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BLOW FLY OVIPOSITION (DIPTERA:
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BLOW FLY OIVPOSITION (DIPTERA: CALLIPHORIDAE) IN MID-
MICHIGAN IN RELATION TO SUNRISE AND SUNSET

By

Kristi Nichole Zurawski

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Entomology

2008

ABSTRACT

“Blow fly oviposition (Diptera: Calliphoridae) in Mid-Michigan in Relation to Sunrise
and Sunset”

By

Kristi Nichole Zurawski
The objective of this research was to determine whether blow flies of mid-Michigan
demonstrate nocturnal oviposition, and if so, under what conditions. In two different field
seasons blow fly oviposition was evaluated in relation to sunset or sunrise. Two
laboratory studies examined oviposition under controlled conditions. During 2006, pigs
were exposed to fly colonization in one hour intervals, beginning two hours before sunset
and ending two hours after sunset, to determine the occurrence of initial oviposition
relative to sunset. In 2007, pigs were placed in the field two hours after sunset and
oviposition was recorded into the following morning. In 2006, oviposition only occurred
in intervals before sunset, but never after dark. During 2007, no oviposition took place
from two hours after sunset to sunrise. A chi square- test using data from both summers
was used to quantify the probability of nocturnal oviposition, which was significantly less
than the observed oviposition rate during daylight hours (X2=10.67; d.f. 1; p<0.01). A
laboratory experiment using bait either hanging 22 cm above or directly on the ground in
a completely dark room found that oviposition never occurred. Another study observed
Lucilia sericata (Meigen) flight activity in the dark and in 13 out of 15 trials blow flies
glided rather than fell to the ground when forced to fly. Based on my studies, when using
insects to help narrow the PMI interval in criminal investigations, nocturnal oviposition

should be considered to occur at very low probabilities or not at all.

ACKNOWLEDGEMENTS

I would like to acknowledge the members of my committee for their support and
guidance through my Masters program. Thanks to Dr. Richard Merritt for financial
support and research advice; Dr. Jim Miller for help with laboratory experiments and
experimental design; Dr. Eric Benbow for statistical advice and data analysis; and Dr.
Neal Haskell for introducing me to forensic entomology and research advice.

Jareé Johnson and Anastasia Legowsky deserve a large amount of thanks for their
help in the field. They were my research assistants, field hands, and friends. Al Snedegar
and his associates at the swine barn have my gratitude for providing me with research
materials. Aaron Tarone deserves thanks for the use of his laboratory strain of Lucilia
sericata blowflies.

Brian Bugajski deserves a significant amount of credit for help in the field,
keeping me sane, building the cages and platforms used in the experiments, and allowing
maggots to be grown on his workbench in the garage. This project wouldn’t have
happened without his support.

Finally, I would like to thank to my Mom and Dad who have supported me both
financially and emotionally for the past 25 years. I wouldn’t have pursued this dream

without their encouragement.

iii

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. v
LIST OF FIGURES ................................................................................. vi

INTRODUCTION .................................................................................... 1

CHAPTER 1:

PUBLICATION ....................................................................................... 5
Abstract ....................................................................................... 5
Introduction ................................................................................... 6
Methods ....................................................................................... 8

2006 Field Season Experiment .................................................. 10

2007 Field Season Experiment ................................................... 10

Laboratory Experiment on Oviposition ......................................... 11

Analysis ............................................................................. 11

Results ....................................................................................... 12
Nocturnal Oviposition ............................................................ 12

2006 Field Season ........................................................ 12

2007 Field Season ......................................................... 12

Laboratory ................................................................... 13

Factors Influencing Oviposition ................................................. 13

2006 Field Season ........................................................ 13

2007 Field Season ......................................................... 13

Laboratory ................................................................... 14

Discussion ................................................................................... 15
APPENDICES .................................................................................... 19
Appendix A- Tables ............................................................... 19

Appendix B- Figures ................................................................ 24

Appendix C- Temperature and Light Graph ................................... 40

Appendix D- Voucher Specimen Information ................................. 57
LITERATURE CITED ............................................................................ 62

iv

LIST OF TABLES

Table 1. Nocturnal oviposition studies found in the primary forensic entomology
literature, providing the design, major results and species studied ............................ 20

Table 2. Environmental conditions measured during the summer 2006 field trials when
oviposition did and did not occur during each time interval. The species of fly that was
found to oviposit are also given. T-2 = 2 h before sunset; T-l = 1 h before sunset; T =
Sunset; T+1 = 1 h after sunset; T+2 = 2 h after sunset. ........................................ 21

Table 3. Environmental conditions during the summer of 2007 for the time of first fly
appearance in the field as well as the time of oviposition. Predicted number of eggs of
oviposition and species of those eggs is also given .............................................. 22

Table 4. Results of t-tests comparing abiotic factors in trials with and without oviposition.
Bold p-values indicates significance .............................................................. 23

LIST OF FIGURES

Figure 1. An aerial map of research site from Google maps. The site was on the property
of the Michigan State University Entomological Research Station, seen in the lower
lefihand corner of the photo ....................................................................... 25

Figure 2. Th Linear regression model of Lucilia sericata egg mass weight and egg
number. R2 = 0.98. The equation for the line is: y = 3275.9x + 17.778 .................... 26

Figure 3. The percent oviposition during each time interval over the summer of 2006.
Data were compiled from eggs collected during each of four trials over the summer. ....27

Figure 4. The percent of each species composition of eggs collected during three time
intervals over the summer of 2006. Eggs were collected at the end of each one hour time
interval and reared to the third larval post feeding instar for identification ................. 28

Figure 5. The percent of each species composition of eggs collected during three trials
over the summer of 2006. Eggs were collected at the end of each one hour time interval

and reared to the third larval post feeding instar for identification ........................... 29

Figure 6. Percent species compositions of adults found on the control pig one week after
trials in the summer of 2006 ....................................................................... 30

Figure 7. The average temperature (°C) (taken at conclusion of trial) where oviposition
did and did not occur. Error bars represent plus and minus 5% error ........................ 31

Figure 8. Average Lux readings (taken at conclusion of the trial) where oviposition did
and did not occur. Error bars represent plus and minus 5% error ............................ 32

Figure 9. Average % Relative humidity for trial dates when oviposition did and did not
occur. Error bars represent plus and minus 5% error .......................................... 33

Figure 10. Average rainfall (cm) for trial days when oviposition did and did not occur.
Error bars represent plus and minus 5% error .................................................. 34

Figure l 1. Wind speed (km/day) for trial dates when oviposition did and did not occur.
Error bars represent plus and minus 5% error .................................................. 35

Figure 12. The percent species composition of all eggs collected from test pigs over the
summer of 2007 .................................................................................... 36

vi

Figure 13. Species composition of eggs collected in five trials over the summer of
2007 .................................................................................................. 37

Figure 14. The percent species composition of adult flies collected during the summer of
2007 .................................................................................................. 38

Figure 15. Percent composition of adult flies collected from test pigs in eight trials during
the summer of 2007. Underlined dates indicate that the trial took place but oviposition
did not occur ........................................................................................ 39

Figure 16. Graph depicting temperatures in the field during the evening of June 29, 2006
during which oviposition occurred ............................................................... 41

Figure 17. Graph depicting temperatures in the field during the evening of August 2,
2006 during which oviposition occurred ......................................................... 42

Figure 18. Graph depicting temperatures in the field during the evening of August 16,
2006 during which oviposition occurred ......................................................... 43

Figure 19. Graph depicting temperatures in the field during the evening of June 14, 2007
until oviposition occurred on June 15, 2007 ..................................................... 44

Figure 20. Graph depicting temperatures in the field during the evening of July 5, 2007
until oviposition occurred on July 6, 2007 ....................................................... 45

Figure 2]. Graph depicting temperatures in the field from the evening of August 16, 2007
until oviposition occurred on August 17, 2007 .................................................. 46

Figure 22. Graph depicting temperatures in the field during the evening of September 12,
2007 and the morning of September 13, 2007 ................................................... 47

Figure 23. Graph depicting temperatures in the field during the evening of September 26,
2007 until oviposition occurred on September 27, 2007 ....................................... 48

Figure 24. Light Readings for June 26, 2006 starting two hours before sunset and ending
two hours after sunset .............................................................................. 49

Figure 25. Light Readings for August 2, 2006 starting two hours before sunset and ending
two hours after sunset ............................................................................. 50

Figure 26. Light Readings for August 16, 2006 starting two hours before sunset and
ending two hours after sunset ..................................................................... 51

Figure 27. Light Readings for June 15, 2007 starting at sunrise and ending when

oviposition occurred. Light readings are in Lux. The leveling off represents the upper
threshold of the light meter (20,000 lux) ......................................................... 52

vii

Figure 28. Light Readings for July 6, 2007 starting at sunrise and ending when
oviposition occurred. Light readings are in Lux. The leveling off represents the upper
threshold of the light meter (20,000 lux) ......................................................... 53

Figure 29. Light Readings for August 17, 2007 starting at sunrise and ending when
oviposition occurred. Light readings are in Lux. The leveling off represents the upper
threshold of the light meter (20,000 lux). The severe jumps in the data are due to cloud
cover ................................................................................................... 54

Figure 30. Light Readings for September 13, 2007 starting at sunrise and ending when
oviposition occurred. Light readings are in Lux. The leveling off represents the upper
threshold of the light meter (20,000 lux) ......................................................... 55

Figure 31. Light Readings for September 27, 2007 starting at sunrise and ending when
oviposition occurred. Light readings are in Lux. The leveling off represents the upper
threshold of the light meter (20,000 lux) ......................................................... 56

viii

Introduction

Forensic entomology uses data derived from insects to assist the criminal justice
system (Catts and Haskell, 1990; Byrd and Castner 2000; Greenberg 1991). This field
dates back to 13th century China when flies were used to assist in solving a murder
involving the use of a farmer’s sickle. The day after the murder the investigator asked the
workers to come together and lay their sickles on the ground. Blow flies were drawn to
one of the sickles and the science of forensic entomology was born (Sung 1981). The
science has substantially developed since then, and continues to attract leading
entomologists into the field.

There are three main types of forensic entomology: urban, stored product pests,
and medico-legal (Catts and Goff 1992). Urban forensic entomology involves insects that
affect man-made structures and other aspects of the human environment (Catts and
Haskell, 1990). Stored product entomology involves insects that infest stored
commodities in some way (Catts and Haskell, 1990).

Medico-legal forensic entomology is the use of insects in determining the amount
of time that has passed since insect colonization, usually within a time period that is
within a few hours of the postmortem interval (PMI) (Catts and Haskell, 1990). Insect
colonization can be used in cases of suicide, homicide and other violent crimes, including
cases of neglect. Many factors can affect the PMI (Catts 1992; Hall and Doisy 1993),
such as temperature (Ames and Turner 2003), disturbance to the body, chemicals (Goff
1993), weather (Mann et a1. 1990) and nocturnal oviposition (Greenberg 1990; Singh and

Bharti 2001; Baldridge et al. 2006; Amendt et al. 2007). Several studies have addressed

the probability of nocturnal oviposition under a variety of conditions, demonstrating
some disagreement among reported findings (Table 1).

The PMI can be calculated using a system of accumulated degree hours (ADH),
or accumulated degree days (ADD). Based on life history characteristics and larval
development times of different fly species, ADH (or ADD) is a measure of thermal
energy required for insect larvae to reach a specific life stage. The ADH’s can be applied
to determine approximate time since death, when fly oviposition initially occurred
(Kamal 1958; Anderson 2000; Byrd and Allen 2001 ). Calculating an ADH usually
assumes nocturnal oviposition does not occur (Catts and Haskell 1990). If blow flies do
indeed oviposit nocturnally, these calculations could be affected by up to 12 hours, which
could be the difference between convicting or acquitting a suspect based on alibis
(Greenberg 1990).

Greenberg (1990) conducted the first study to examine nocturnal oviposition. The
study was conducted in Chicago, Illinois, using rats and ground beef as bait (Greenberg
1990). The species that reportedly oviposited during the night were Lucilia sericata
(Meigen), Calliphora vicina (Robineau-Desvoidy) and Phormia regina (Meigen).
Nocturnal oviposition (from 0100 to 0400) occurred in 33% of trials, and some
oviposition was reported on rat carcasses one hour after sunset but not after.

The experimental design of this study raised a few questions. First, it was conducted in an
urban setting, having substantial artificial lighting. Second, the food source was placed on
the ground near bushes that may have allowed flies to walk rather than fly to the bait.

A second relevant study by Singh and Bharti (2001) examined nocturnal

oviposition of blow flies-in Punjab, India, by placing mutton on a 2 meter high wooden

platform. Oviposition occurred five times at ambient light intensities between 0.6-0.8 lux.
The species reported to oviposit were C. vicina, Chrysomya megacephala (F abricius) and
Chrysomya rufifacies (Macquart). Nocturnal oviposition (from 2200 to 0300) occurred in
33% of trials, which was identical to Greenberg’s (1990) findings. This study supported
the hypothesis that the colonizers flew rather than crawled to the illuminated food.

Baldridge et a1. (2006) evaluated the effect of light on fly ovipositional behavior
in Texas using a variety of baits. Nocturnal oviposition occurred only once in over 200
hours of nocturnal bait presentation. The only oviposition occurred on a pig within an
hour of sunset. Flies were not observed between 2200 and 0600.

Amendt et al. (2007) conducted the most recent study in Munich, Germany using
hedgehogs and beef liver as bait. Oviposition never occurred at night in 51 field trials ,
but was reported to occur under darkened conditions in 33% of laboratory trials (Amendt
et al. 2007).

Woodridge et al. (2007) conducted a study on the flight patterns of L. sericata and
C alliphora vomitoria (Linnaeus) under reported darkness. Using a wind tunnel they
found that fly activity was correlated with light intensity and the probability of oriented
flight leading to oviposition on a corpse in the dark was low. However, they were not
able to conclude with certainty that the wind tunnel was absolutely dark.

The objectives of this study were to: (1) describe nocturnal oviposition in relation
to sunrise and sunset in a rural Michigan setting; and (2) evaluate abiotic variables that
were hypothesized to affect oviposition timing, magnitude and species composition after
sunrise. The hypothesis was that blow flies would not be found to oviposit under natural

conditions after dark and that the initial timing, duration and magnitude of oviposition

occurring after sunrise would vary based on environmental conditions such as

temperature and precipitation.

Chapter One: “Blow fly oviposition (Diptera: Calliphoridae) in Mid-Michigan in
Relation to Sunrise and Sunset”
Abstract-

The most common application of forensic entomology involves establishing a
post mortem interval (PMI) to aid investigators in determining the time since initial insect
colonization of a corpse. In most instances, it is calculated on the assumption that blow
flies (Diptera: Calliphoridae) do not oviposit during the night. The objective of this
research was to determine whether blow flies of mid-Michigan demonstrate nocturnal
oviposition, and if so, under what conditions. In two different field seasons (summers of
2006 and 2007), blow fly oviposition was evaluated in relation to sunset or sunrise. In
addition, two laboratory studies examined oviposition under controlled conditions.

During summer of 2006, pigs were exposed to fly colonization in one hour
intervals, beginning two hours before sunset and ending two hours after sunset, to
determine the occurrence of initial oviposition relative to sunset. In 2007, pigs were
placed in the field two hours after sunset and oviposition was recorded into the following
morning. Temperature and light conditions were monitored during both experiments. In
the first summer, oviposition only occurred in intervals before sunset, but never after
dark. During the second summer, no oviposition took place from two hours after sunset to
sunrise. On average, adult flies arrived at the carcasses 50 min after sunrise but did not
oviposit until at least four hours later. A chi square- test using data from both summers
was used to quantify the probability of nocturnal oviposition, which was significantly less

than the observed oviposition rate during daylight hours (X2=10.67; d.f. 1; p<0.01).

A laboratory experiment using bait either hanging 22 cm above or directly on the
ground in a completely dark room found that oviposition never occurred. A second
laboratory study observed Lucilz'a sericata (Meigen) flight activity in the dark and in 13
out of 15 trials blow flies glided rather than fell to the ground when forced to fly. Based
on my studies, when using insects to help narrow the PMI interval in criminal
investigations, nocturnal oviposition should be considered to occur at very low
probabilities or not at all.

Introduction-

Forensic entomology uses data derived from insects to assist the criminal justice
system (Catts and Haskell, 1990; Byrd and Castner 2000; Greenberg 1991). MedicoJegal
forensic entomology uses insects to determine the amount of time that has passed
between death and insect colonization, referred to as the postmortem interval or PMI
(Catts and Haskell, 1990). Many factors can affect the PMI (Hall and Haskll 1995; Catts
1992; Hall and Doisy 1993), such as temperature (Ames and Turner 2003), disturbance to
the body, chemicals (Goff 1993), weather (Mann et al. 1990) and nocturnal oviposition
(Greenberg 1990; Singh and Bharti 2001; Baldridge et al. 2006; Amendt et al. 2007). The
PMI can be calculated using a method of accumulated degree hours (ADH), or
accumulated degree days (ADD). Based on life history characteristics and larval
development times of different fly species, ADH is a measure of thermal energy required
for larvae to reach their present life stage. The ADH’s can be applied to determine
approximate time since death (Catts 1992; Anderson 2000; Byrd and Allen 2001).
Calculating an ADH usually assumes that blow flies do not oviposit at night (Catts and

Haskell 1990). If blow flies oviposit nocturnally, these calculations could be affected by

up to 12 hours, which could be the difference between convicting or acquitting a suspect,
based on alibi’s (Greenberg 1990). Several studies have addressed the probability of
nocturnal oviposition under a variety of conditions, demonstrating some disagreement
among reported findings (Table 1).

Greenberg (1990) conducted the first study to examine nocturnal oviposition. The
study was conducted in Chicago, Illinois using rats and ground beef as bait (Greenberg
1990). The species that reportedly oviposited during the night were Lucilia sericata
(Meigen), Calliphora vicina (Robineau-Desvoidy) and Phormia regina (Meigen).
Nocturnal oviposition (from 0100 to 0400) occurred in 33% of trials, and some
oviposition was reported on rat carcasses within one hour of sunset. The experimental
design in this study raised some questions. First, it was conducted in an urban setting,
having substantial artificial lighting, and second, the food source was placed on the
ground near bushes which may have have allowed flies to walk rather than fly to the bait.

A second relevant study by Singh and Bharti (2001) examined nocturnal
oviposition of blow flies-in Punjab, India, by placing mutton on a 2 meter high wooden
platform. Oviposition occurred five times at ambient light intensities between 0.6-0.8 lux.
The species reported to oviposit were C. vicina, Chrysomya megacephala (Fabricius),
and Chrysomya rufifacies (Macquart). Nocturnal oviposition (from 2200 to 0300)
occurred in 33% of trials, which was identical to Greenberg’s (1990) findings. This study
supported the hypothesis that the colonizers flew rather than crawled to the illuminated
food.

Baldridge et al. (2006) evaluated the effect of light on fly ovipositional behavior

in Texas using a variety of baits. Nocturnal oviposition occurred only once in over 200

hours of nocturnal bait presentation. The only oviposition that occurred was within an
hour after sunset. Flies were not observed between 2200 and 0600.

Amendt et al. (2007) conducted the most recent study in Munich, Germany using
hedgehogs and beef liver as bait. Oviposition never occurred at night in 51 field trials, but
was reported during the night in 33% laboratory trials (Amendt et al. 2007).

Woodridge et al. (2007) conducted a study on the flight patterns of L. sericata and
Calliphora vomitoria (Linnaeus) in supposed darkness. Using a wind tunnel they found
that fly activity was correlated with light intensity and the probability of oriented flight
leading to oviposition on a corpse in the dark was low. However, they were not able to
conclude with certainty that the wind tunnel was absolutely dark so we studied blow fly
flight activity in complete darkness.

The objectives of this study were to: (1) describe nocturnal oviposition in relation
to sunrise and sunset in a rural Michigan setting; and (2) evaluate abiotic variables that
were hypothesized to affect oviposition timing, magnitude and species composition after
sunrise. The hypothesis was that blow flies would not oviposit under natural conditions
after dark and that the initial timing, duration and magnitude of oviposition occurring
after sunrise would vary based on environmental conditions such as temperature and
precipitation.

Methods-
2006 Field Season Experiment-

Six pigs weighing an average of 25 kg were obtained from the Michigan State
University Swine Research Facility. Each pig was euthanized by lethal injection at

approximately 1600 hours and transferred to a black plastic garbage bag immediately

following death. Each bag was tightly tied off and placed into a second bag to prevent
insect access. The pigs were immediately transported to the Michigan State University
Entomological Field Research Center, approximately 0.8 km from the Michigan State
University Swine Research Facility and stored inside of a barn for approximately 4 h. A
Hobo© temperature data logger (set at 1 min intervals) was placed next to the bagged pigs
in the barn and moved with the pigs into the field for the duration of the experiment.

Two wooden platforms, 15 cm high were erected 1.6 m apart in a grassy field 183
m from the barn (Figure 1). Tanglefoot©, an adhesive paste, was generously applied to
each leg of both platforms to capture any crawling insects. Beginning two hours before
sunset, one pig was removed from the barn and transported to the field. Pigs were
removed from the bags and placed on a platform facing north. After one hour of exposure
the pig was removed and replaced with another pig from the barn. This process was
repeated every hour until two hours after sunset for a total of five l-hour time intervals.

After exposure, the pigs were carefully examined for the presence of fly eggs,
which were removed using a small paintbrush and placed in a styrofoam cup containing a
piece of beef liver on top of a moistened paper towel (Tarone and Foran 2007). After the
last pig was examined, the styrofoam cups were transported to the laboratory. Hatched
larvae were fed 28g of beef liver each day until reaching the third larval instar post-
feeding stage, after which they were preserved in 70% EtOH and identified to species
(Stoganovich et a1 1962).

In 2006 and 2007, a Hobo© temperature data logger and a TBS-1336 Data logging
light meter were centrally located between the platforms. Both data loggers logged at 1-

min intervals for the duration of each trial. Sunset and sunrise were determined using the

Weather Channel website (www.weather.com), which coincides with data on NOAA’s

 

website, but the readings were closer to the site.

During this first summer a pig was placed in the field on the second platform two
hours before sunset. A wood-framed cage wrapped with chicken wire was placed over
this pig. The pig was left exposed for one week, and the cage prevented predators from
contacting the carcass. Adult flies were collected in the days following the initial
experiment to acquire information about the local species that were present at the time of
the experiment. These flies were later pinned and identified to species using the
taxonomic key of Whitworth (2006).

2007 Field Season Experiment-

The same experimental design of 2006 was repeated in 2007 except for the
following conditions: 1) three replicate pigs were obtained for each of eight trials; 2)
replicate pigs were exposed from two hours after sunset until oviposition occurred after
sunrise the following morning; and 3) the pigs were placed on 15 cm high platforms that
were 10 m apart, examined every hour after sunset until sunrise, and thereafter every half
hour. Examination involved a thorough inspection of the body, with a concentration on
mucus membranes and orifices. Egg collection and rearing were performed as in 2006,
except adult flies were collected from the three pigs during the morning of each trial.

A regression model was developed to predict egg number from egg mass during
the 2007 field season in order to determine the magnitude of oviposition during each trial
(Figure 2). In five separate instances, a piece of beef liver was placed into a cage that

contained a laboratory strain of L. sericata, and after 2 h the liver was removed from the

10

cage and the egg masses were collected, separated into sixteen different egg counts
ranging from 5 to 250 g, and weighed on a Sautorius balance.
Laboratory Experiment on Oviposition-

Ten adult flies in a 60:40 female/male ratio of laboratory reared L. sericata were
placed into a 473 mL glass mason jar and then into a light tight box for 1 h to acclimate
to darkness. After 1 h, the jar was set on its side on the floor in a completely dark room
(1.2 m by 4.2 m), and the lid was removed. A weighing dish containing 28 g of beef liver
was either placed in a small basket that was hung from the ceiling of the room 22 cm
above the floor or directly on the floor. The flies were left overnight for eight h and the
liver was checked in the morning for eggs. Each experiment was repeated five times and
five trials also were performed with the lights on for both bait placements.

A second observational laboratory experiment involved 15 L. sericata adults that
were acclimated to darkness and placed into 10mL glass tubes with a piece of cotton
covering the opening. The flies were then held at eye level, and launched into the air by
flicking the tube. After 30 seconds the lights were turned on and the flies were located
and collected. Prior to launching the live flies, 15 plastic flies were launched to form an
area where flies would land if they fell straight to the ground as opposed to where they
would land if they were actively flying or simply gliding.

Analysis-

The probability of nocturnal oviposition was analyzed using a chi square test.
Using data from both field seasons, the probability of observed diurnal oviposition was
compared to observed nocturnal oviposition that occurred in both the field and laboratory

experiments. T-tests were used to evaluate environmental and climatic differences

ll

between dates when oviposition occurred compared to dates when there was none.
Pearson Product Moment correlation analyses were used to evaluate the relationship
between the number of eggs oviposited and the environmental variables. The relative
species composition of blow fly adults and larvae were to describe the community
composition across the field seasons.

Results-

Nocturnal Oviposition
2006 Field Season

Oviposition occurred 2 hours before sunset in 50% of the trials, 1 hour before
sunset in 75% of the trials, and at sunset in 50% of the trials. Oviposition did not occur 1
and 2 hours after sunset (Table 2, Figure 3). No insects were found crawling up the

platform and on to the pigs in 2006 or 2007.

2007 Field Season

Adult flies were never observed after sunset, and no nocturnal oviposition was
documented on any pig in any trial. Flies were first observed on average 50 minutes after
sunrise and oviposited 4 hours later. The earliest that flies arrived and oviposited were 8
minutes and 3 ‘/2 hours after sunrise, respectively (Table 3). The average lux reading in
trials when oviposition occurred was 19193 compared to 11805 when oviposition was not
seen. Average temperatures were also higher when oviposition occurred (32° C) than
when it did not (23° C).

No oviposition occurred at night, but oviposition did occur in 33% of the trials

during the daylight. Using this 33% oviposition rate as the probability of oviposition at

12

any time, the probability of nocturnal oviposition was significantly lower than oviposition
during daylight hours ( X2 = 10.67; d.f. = 1, p<0.01).
Laboratory

In both laboratory experiments no oviposition occurred in the dark. Under lighted
conditions, oviposition occurred in 60% of the trials when the bait was hanging from the
ceiling and 80% when on the floor. This resulted in an average 70% probability of
oviposition under laboratory lighted conditions which was significantly greater than
oviposition under dark conditions (X2 = 7.0; d.f. = 1, p<0.01)

Factors influencing oviposition:
2006 Field Season:

In the hours before sunset Lucilia coeruleiviridis (Macquart) was the most
common blow fly species found ovipositing compared to P. regina which was the most
common species found to oviposit during the sunset time interval (Figure 4); however, C.
vomitoria made up from about 20 — 35% of the eggs collected during the trials. L.
coeruleiviridis was the most frequent species ovipositing in early summer compared to C.
vomitoria at the end of the season (Figure 5). P. regina was the most frequent adult fly
collected from pigs after one week of exposure among all trials (Figure 6).The species
composition changed not only by time interval, but also by the date (Figures 3-5). P.
regina was the most prevalent blow fly ovipositing at sunset.

2007 Field Season:

On days when oviposition occurred, temperature was significantly greater (t =

3.46; d.f. = 6; p= 0.014) than days when no oviposition occurred (Figure 7). Light was

also significantly greater (t= 9.81; d.f.: 6; p<0.001) on days when oviposition (Figure 8).

13

There were no statistically significant differences in percent relative humidity (t=0.7l;
d.f.: 6; p=0.50), wind speed (t= 1.62;d.f.= 6; p=0.16), or precipitation (t=1.21; d.f.=6 ;
p=0.27), between dates of no oviposition and those with detected oviposition (Figures 9—
1]. Correlation analyses were found to be insignificant between number of eggs and
temperature (p= -0.05; d.f. 3; p=0.47), light (p= 0.37; d.f. 3; p=0.29), wind speed (p= -
0.34; d.f. 3; p=0.31), relative humidity (p= 0.038; d.f. 3; p=0.48), rainfall (p= 0.45; d.f. 3;
p =0.25), and time of oviposition (p= -0.62; d.f. 3; p=0.15).
L. sericata made up the highest percentage of blow fly species eggs collected from pigs
during the 2007 summer; however, this varied by date (Figures 12, 13). P. regina was the
most common adult fly species when averaged across the season; but this also varied by
date (Figures 14, 15). Similar to the 2006 summer, L. coeruleiviridis was most abundant
early in the summer, but the community changed to almost entirely L. sericata in July
and August and then was replaced by C. vicina and P. regina in September. There was a
higher species richness in the adults collected, compared to the eggs (Sarcophagidae and
Pollenia rudis (Fabricius) were only collected as adults) (Figures 12-15). It is interesting
to note that P. rudis is a known parasite of earthworms and most likely was at the carcass
incidentally, but was present at the study site. P. regina adults were found in all trials
except on 16 August 2007 when only L. sericata was collected; however, Cochliomyia
macellaria (Fabricius) eggs were found in that trial. Although L. sericata had the highest
oviposition rate after sunrise, P. regina was the most prevalent adult collected.
Laboratory:

When stimulated for flight, flies were observed gliding to the ground, rather than

flying directly, or simply falling. In 15 trials, 13 flies glided which suggested that they

14

may have some control over movement when provoked to fly but not enough to take off
into flight. The other two flies simply fell to the ground. If flies will not initiate flight
when stimulated in the dark, it is unlikely that the will fly to a carcass at night. If flies are
not flying, then the likelihood of oviposition is very low. The flies’ behavior was
consistent between trials suggesting an innate reaction.

Discussion:

In twenty-two separate trials, including both field and laboratory conditions,
nocturnal oviposition was not found to occur once. These findings are in contrast to
those of Greenberg (1990) and Singh and Bharti (2000), where both reported nocturnal
oviposition in 33% of their trials. However, it is consistent with the findings of Amendt et
al. (2007) and Baldridge et al. (2006). Baldridge et al. (2006) recorded oviposition only
once and it was approximately 30 min after sunset. This agrees with my field results in
2006 that showed oviposition occurred when there was still ambient light during sunset,
but not during the dark hours. These data suggest that if a body is exposed after sunset, it
will not be colonized until the following morning or later which is consistent with what
Smith (1986) described in his manual on forensic entomology. My laboratory results
suggest that under dark conditions, flies do not engage in active flight, providing a
plausible reason why nocturnal oviposition was not documented in this field study, or
others (see above), under naturally dark conditions (i.e., no artificial lighting).

P. regina has been reported to arrive later on remains than other blowflies, such as
L. sericata, and C. vicina (Hall and Doisy 1993; Byrd and Castner 2001; Lord and Burger
1984; Demo and Cothran 1976). Anderson and VanLaerhoven (1996) collected adult P.

regina within 24 h of death, but eggs were not laid until 48 hours after death. The results

15

of my 2006 field season demonstrated that P. regina adults and eggs were collected less
than 24 hours after death, but before or during sunset time periods and never after dark.

In the 2007 field season, the earliest that flies appeared at the carcass was 15 min
after sunrise, but oviposition by those flies did not occur for at least another 3 hours. This
is important in PMI calculations because it should not be assumed that flies will oviposit
immediately following sunrise. The abiotic factors of temperature and light were
significantly higher in trials where oviposition occurred, compared to those trials when it
did not (Table 4, Figures 7, 8). Temperature and light have been shown to have be
positively correlated with oviposition for other flies such as the blueberry maggot
Rhagoletis mendax (Curran), but negatively correlated with the Carribean fruit fly
Anastrepha suspensa (Loew) (Smith and Prokopy 1981; Burk 1983). Wind speed,
humidity and precipitation were not statistically significant between trials with and
without oviposition, although all average values were lower in trials with oviposition
(Table 4). The relationship between number of eggs laid and abiotic factors were
evaluated and no significant associations were found. However, because of the low
sample size (i.e., number of trial of no oviposition), the statistical power was low and
may have limited my ability to detect significant differences in these variables.

This study differs from previous studies on several important points. First, with
the exception of a few trials by Baldridge et al. (2006), this is one of the first studies to
use pig carcass models, which have been shown to be the best substitute for humans
(Goff 2000, Byrd and Castner 2001, Schoenly et al. 2007). Second, I remained at the
field site through the duration of the trials and made continual, hourly observations as

opposed to leaving the bait overnight and documenting oviposition the next morning.

16

This allowed for important observations about blowfly behavior to be recorded, namely
the time of adult fly’s arrival, their disappearance around sunset, and the fact that it took
at least 3 hours after sunrise to oviposit after appearing at the carcass. In addition, species
composition of adults and larvae were recorded in both field seasons and the most
prevalent adult species were not necessarily the species that were most often found to
have oviposited. This indicated that adults may be attracted to the carcass, but oviposition
conditions may not be optimal at that particular time. If eggs are collected from a body
and one is trying to determine the PMI, they should not assume that eggs are the same
species as the adults that were collected at the same time.

A criticism of Greenberg’s (1990) study was that perhaps flies had crawled to
oviposit on the bait instead of flying to it. My laboratory studies did not support this
hypothesis since no oviposition occurred under darkness when bait was placed directly on
the floor. Another criticism of Greenberg (1990) was that there was a streetlight nearby
and the light could have affected the fly’s behavior. My findings suggest that the
streetlight may have been necessary for oviposition in the Greenberg (1990) study. I
found that a significant relationship exists between light levels in the field and whether or
not oviposition occurs. I also found that no nocturnal oviposition occurred in field studies
that took place in a rural environment with little nearby artificial lighting. If there is
enough artificial ambient light, then nocturnal oviposition may be possible. Future studies
should test oviposition under controlled lighting conditions in the field.

The results of this study suggest that if a body is exposed to the environment after
sunset, it will not be colonized on average until at least three hours afier sunrise the

following morning, provided temperature and light conditions are favorable. In

17

conclusion, my studies demonstrate that nocturnal oviposition is unlikely and improbable
even under favorable weather, temperature and light conditions. In criminal
investigations using insects to help narrow the PMI interval, it should be noted that there
are time intervals immediately after sunset and sunrise where oviposition is unlikely, and
when oviposition does occur after sunrise, that identified adult flies associated with a
corpse may not be the species ovipositing at that time. Both of these factors should be
considered when developing entomological—based PMI estimates during criminal

investigations.

18

APPENDIX A

TABLES

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56

APPENDIX D

VOUCHER SPECIMEN INFORMATION FOR THESIS

57

Appendix D
Record of Deposition of Voucher Specimens"
The specimens listed on the following sheet(s) have been deposited in the named museum(s) as
samples of those species or other taxa, which were used in this research. Voucher recognition
labels bearing the Voucher No. have been attached or included in fluid-preserved specimens.
Voucher No.: 2008-02

Title of thesis or dissertation (or other research projects):

“Nocturnal Oviposition Behavior of Blowflies in Relation to Sunrise and Sunset”

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

lnvestigator’s Name(s) (typed)
Kristi Zurawski
Date: March 2008

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix 1 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator, Michigan State
University Entomology Museum.

58

Appendix D

Voucher Specimen Data

Page __1_ of 3 Pages

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59

Appendix D

Voucher Specimen Data

3 Pages

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60

Appendix D

Voucher Specimen Data

Page _3_ of 3 Pages

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61

LITERATURE CITED

62

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65

   

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