2001;

Sharonowski, et al., 2008). Essential to current forensic science is understanding that the

succession of insect visitation to carrion corresponds specifically to stage in the

142 decomposition of the flesh, which allows a relative post-mortem interval to be established

upon observation of the developmental stages of the insects present on the body, the level of decay, amount of material present on the decaying carcass, and the VOC profile of decaying carrion (Campobasso, et al., 1002; Hensage, et al., 1995).

Seeing the importance of such data in law proceedings, in response to the Daubert

v. Merrell Dow Pharmaceuticals mandate that scientific evidence be testable, have a known error rate, to be peer-reviewed and accepted practice within the scientific

community, the National Research Council in 2009 expressed the need for the forensic

sciences, including the specialty of forensic entomology, to improve its standards and

accuracy (Tomberlin, et al., 2011; Tomberlin, et al., 2011b). Forensic entomology

currently relies heavily on fieldwork; the collection of larvae from carcasses, as well as visual identification of species and larval stage (Burke and Gold, 1997). One major way

of increasing precision is to rely less on the currently accepted standards of observation

and measurement, and to begin performing genetic tests to uncover markers unique to

each larval and adult age, which would allow the establishment of a more accurate PMI

(Tomberlin, et al., 2011a, b; Tarone, et al., 2007). A primary barrier to the genetic

identification and testing of the forensically important L. sericata, is that the genome

remains unknown.

Still wholly unsequenced, the Lucilia sericata genome continues to be a mystery, despite a large current focus, both genetic and behavioral (Frederickx, et al., 2012;

Stocker, 1994; Sze, et al., 2012) on this particular organism as a representative of the family, Calliphoridae. Seeking to add context to the transciptome sequenced by Tarone, et al., as well as to the base of knowledge for this organism and others, Christine Picard, has begun to sequence the genome of Chrysomya rufifaces and Chrysomya macellaria

143 (Sze, et al., 2012). Of primary interest to the proposed work, is the sequencing of genes associated with sensing.

A natural biosensor, blow flies sense volatiles released by necrosis and decay, and

alter behavior accordingly. L. sericata’s gustatory and olfactory abilities are vital to the

association between behavior and sensing (Jacquin-Joly and Merlin, 2004). This

connection is aided by odorant binding proteins (OBPs) and odorant and gustatory neuron

receptors (ORNs, GRNs) contained within the blow fly sensory organs (Hansson, 1995).

Crossing through the cuticle of the sensilla, the hydrophobic odorant molecule is bound

by a hydrophilic OBP, after which it crosses the hemolymph to connect with either an

olfactory receptor neuron or a gustatory receptor neuron (Amrein and Thorne, 2005;

Galindo and Smith, 2001; Goleiowski, et al., 2007; Hansson, 1995; Hensage, et al., 1995;

Jacquin-Joly and Merlin, 2004; Mustaparta, et al., 1996). This attachment then triggers a

signal transduction pathway that allows translation of this external chemical message into

activation of a neurological pathway eventually resulting in specific blow fly migration

toward the scent source (Fuss and Ray, 2009; Frederickx, et al., 2012).

In the natural environment, it is the broad spectrum of volatiles released during

the process of tissue and bodily decay that attracts blow flies43, signaling available

nutrients essential for both larval and adult growth and development (Browne, et al.,

1990; Browne, 1993; Hobson, 1995; Sharonowski, et al., 2008). To reach sexual

maturity and achieve vitellogenesis (egg formation), dietary protein is required for

females (Browne, et al., 1990). The preference of females for different types of food is

highly influenced by their reproductive state, which in turn affects the food-seeking

behaviors, leading L. sericata to necrotic and decomposing tissue (Browne, et al., 1987,

1990; Browne, 1993; Huntington and Higley, 2012).

144 Not only is L. sericata of primary importance in forensic investigations, but in

New Zealand and Australia Lucilia cuprina, a very closely related sister species, is more

commonly known as an agricultural pest (Dallwitz, et al., 1984). “Fly Strike,” or myiasis

of livestock dermal tissue, poses a sizable problem to the wool industry, which in

Australia alone, accounts for $2.4 billion in exports (Dallwitz, et al., 1984). During

seasons with particularly heavy rain and flooding, it is normal for the fleece to become

moldy and to rot, which causes an infection of the underlying tissue, dematophilosis

(Colditz, et al., 1992). While fleece rot can heavily affect regions of the lower limbs and

underbelly, the hindquarters (known as the “breech”) with feces-matted fleece are the

most common infected areas (Phillips, 2009). The secretions from these infections mixed

with the fleece rot exude scents similar to those of decaying flesh, which in turn attracts blow flies to the contaminated areas where eggs are laid, larvae hatch, and myiasis results.

The array of volatile organic compounds (VOCs) associated with fleece rot and decomposition may not be distinguishable by the human nose, but Lucilia sericata, one of the most widely examined species of blow fly, easily discriminates between fecal odors and the volatile profile of decaying or infected organic material through pattern recognition (Jacquin-Joly and Merlin, 2009; Morita, 1992). Similar to the way in which musical chords are played on a piano, a certain set of neurons triggered in the fly brain is

translated as a particular odor, and if this odor is associated with decomposition, behavior is then altered (Stocker, 1994).

Despite being sister species, populations of Lucilia cuprina endogenous to

Australia and New Zealand are more likely to respond to volatiles associated with wool- rot and dematophilosis, than to those associated with a decomposing carcass, to which the

North American population primarily respond (Tourle, et al., 2009). This marked

145 difference in the sense-and-response cascade within a single species, adds new depth to

the debated topic of Dipteran sensing: is the behavior determined by volatile selection at

the lower odorant binding protein (OBP) level, or at the post-binding neurologic level?

Comparing the OBP gene levels of two Lucilia populations given the exposure to volatile organic compounds (VOCs), and knowing that one responds more readily than the other

to decomposition-specific VOCs, will not only shed light on the forensically important

blow fly sensing, but will also allow existing theories for Dipteran sensing to be

enlightened (Golebiowski, et al., 2007; Rodrigues and Siddiqi, 1981).

The proposed work will contribute significantly to the understanding of the similarities and differences between the genetic basis for olfactory and gustatory sensing systems of North American and Australian blow fly populations, as well how and why male and female blow flies, respectively, sense VOC profiles and migrate toward decomposing matter. This understanding will allow characterization of the link between gene up- or down-regulation, and scent perception and the subsequent feeding, mating, and egg-laying behaviors in Lucilia sericata. Also, comparisons regarding the genetic and physical characteristics of the olfactory and gustatory organs between these species of blow fly will be produced, contributing to the growing amount of knowledge about these organisms.

Creating a bio-mimetic system employing this knowledge of blow fly sensing mechanisms, will allow the rapid localization of decedents in the event of a large-scale mass disaster, such as Hurricanes Katrina and Sandy, preventing the spread of disease and water contamination; or on a smaller scale, such as a local homicide or missing- persons investigation. Currently the blow fly is utilized by forensic scientists for its

146 highly sensitive gas-sensing ability, this work provides for a method of acquiring and utilizing its biological mechanism for broad application in sensing airborne volatiles.

Significance, Research Objectives, Methods

Casey Anthony, and Anthony Sowell are just two names that bring to mind

lengthy, horrific murder trials, whose outcomes hinged on forensic evidence. Admitted

to body of proof in both of these cases was testimony of the smells associated with the

crimes. Oak Ridge National Laboratory found that “a portion of the total odor signature

from [Casey Anthony’s] trunk ‘is consistent’ with a decomposing body that could be

human.” Although this evidence points toward the possibility that a corpse could have

occupied the car, the fact that only five of the possible four hundred compounds

associated with decomposition were discovered did not evince to the jury that the source

of the scents was little Caylee’s body. For years, Ray’s Sausage Factory in Cleveland,

Ohio was blamed for the stench that permeated the neighborhood, resulting in fines from

the Health Inspector and mandatory replacement of the facility’s interior plumbing and

grease trap. Five years later, the public now knows that that stench was not poor

practices of the factory, but the eleven rotting and buried bodies of women found on

Anthony Sowell’s property next door. As the Sowell and Anthony trials have shown,

individuals can be convicted or acquitted based on the validity and availability of forensic

data presented in court, for which, as stated by the National Research Council in 2009,

collection and processing methods are still lacking. If the officials in both cases could

have better discriminated the decomposition-related volatile organic compounds by using

a mechanical biosensor utilizing distinct pattern recognition, it is possible that Sowell’s

victims could still be alive, and that the Anthony verdict could have been resolved.

147 Knowing the sense-and-response cascade in the blow fly, and how important

harnessing the information for applications in the forensic sciences, the main objective of

this proposed work is to gain a better understanding of how the possible genotype differences of Lucilia sericata populations from North America or Australia affects phenotypic traits, such as attraction to either wool rot of carrion, and applying this acquired knowledge to help create tools to enhance forensic investigation.

Objective 1: Compare the gene sequences and expression of L. sericata odorant binding

proteins, gustatory receptor neurons, and odorant receptor neurons in adults from both

North American and Australian populations, focusing on molecular markers and SNPs.

Methods: Sequence and data mining using BLAST and SeqGene (Deng, 2011). Gene

expression studies probing c-DNA libraries specific to sensing genes and proteins, using

Q-RT-PCR (quantitative reverse-transcription polymerase chain reaction), followed by

Western blotting to establish presence of up- or down-regulated protein. Isolation of up- regulated odorant binding proteins or odorant receptors, and modeling of binding sites.

Objective 2: Expose any behavioral or neurologic response difference to decomposition-

related amino acids and volatiles in adults from both North American and Australian

populations.

Methods: Proboscis extension reflex (PER) assay to determine gustatory response, and

electroantennogram (EAG) paired with a dual-choice olfactometer bio-assay to probe the

olfactory response of the populations (Amrein and Thorne, 2005; Jenkins, et al., 2012;

Kendra, et al., 2005). Organisms exposed to decomposition-related volatiles will also be

subject to the gene expression studies delineated in Objective 1.

148 Objective 3: Using both the genetic and behavioral knowledge acquired, design a

mechanical biosensor to be deployed in forensic investigations, mass casualty incidents, and health promotion.

Methods: After choosing one or more appropriate proteins with high affinity for

decomposition VOCs, as established through both the genetic and behavioral studies

comparing the two populations L. sericata populations, the biochemistry of OBP or OR binding sites will then be subjected to SELEX to develop apatamers for device application and building (Burke and Gold, 1997; Diekmann, et al., 1996; Huizenga and

Szostak, 1995).

By using an interdisciplinary approach to understand how the blow fly recognizes volatiles at the molecular level, and how this effects divergent behavior in two separate populations, a bio-inspired mechanisms for sensing airborne volatiles will be designed to support the forensic efforts of the United States Food and Drug Administration (USFDA),

United States Department of Agriculture (USDA), the Department of Justice, and

Department of Defense (DOD), in addition to similar organizations from around the world.

Training Objectives

As mentioned previously, the main aim of this proposed work is to gain a better

understanding of how the possible genotype differences of Lucilia sericata populations from North America or Australia affects phenotypic traits, such as attraction to either wool rot of carrion, and applying this acquired knowledge to help create tools to enhance

forensic investigation. As stated this objective seems specific and narrow, but when

placed in the context of the state of current knowledge of both the Calliphorid family, and

149 more broadly, that of the Dipteran order, this proposed work has the potential of not only

contributing to the applied forensic sciences, but also the understanding of insect sensing.

Training Objective 1: Learn and become competent on data- and sequence-mining

software, including SeqGene.

To this end, the first item to be addressed will be the genetic basis of Luilia sericata

sensing. Sequences already discovered by Christine Picard, will be mined for genetic

markers, functional genes, short nucleotide polymorphisms, and microsatellites with the

hope of better informing the forensic science community. Genes associated with sensing,

such as Odorant Receptor 83b (Or83b) and Drosophila Odorant Receptor 43a (DOR43a),

which are expressed in both blow flies and the model Dipteran organism, Drosophila

melanogaster, will be the primary focus, along with those sharing OR and OBP-specific

consensus sequences (Fuss and Ray, 2009; Krieger and Breer, 1999; Krieger et al., 2003).

Training Objective 2: Bring the PER, EAG, and Y-tube bio-assays to the university; gain

experience and streamline training both undergraduate and graduate students in those

techniques, as all three are under-utilized within the specialty of forensic entomology.

Upon gathering a collection of odorant binding protein, odorant receptor, and gustatory receptor genes, the next step will be to perform bioassays to establish a baseline of

response in both the North American and Australian populations. Both the proboscis

extension reflex assay (PER), which examines the gustatory response, and electro-

antennogram (EAG), which shows the excitation of the olfactory receptor neurons, will

be used to assess whether a difference in response to decomposition-related amino acids

and volatiles exists between the two populations.

150 Training Objective 3: Come to a deeper understanding of protein modeling, and selection

of high-affinity binding sites for aptamer production.

Once the standard responses of both populations to decomposition-related

volatiles and amino acid solutions are determined, gene-expression studies will

commence, looking at the up- or down-regulation of odorant binding protein (OBP),

odorant receptor (OR), and gustatory receptor (GR) genes post-exposure.

Training Objective 4: Experience and become trained on the SELX process and aptamer

selection, as well as understanding device choice and design.

Both the up-regulated OBP protein and binding site modeling, will then be subject

to the SELEX (systematic evolution of ligands by exponential enrichment) process, to develop aptamers specific for decomposition-related volatile organic compounds, such as indole, cadaverine, and putrescine. Creating an aptamer through this process begins with

the creation a large library of oligonucleotides is created to simulate the binding capabilities of the OBP, all of which are then subjected to repeated selection events, including the repeated exposure to the volatiles of interest—those oligonucleotides which most tightly bind to the volatile and mimic the original biology are then selected.

Career Development

While the research goals detailed herein remain paramount, it is also important

that the process of research and the performing of experiments contribute to the education

and teaching of younger scientists. As a primary investigator develops new protocol or

technique it is imperative that the process be recorded and passed onto to rising scholars.

Without this distribution of and access to innovation, Science will become stagnant,

151 ceasing to move forward in the current of discovery. This tradition will continue to pervade the proposed post-doctoral research.

Not only will the scope of this research allow development of future scientists, but also allow the primary scientist an expanded understanding of biosensor design and development. Having taken and enjoyed four seminar courses on this topic, now participating in applying basic biology to the creation of a field-deployable system will fully enhance the biosensor education begun as a doctoral student.

Timetable

Fall: Begin mining of Christine Picard’s L. sericata genome data, using A. Tarone’s transcriptome as a guide, as well as already known D. melanogaster sequences, to establish plausible odorant binding protein (OBP), odorant receptor (OR), and gustatory receptor (GR) sequences.

Spring: Perform bioassays—the proboscis extension reflex (PER) assay, electroantennogram (EAG) and dual choice olfactometer studies (Y-tube)—to establish the baseline of behavioral response to decomposition-related amino acids and volatile organic compounds (VOCs) for both North American (NA) and Australian (A) populations of L. sericata, noting both similarities and differences. Start gene expression studies. It is expected that while the (NA) population will have high responses at both the gustatory and olfactory levels to decomposition-related analytes and vapors, the (A) population will have a much lower gustatory response, as this population prefers to feed on infected tissue rather than carrion, as well as lower neurological responses as assessed by EAG. If both the NA and A populations have the same or similar EAG results, contrary to expectations, this would suggest that behavior following volatile exposure is

152 mediated at the higher neurological brain center. Whereas if both PER and EAG

responses are low in the Australian population, this would suggest that the sensing

connection to behavior is mediated at the molecular, possibly OBP, level before the

neuron is excited. Gaining this knowledge will shed new light on the Dipteran sensing

debate, bringing strong evidence to the hypothesis that behavior in response to volatiles

or analytes is mediated at the molecular level, rather than by the insect brain.

Fall: Continue and complete gene expression studies to ascertain the up-regulation in

NA populations of specific OBPs and ORs in response to exposure to decomposition-

related VOCs. The A population should not show the same pattern of gene expression

given the divergence in behavior and volatile attraction. Begin modeling of VOC-

specific OBP binding sites. It is expected that after extended volatile exposure, both OBP

and OR levels will rise in a time-dependent manner, specifically those that are necessary

for decomposition sensing, specifically the homologs for Anopheles gambiaeOBP1

(AgamOBP1), and the D. melanogaster LUSH. After this up-regulation has been established at both the RNA and protein levels, the L. sericata homologs of AgamOBP1 and LUSH will be isolated, sequenced, then modeled to establish clear volatile binding sites.

Spring: Complete and submit the modeled decomposition VOC-specific OBP binding

sites to SELEX and pursue aptamer development. Once the appropriate aptamer has been

ascertained, begin process of mounting on a potentially field-deployable device.

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APPENDIX 1 SUPPLEMENTARY TABLES FOR CHAPTER 3

Supp. Table A1.1: Log-Rank Survival Curve Analysis

Diet Chi Square DF P value Sucrose 36.54 2 < 0.0001 Honey-water (al) 97.26 2 < 0.0001 Honey-water (contr.) 68.21 2 < 0.0001

Sup Table A1.1 Log-Rank (Mantel-Cox Test) comparisons of survival curves within all three variations of diets for each sugar source (sugar source only, sugar source plus milk, sugar source plus liver)

177 Supp. Table A1.2: Linear Regression Model of Female and Egg Number

Diet DF F R2 P Value Slope of Best-Fit Honey-water al Liver Only 48 2.173 0.04331 0.1470 6.290 ± 4.267 Liver plus Milk 33 7.346 0.1821 0.0106 19.18 ± 7.078 Sucrose al Liver Only 29 0.9643 0.03218 0.3342 3.847 ± 3.917 Liver plus Milk 19 6.646 0.2591 0.0184 -21.70 ± 8.41

Sup Table A1.2 Linear regression model statistics of the relationship between female number and number of eggs laid for Honey-water and Sucrose ad libitum diets supplemented with either liver only or milk plus liver.

178

Supp. Table A1.3: Two-Way ANOVA of Fly Age and Egg Area

Source of % of Total Variation DF SSq MS F variation P Value Interaction 33 2.783 0.0843 47.15 56.25 < 0.0001 Diet 3 0.5729 0.1910 106.8 11.58 < 0.0001 Day 11 0.7561 0.0687 38.43 15.28 < 0.0001

Honey-water al Sucrose al Liver Only Liver, Milk Liver Honey-water al Liver Only Liver plus Milk 29***, 31***, 33***, 39***, 47***, 49***, 55*** Sucrose al Liver Only 21***, 29***, 21***, 29***, 39***, 41***, 31***, 33***, 43***, 51***, 41***, 43***, 55*** 47***, 49***, 51*** Liver plus Milk 21***, 27***, 21***, 27***, 21***, 27***, 29***, 33***, 31***, 49*** 33***, 41***, 47***, 55*** 43***, 47***, 51*** P < 0.05*, P < 0.01**, P < 0.001*** Supp. Table A1.3 Two-Way ANOVA statistics of the relationship between fly age and area of eggs laid, compared between diets (A). Bonferroni Post-Tests to determine significance between diets of egg area according to fly age (B).

179 Sup Table A1.4: Two-Way ANOVA of Fly Age and Egg Number

Source of % of Total Variation DF SSq MS F variation P Value Interaction 36 78086 2169 0.9897 32.97 0.5059 Diet 3 29038 9679 4.417 12.26 0.0077 Day 12 15759 1313 0.5992 6.65 0.8328

Honey-water al Sucrose al Liver Only Liver, Milk Liver Honey-water al Liver Only Liver plus Milk ns Sucrose al Liver Only 21* 21* Liver plus Milk ns ns 21* ns = Not Significant, P < 0.05*, P < 0.01**, P < 0.001***

Sup Table A1.4 Two-Way ANOVA statistics of fly age and number of eggs laid,

compared between diets (A). Bonferroni Post-Tests to determine significance between

diets of egg area according to fly age (B).

180 APPENDIX 2 SUPPLEMENTARY TABLES FOR CHAPTER 4

Supp. Table A2.1: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and

Weight of HWP fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.00018060.00004516 7.576 10.20 < 0.0001 Sex 1 0.00040820.0004082 68.47 23.06 < 0.0001 Day 4 0.00067470.0001687 28.29 38.11 < 0.0001

Abdominal Area of HWP fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 4.326 1.082 1.851 3.63 0.1264 Sex 1 53.98 53.98 92.40 45.27 < 0.0001 Day 4 10.70 2.675 4.578 8.97 0.0021

Wing Area of HWP fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 2.744 0.6860 0.8912 3.32 0.4732 Sex 1 17.13 17.13 22.26 20.73 < 0.0001 Day 4 1.197 0.2992 0.3887 1.45 0.8162

181 Supp. Table A2.1: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and

Weight of HWP fed Flies

(Continued)

Day Weight Abdominal Area Wing Area 0 P > 0.05 P<0.001 P > 0.05

5 P > 0.05 P < 0.05 P > 0.05

10 P<0.001 P < 0.05 P > 0.05

15 P<0.001 P<0.001 P < 0.05

20 P<0.001 P<0.001 P < 0.05

Supp. Table A2.1 Two-way ANOVA statistical analysis of the effect of diet on the mean abdominal area, wing area, and weight of honey-water and protein fed flies (A).

Statistical significance determined by Bonferroni post-tests (B).

182 Supp. Table A2.2: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and

Weight of HWO fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.00009690.0000242 2.443 6.06 0.0536 Sex 1 0.00021520.0002152 21.71 13.46 < 0.0001 Day 4 0.00051350.0001284 12.95 32.11 < 0.0001

Abdominal Area of HWO fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 5.816 1.454 1.220 4.87 0.3094 Sex 1 14.19 14.19 11.91 11.89 0.0009 Day 4 10.03 2.509 2.106 8.40 0.0885

Wing Area of HWO fed Flies

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 20.48 5.120 3.179 10.58 0.0178 Sex 1 25.34 25.34 15.73 13.08 0.0002 Day 4 20.60 5.149 3.197 10.64 0.0173

183 Supp. Table A2.2: Two-Way ANOVA of Mean Abdominal Area, Wing Area, and

Weight of HWO fed Flies

(Continued)

Day Weight Abdominal Area Wing Area 0 P > 0.05 P > 0.05 P > 0.05

5 P > 0.05 P > 0.05 P > 0.05

10 P<0.01 P > 0.05 P > 0.05

15 P < 0.05 P < 0.05 P > 0.05

20 P > 0.05 P > 0.05 P<0.001

Supp. Table A2.2 Two-way ANOVA statistical analysis of the effect of diet on the mean abdominal area, wing area, and weight of honey-water only fed flies(A). Statistical significance determined by Bonferroni post-tests (B).

184 Supp. Table A2.3: Linear Regression Model Statistics for HWP Flies

slope y-intercept F DF R2 P value Abdominal Area Male 0.01460 5.960 0.1224 1, 3 0.03920 0.7496 Female 0.02524 7.356 9.608 1, 3 0.7620 0.0533 Wing Area Male -0.006962 11.90 1.482 1, 3 0.3307 0.3104 Female 0.03805 12.33 23.47 1, 3 0.8867 0.0168 Weight Male 0.0001438 0.01058 3.239 1, 3 0.5192 0.1697 Female 0.0005202 0.01101 13.93 1, 3 0.8228 0.0335

Supp. Table A2.3 Linear regression model statistics of the abdominal area, wing area and weight of both male and female honey-water and protein fed flies. Age post-eclosion is a statistically significant predictor of both female wing area and female weight.

185 Supp. Table A2.4 Two-Way ANOVA of the Age Effect of HWP Flies

A. Abdominal Area

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 4.326 1.082 1.851 3.63 0.1264 Day (Age) 4 10.70 2.675 4.578 8.97 0.0021 Sex 1 53.98 53.98 92.40 45.27 < 0.0001

Wing Area

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 2.744 0.6860 0.8912 3.32 0.4732 Day (Age) 4 1.197 0.2992 0.3887 1.45 0.8162 Sex 1 17.13 17.13 22.26 20.73 < 0.0001

Weight

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.00018060.00004516 7.576 10.20 < 0.0001 Day (Age) 4 0.0006747 0.0001687 28.29 38.11 < 0.0001 Sex 1 0.00040820.0004082 68.47 23.06 < 0.0001

Supp. Table A2.4 Two-way ANOVA statistical analysis of the effect of age on honey- water and protein fed male and female blow flies on the abdominal area, wing area and weight (A). Statistical significance determined by Bonferroni post-test (B).

186 Supp. Table A2.4 Two-Way ANOVA of the Age Effect of HWP Flies (continued)

Day 0 v 5 0 v 10 0 v 15 0 v 20 5 v 10 5 v 15 5 v 20 10 v 15 10 v 20 15 v 20

Abdominal Area

Male P<0.001 P<0.001 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Wing Area

Male P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Weight

Male P < 0.05 P<0.01 P<0.01 P<0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P<0.001 P<0.001 P<0.001 P<0.001 P > 0.05 P<0.01 P<0.001 P > 0.05 P > 0.05 P > 0.05

187 Supp. Table A2.5 Two-Way ANOVA of the Age Effect of HWO Flies

A. HWO Weight

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.000096870.00002422 2.443 6.06 0.0536 Day (Age) 4 0.0005135 0.0001284 12.95 32.11 < 0.0001 Sex 1 0.0002152 0.0002152 21.71 13.46 < 0.0001

HWO Wing Area

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 20.48 5.120 3.179 10.58 0.0178 Day (Age) 4 20.60 5.149 3.197 10.64 0.0173 Sex 1 25.34 25.34 15.73 13.08 0.0002

HWO Abdominal Area

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 12.07 3.017 2.569 8.49 0.0444 Day (Age) 4 17.32 4.331 3.687 12.19 0.0084 Sex 1 21.08 21.08 17.95 14.84 < 0.0001

Supp. Table A2.4 Two-way ANOVA statistical analysis of the effect of age on honey-

water only fed male and female blow flies on the abdominal area, wing area and weight

(A). Statistical significance determined by Bonferroni post-test (B).

188 Supp. Table A2.5 Two-Way ANOVA of the Age Effect of HWO Flies (continued)

Day 0 v 5 0 v 10 0 v 15 0 v 20 5 v 10 5 v 15 5 v 20 10 v 15 10 v 20 15 v 20

Abdominal Area

Male P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P > 0.05 P > 0.05 P < 0.05 P<0.001 P > 0.05 P < 0.05 P<0.001 P > 0.05 P < 0.05 P > 0.05

Wing Area

Male P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P > 0.05 P > 0.05 P > 0.05 P<0.001 P > 0.05 P > 0.05 P<0.001 P > 0.05 P<0.01 P<0.01

Weight

Male P > 0.05 P < 0.05 P > 0.05 P<0.01 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Female P<0.001 P<0.001 P<0.001 P<0.001 P < 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

189 APPENDIX 3 SUPPLEMENTARY TABLES FOR CHAPTER 6

Supp. Table A3.1: Two-Way ANOVA of the PER for HWO fed Male and Female

Flies

A. Day 1

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 12.42 1.774 3.875 1.26 0.0007 Sex 1 0.0 0.0 0.0 0 1 Treatment 7 916.1 130.9 285.8 92.81 < 0.0001

B. Day 5

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 90.55 12.94 11.98 10.22 < 0.0001 Sex 1 15.45 15.45 14.31 1.74 0.0002 Treatment 7 642.3 91.75 84.99 72.45 < 0.0001

C. Day 7

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 14.46 2.065 6.011 4.81 < 0.0001 Sex 1 49.12 49.12 142.9 16.33 < 0.0001 Treatment 7 209.8 29.97 87.22 69.73 < 0.0001

190 Supp. Table A3.1: Two-Way ANOVA of the PER for HWO fed Male and Female Flies (Continued)

D. Day 9

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 54.30 7.757 8.908 16.08 < 0.0001 Sex 1 0.9184 0.9184 1.055 0.27 0.3064 Treatment 7 170.9 24.42 28.04 50.63 < 0.0001

E. Bonferroni Post-Tests

Day 1 Day 5 Day 7 Day 9 HW P > 0.05 P > 0.05 P > 0.05 P > 0.05

Lys P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Trp P > 0.05 P<0.01 ** P<0.001 *** P > 0.05

Tyr P > 0.05 P<0.01 ** P > 0.05 P > 0.05

Phe P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Met P > 0.05 P<0.001 *** P<0.001 *** P > 0.05

Pro P > 0.05 P<0.001 *** P<0.001 *** P > 0.05

Suc P<0.001 *** P<0.001 *** P > 0.05 P<0.001 ***

Supp. Table A3.1 Two-Way ANOVA statistics of the mean (±SE) proboscis extension

reflex comparing the honey-water only fed male and female response according to day or age

post-eclosion (A-D) and treatment (100uM amino acid or sugar solution). Bonferroni post-

tests to determine significance between sexes for each amino acid according to day (E).

191 Supp. Table A3.2: Two-Way ANOVA of the PER for HWL fed Male and Female

Flies

A. Day 1

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 2.641 0.3773 0.8774 0.52 0.5294 Sex 1 2.032 2.032 4.725 0.40 0.0334 Treatment 7 472.8 67.54 157.1 93.62 < 0.0001

B. Day 5

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 0.3980 0.056860.06357 0.07 0.9996 Sex 1 0.049450.04945 0.05529 0.01 0.8147 Treatment 7 486.5 69.51 77.71 87.11 < 0.0001

C. Day 7

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 4.199 0.5998 0.8400 0.95 0.5572 Sex 1 0.8046 0.8046 1.127 0.18 0.2914 Treatment 7 373.2 53.31 74.66 84.62 < 0.0001

192

Supp. Table A3.2: Two-Way ANOVA of the PER for HWL fed Male and Female Flies (continued)

E. Bonferroni Post-Tests

Day 1 Day 5 Day 7 HW P > 0.05 P > 0.05 P > 0.05

Lys P > 0.05 P > 0.05 P > 0.05

Trp P > 0.05 P > 0.05 P > 0.05

Tyr P > 0.05 P > 0.05 P > 0.05

Phe P > 0.05 P > 0.05 P > 0.05

Met P > 0.05 P > 0.05 P > 0.05

Pro P > 0.05 P > 0.05 P > 0.05

Suc P > 0.05 P > 0.05 P > 0.05

Supp. Table A3.2 Two-Way ANOVA statistics of the mean (±SE) proboscis extension

reflex comparing the honey-water plus liver fed male and female response according to day or age post-eclosion (A-D) and treatment (100uM amino acid or sugar solution). Bonferroni post-tests to determine significance between sexes for each amino acid according to day

(E).

193

Supp. Table A3.3: Two-Way ANOVA of the PER of HWO- and HWL- fed Females

A. Day 1

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 1.520 0.2171 0.2789 0.24 0.9608 Sex 1 2.253 2.253 2.894 0.36 0.0922 Treatment 7 555.4 79.35 101.9 87.61 < 0.0001

B. Day 5

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 40.99 5.856 4.265 6.78 0.0004 Sex 1 26.63 26.63 19.39 4.41 < 0.0001 Treatment 7 394.0 56.29 40.99 65.19 < 0.0001

C. Day 7

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 52.74 7.535 20.36 13.27 < 0.0001 Sex 1 29.76 29.76 80.42 7.49 < 0.0001 Treatment 7 279.7 39.95 108.0 70.39 < 0.0001

D. Day 9

Source of % of Total Variation DF SSq MS F variation P Value Interaction 7 22.13 3.162 6.758 14.52 < 0.0001 Sex 1 0.049260.04926 0.1053 0.03 0.7464 Treatment 7 92.76 13.25 28.32 60.88 < 0.0001

194

Supp. Table A3.3: Two-Way ANOVA of the PER of HWO- and HWL- fed Females (continued)

E. Bon Ferroni Post-Tests

Day 1 Day 5 Day 7 Day 9 HW P > 0.05 P > 0.05 P<0.01 ** P < 0.05 *

Lys P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Trp P > 0.05 P < 0.05 * P<0.001 *** P > 0.05

Tyr P > 0.05 P < 0.05 * P > 0.05 P > 0.05

Phe P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Met P > 0.05 P < 0.05 * P<0.001 *** P > 0.05

Pro P > 0.05 P < 0.05 * P<0.001 *** P > 0.05

Suc P > 0.05 P > 0.05 P<0.001 *** P<0.001 ***

Supp. Table A3.1 Two-Way ANOVA statistics of the mean (±SE) proboscis extension

reflex comparing the response of honey-water only fed females and honey-water plus liver

fed females according to day or age post-eclosion (A-D) and treatment (100uM amino acid or

sugar solution). Bonferroni post-tests to determine significance between females fed

different diets for each amino acid according to day (E).

195 Supp. Table A3.4: Age Effect Two-Way ANOVA of the PER of HWO- fed Males

Source of % of Total Variation DF SSq MS F variation P Value Interaction 15 0.4946 0.0329837.13 39.51 < 0.0001 Day (Age) 3 0.4073 0.1358 152.9 32.54 < 0.0001 Amino Acid 5 0.1954 0.03907 43.99 15.61 < 0.0001

Day 1 vs 5 1 vs 7 1 vs 9 5 vs 7 5 vs 9 7 vs 9 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Lys P > 0.05 *** *** *** *** *** P<0.001 P<0.001 P<0.001 Trp P > 0.05 P > 0.05 P > 0.05 *** *** *** P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Tyr P > 0.05 *** *** *** *** *** P<0.001 P<0.01 P<0.001 P<0.001 Phe P > 0.05 P > 0.05 *** ** *** *** P<0.001 P<0.001 P<0.001 Met P > 0.05 P > 0.05 P > 0.05 *** *** *** P<0.001 P<0.001 P<0.001 P<0.001 Pro P > 0.05 P > 0.05 *** *** *** ***

Supp. Table A3.4 Two-way ANOVA statistical analysis of the effect of age on honey- water only fed males on the proboscis extension reflex according to amino acid treatment

(A). Statistical significance determined by Bonferroni post-test (B).

196 Supp. Table A3.5: Age Effect Two-Way ANOVA of the PER of HWO- fed Females

Source of % of Total Variation DF SSq MS F variation P Value Interaction 15 33.92 2.261 64.86 26.33 < 0.0001 Day (Age) 3 77.18 25.73 737.8 59.91 < 0.0001 Amino Acid 5 11.66 2.332 66.88 9.05 < 0.0001

Day 1 vs 5 1 vs 7 1 vs 9 5 vs 7 5 vs 9 7 vs 9 P<0.01 P<0.001 P<0.001 P<0.001 P<0.01 P<0.001 Lys ** *** *** *** ** *** P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Trp *** *** *** *** *** *** P<0.001 P<0.001 P<0.001 Tyr P > 0.05 P > 0.05 P > 0.05 *** *** *** P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Phe P > 0.05 *** *** *** *** *** P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Met P > 0.05 *** *** *** *** *** P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 Pro P > 0.05 *** *** *** *** ***

Supp. Table A3.5 Two-way ANOVA statistical analysis of the effect of age on honey- water only fed males on the proboscis extension reflex according to amino acid treatment

(A). Statistical significance determined by Bonferroni post-test (B).

197 APPENDIX 4 SUPPLEMENTARY TABLES FOR CHAPTER 7

Supp. Table A4.1: Two-Way ANOVA of the EAG Response of HWO- and HWL-fed

Female Flies

A. Putrescine – F

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.05596 0.01399 4.213 19.23 0.0047 Diet 1 0.00041270.0004127 0.1243 0.14 0.7257 Day 4 0.04866 0.01216 3.664 16.72 0.0101

B. Cadaverine – F

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.02903 0.007258 9.455 25.45 < 0.0001 Diet 1 0.00093770.0009377 1.222 0.82 0.2741 Day 4 0.04418 0.01105 14.39 38.73 < 0.0001

C. Butyric Acid – F

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.2770 0.069244.008 15.35 0.0058 Diet 1 0.1018 0.1018 5.891 5.64 0.0180 Day 4 0.3200 0.080004.631 17.73 0.0024

198 Supp. Table A4.1: Two-Way ANOVA of the EAG Response of HWO- and HWL-fed Female Flies (continued)

D. Dimethyl Disulfide – F

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.06469 0.01617 8.992 21.56 < 0.0001 Diet 1 0.0076540.007654 4.256 2.55 0.0438 Day 4 0.1270 0.03174 17.65 42.32 < 0.0001

E. Phenol – F

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.0036230.0009057 1.028 5.52 0.4027 Diet 1 0.0000030.000003 0.0034360.00 0.9535 Day 4 0.02147 0.005367 6.094 32.72 0.0005

F. Bon Ferroni Post-Tests

Dimethyl Putrescine Cadaverine Butyric Acid Disulfide Phenol Day2 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day4 P < 0.05 * P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05

Day10 P < 0.05 * P<0.001 *** P > 0.05 P > 0.05 P > 0.05

Day12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

199 Supp. Table A4.2: Two-Way ANOVA of the EAG Response of HWO- and HWL-fed

Male Flies

A. Putrescine – M

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.3784 0.0630749.05 40.98 < 0.0001 Diet 1 0.020870.02087 16.23 2.26 0.0001 Day 6 0.4328 0.0721356.10 46.87 < 0.0001

B. Cadaverine – M

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.4844 0.08074111.0 44.77 < 0.0001 Diet 1 0.022230.02223 30.56 2.05 < 0.0001 Day 6 0.5266 0.08777120.7 48.67 < 0.0001

C. Butyric Acid – M

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.3496 0.05827 5.022 26.48 0.0002 Diet 1 0.0062930.006293 0.5424 0.48 0.4639 Day 6 0.1406 0.02343 2.020 10.65 0.0742

D. Dimethyl Disulfide – M

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.2110 0.03516 10.96 36.72 < 0.0001 Diet 1 0.0042540.004254 1.326 0.74 0.2535 Day 6 0.1380 0.02300 7.168 24.01 < 0.0001

E. Phenol – M

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.07958 0.01326 11.12 35.99 < 0.0001 Diet 1 0.00027620.0002762 0.2316 0.12 0.6321 Day 6 0.06849 0.01141 9.569 30.97 < 0.0001

200 Supp. Table A4.2: Two-Way ANOVA of the EAG Response of HWO- and HWL-fed

Male Flies

(continued)

F. Bonferroni Post-Tests

Dimethyl Putrescine Cadaverine Butyric Acid Disulfide Phenol Day2 P > 0.05 P < 0.05 P > 0.05 P > 0.05 P < 0.05 *

Day4 P > 0.05 P<0.001 *** P<0.01 ** P<0.001 *** P > 0.05

Day6 P<0.001 *** P<0.001 *** P > 0.05 P > 0.05 P<0.001 ***

Day8 P<0.001 *** P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Day10 P > 0.05 P > 0.05 P > 0.05 P<0.001 *** P > 0.05

Day12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

201 Supp. Table A4.3: Two-Way ANOVA Analysis of the EAG Response of HWL-fed

Male and Female Flies

A. Putrescine

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.01454 0.002423 0.7189 3.72 0.6355 Sex 1 0.00000060.0000006 0.0001908 0.00 0.9890 Day 6 0.1167 0.01945 5.773 29.87 < 0.0001

B. Cadaverine

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.01971 0.003286 2.801 7.91 0.0167 Sex 1 0.00028910.0002891 0.2465 0.12 0.6211 Day 6 0.1459 0.02431 20.72 58.54 < 0.0001

C. Butyric Acid

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.049290.008214 0.5231 2.31 0.7893 Sex 1 0.010960.01096 0.6981 0.51 0.4058 Day 6 0.7562 0.1260 8.027 35.41 < 0.0001

D. Dimethyl Disulfide

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.03939 0.006565 2.092 6.68 0.0643 Diet 1 0.00090280.0009028 0.2877 0.15 0.5933 Day 6 0.3175 0.05291 16.86 53.81 < 0.0001

E. Phenol

Source of % of Total Variation DF SSq MS F variation P Value Interaction 6 0.01298 0.002164 2.304 8.60 0.0445 Diet 1 0.0024700.002470 2.630 1.64 0.1097 Day 6 0.07456 0.01243 13.23 49.35 < 0.0001

202

Supp. Table A4.3: Two-Way ANOVA Analysis of the EAG Response of HWL-fed

Male and Female Flies (continued)

F. Bonferroni Post-Tests

Dimethyl Putrescine Cadaverine Butyric Acid Disulfide Phenol Day2 P > 0.05 P<0.01 P > 0.05 P > 0.05 P<0.01

Day4 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day6 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day10 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

203 Supp. Table A4.4: Two-Way ANOVA Analysis of the EAG Response of HWO-fed

Male and Female Flies

A. Putrescine

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.01133 0.002831 2.262 10.37 0.0750 Sex 1 0.005388 0.005388 4.304 4.93 0.0430 Day 4 0.02737 0.006844 5.467 25.07 0.0009

B. Cadaverine

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.06667 0.01667 17.46 33.59 < 0.0001 Sex 1 0.008732 0.008732 9.148 4.40 0.0039 Day 4 0.07347 0.01837 19.24 37.01 < 0.0001

C. Butyric Acid

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.059290.01482 1.447 8.24 0.2311 Sex 1 0.027940.02794 2.727 3.88 0.1044 Day 4 0.068820.01720 1.679 9.56 0.1680

D. Dimethyl Disulfide

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.009000 0.002250 1.582 3.12 0.1923 Sex 1 0.005909 0.005909 4.155 2.05 0.0464 Day 4 0.1970 0.04925 34.63 68.24 < 0.0001

E. Phenol

Source of % of Total Variation DF SSq MS F variation P Value Interaction 4 0.003921 0.00098013.410 15.77 0.0163 Sex 1 0.00047490.0004749 1.652 1.91 0.2054 Day 4 0.007816 0.001954 6.798 31.44 0.0002

204

Supp. Table A4.4: Two-Way ANOVA Analysis of the EAG Response of HWO-fed

Male and Female Flies (continued)

F. Bonferroni Post-Tests

Dimethyl Putrescine Cadaverine Butyric Acid Disulfide Phenol Day2 P > 0.05 P<0.001 P > 0.05 P > 0.05 P > 0.05

Day4 P > 0.05 P<0.001 P > 0.05 P > 0.05 P > 0.05

Day10 P<0.01 P<0.001 P > 0.05 P < 0.05 P > 0.05

Day12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Day14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

205 Supp. Table A4.5: Two-Way ANOVA of the Age Effect of the EAG of HWO-fed

Females

Source of % of Total Variation DF SSq MS F variation P Value Interaction 16 0.038670.002417 1.034 7.54 0.4255 Day 4 0.1258 0.03144 13.45 24.53 < 0.0001 Volatile 4 0.032620.008154 3.488 6.36 0.0096

Dimethyl Day Putrescine Cadaverine Butyric Acid Phenol Disulfide 2 v 4 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

2 v 10 P > 0.05 P > 0.05 P < 0.05 * P<0.01 ** P > 0.05

2 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

2 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

4 v 10 P < 0.05 P > 0.05 P<0.01 ** P<0.01 ** P > 0.05

4 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

4 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

10 v 12 P<0.01 ** P < 0.05 * P > 0.05 P<0.001 ** P > 0.05

10 v 14 P<0.01 ** P < 0.05 * P<0.01 ** P<0.001 *** P > 0.05

12 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

Supp. Table A4.5 Two-way ANOVA statistical analysis of the effect of age on the mean electroantennogram response of honey-water only fed female flies to decomposition- related volatiles (A). Statistical significance determined by Bonferroni post-test (B).

206 Supp. Table A4.6: Two-Way ANOVA of the Age Effect of the EAG of HWL-fed

Females

Source of % of Total Variation DF SSq MS F variation P Value Interaction 24 0.3183 0.01326 1.867 12.98 0.0113 Day 6 0.5071 0.08451 11.90 20.68 < 0.0001 Volatile 4 0.2770 0.06925 9.749 11.30 < 0.0001

Dimethyl Day Putrescine Cadaverine Butyric Acid Phenol Disulfide 2 v 4 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 P > 0.05 2 v 6 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 10 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 4 v 6 P > 0.05 P > 0.05 P<0.001*** P > 0.05 P > 0.05 4 v 8 P > 0.05 P > 0.05 P<0.001*** P > 0.05 P > 0.05 4 v 10 P > 0.05 P > 0.05 P<0.01 ** P > 0.05 P > 0.05 4 v 12 P > 0.05 P > 0.05 P<0.001 *** P < 0.05 * P > 0.05 4 v 14 P > 0.05 P > 0.05 P<0.001 *** P < 0.05 * P > 0.05 6 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 6 v 10 P > 0.05 P > 0.05 P < 0.05 * P > 0.05 P > 0.05 6 v 12 P > 0.05 P > 0.05 P > 0.05 P < 0.05 * P > 0.05 6 v 14 P > 0.05 P > 0.05 P > 0.05 P < 0.05 * P > 0.05 8 v 10 P > 0.05 P > 0.05 P < 0.05 * P > 0.05 P > 0.05 8 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 8 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 10 v 12 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 P > 0.05 10 v 14 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 P > 0.05 12 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

207

Supp. Table A4.6: Two-Way ANOVA of the Age Effect of the EAG of HWL-fed

Females (continued)

Supp. Table A4.6 (continued from previous page) Two-way ANOVA statistical analysis of the effect of age on the mean electroantennogram response of honey-water and liver fed female flies to decomposition-related volatiles (A). Statistical significance determined by Bonferroni post-test (B).

208 Supp. Table A4.7: Two-Way ANOVA of the Age Effect of the EAG of HWO-fed

Males

Source of % of Total Variation DF SSq MS F variation P Value Interaction 24 0.6580 0.02742 6.641 24.12 < 0.0001 Day 6 1.293 0.2155 52.20 47.41 < 0.0001 Volatile 4 0.1239 0.03099 7.505 4.54 < 0.0001

Dimethyl Day Putrescine Cadaverine Butyric Acid Phenol Disulfide 2 v 4 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 6 P<0.001 *** P<0.001 *** P<0.01 ** P > 0.05 P > 0.05 2 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 10 P > 0.05 P > 0.05 P > 0.05 P<0.01 ** P > 0.05 2 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 2 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 4 v 6 P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05 P > 0.05 4 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 4 v 10 P > 0.05 P > 0.05 P < 0.05 * P<0.001 *** P > 0.05 4 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 4 v 14 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 P > 0.05 6 v 8 P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05 P<0.01 ** 6 v 10 P<0.001 *** P<0.001 *** P<0.01 ** P > 0.05 P<0.01 ** 6 v 12 P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05 P<0.01 ** 6 v 14 P<0.001 *** P<0.001 *** P > 0.05 P > 0.05 P < 0.05 * 8 v 10 P > 0.05 P > 0.05 P<0.01 ** P<0.001 *** P > 0.05 8 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 8 v 14 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 P > 0.05 10 v 12 P > 0.05 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 10 v 14 P > 0.05 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 12 v 14 P > 0.05 P > 0.05 P<0.01 ** P > 0.05 P > 0.05

209

Supp. Table A4.7: Two-Way ANOVA of the Age Effect of the EAG of HWO-fed

Males (continued)

Supp. Table A4.6 (continued from previous page) Two-way ANOVA statistical analysis of the effect of age on the mean electroantennogram response of honey-water only fed male flies to decomposition-related volatiles (A). Statistical significance determined by

Bonferroni post-test (B).

210 Supp. Table A4.8: Two-Way ANOVA of the Age Effect of the EAG of of HWL-fed

Males

Source of % of Total Variation DF SSq MS F variation P Value Interaction 24 0.1807 0.007529 2.250 12.99 0.0014 Day 6 0.4761 0.07935 23.72 34.22 < 0.0001 Volatile 4 0.1292 0.03230 9.655 9.28 < 0.0001

Dimethyl Day Putrescine Cadaverine Butyric Acid Phenol Disulfide 2 v 4 P > 0.05 P > 0.05 P<0.001 *** P<0.01 ** P > 0.05 2 v 6 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 * 2 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P < 0.05 * 2 v 10 P > 0.05 P<0.01 ** P > 0.05 P > 0.05 P < 0.05 * 2 v 12 P > 0.05 P<0.01 ** P > 0.05 P < 0.05 * P < 0.05 * 2 v 14 P > 0.05 P<0.01 ** P > 0.05 P < 0.05 * P < 0.05 * 4 v 6 P > 0.05 P < 0.05 * P<0.001 *** P<0.001 *** P > 0.05 4 v 8 P > 0.05 P < 0.05 * P<0.001 *** P > 0.05 P > 0.05 4 v 10 P > 0.05 P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05 4 v 12 P > 0.05 P<0.001 *** P<0.001 *** P<0.001 *** P > 0.05 4 v 14 P > 0.05 P<0.01 ** P<0.001 *** P<0.001 *** P > 0.05 6 v 8 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 6 v 10 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 6 v 12 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 6 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 8 v 10 P<0.01 ** P > 0.05 P > 0.05 P < 0.05 * P > 0.05 8 v 12 P<0.01 ** P > 0.05 P > 0.05 P<0.001 *** P > 0.05 8 v 14 P > 0.05 P > 0.05 P > 0.05 P<0.001 *** P > 0.05 10 v 12 P > 0.05 P > 0.05 P < 0.05 * P > 0.05 P > 0.05 10 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 12 v 14 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05

211

Supp. Table A4.8: Two-Way ANOVA of the Age Effect of the EAG of of HWL-fed

Males (continued)

Supp. Table A4.7 (continued from previous page) Two-way ANOVA statistical analysis of the effect of age on the mean electroantennogram response of honey-water and liver fed male flies to decomposition-related volatiles (A). Statistical significance determined by Bonferroni post-test (B).

212 APPENDIX 5

A PHYLOGENETIC TREE OF DIPTERA

213 APPENDIX 5

Figure 5.1 – A Phylogenetic Tree of Diptera – Part 1

214 APPENDIX 5

Figure 5.1 – A Phylogenetic Tree of Diptera – Part 2

215

Figure 5.1: A Phylogenetic Tree of Diptera Adapted from the Tree of Life Project (www.tolweb.org). (Wood & Borkent, 1989;

Oosterbroek & Courtney, 1995; Yeates and Wiegmann, 1999, 2005; Yeates, 2003, 2002; Nagatomi &Lin, 1994; Weigmann, 2003;

Yeates, et al, 2003; Mowton & Wiegmann, 2004; Zloty, 2005; McAlpine & Woods, 1989; Brown, 1992; Cumming, 1995; Disney,

2004; McAlpine, 1990; Wheeler, 2000; Barra, et al., 2007; Rognes, 1997; Pape & Arnaud, 2001)

216

VITA NAME: Allissa Marie Melrose Blystone ADDRESS: 300 College Park Department of Biology University of Dayton 300 College Park Dayton, OH 45469-2320 PHONE: 937.229.2583 FAX: 937.229.2021 EMAIL: [email protected] EDUCATION: B.S., Baylor University, Waco, Texas, 2003 M.A., Baylor University, Waco, Texas, 2004

217