Developmental Stages of the Fly Phormia regina and their use in Forensic Science

1
Developmental Stages of the Fly Phormia regina
and their use in Forensic Science Nathan Allison,
College of Nursing, Marshall University
Abstract Forensic investigations utilize many
techniques to estimate time of death. When a
death occurs, the most obvious post-mortem
indicator is the appearance of flies and other
insects that feed on the carcass. One common
carrion fly is the black blowfly, Phormia regina.
This fly is one of the first to appear and lay
its eggs in the orifices of the decaying carcass.
By examining the larval stages of these flies,
an accurate time of death can be determined. In
this project, second (four day) and third (six
day) instar larval stages were examined using a
SEM. Morphological differences were used to
differentiate between the second and third larval
stages. Specifically, the spiracles (breathing
organs) were imaged. Due to the extensive
cuticular folding that occurred while preparing
the specimens, three processes of preparation
were used. The first process involved only the
dehydration of the larvae in an ethanol series.
The second process involved fixing the larvae in
four percent glutaraldehyde followed by
dehydration in an ethanol series. The third
process involved the use of Karnovskys fixative.
Karnovskys fixative was used because of the
cuticular folding that occurred while using the
other two methods of preparation. The process
with Karnovskys fixative began by placing the
larvae in the fixative for an hour. Then the
larvae were washed with Cocadylate buffer, pH of
7.5. Finally, the larvae where post-fixed in
0.47 OsO4 for an hour and a half. Once this
process was completed the larvae were processed
through an ethanol series. All larvae were
subsequently dried and sputter coated for imaging
with the Jeol 5310 SEM. By observing the larvae
with the SEM, the morphological differences in
spiracle formation were determined. The second
instar had two small slits in each spiracle while
the third instar had three. Since the spiracles
have different features in each larval stage, not
only can the investigator look at the size of the
larvae but the spiracle formation that has taken
place. Therefore, the spiracles can be used as a
tool in order to estimate the time of death of a
specific carcass.
Results and Discussion For over one hundred
years blowflies and other Calliphoridae have been
recognized as primary decomposers of animal
cadavers. In this study we attempt to refine the
practice of using fly colonization of cadavers to
determine time of death. Although size of larvae
can be used to determine which larval stage of
development a specific species of fly is in,
there is a wide range of sizes that each larval
stage can contain. This depends on the amount of
food available to the larvae have and the ambient
and maggot mass temperatures in which they
develop (Joy et al. 2002). This is why
spiracular formation is often used to determine
the stage of development a larva is in. As can
be seen in the low magnification image of the
second instar (Figure 5) larva there are two
slits in each spiracle. On the other hand, the
low magnification image of the third instar
(Figure 6) larva shows there are three slits in
each of the spiracles. It appears that the
number of slits in each spiracle is a more
consistent way of classifying larval development
then just looking at the size of the larvae
itself. The high magnification micrographs of
the second (Figure 7) and third (Figure 8)
instars confirm the results that were obtained by
using low magnification. They also show the many
other characteristics that are found throughout
larval development. These figures show the
peritreme and button that are seen in Figure 3.
Spiracular hairs are also seen surrounding all
the slits that are located in the spiracles. The
hairs appear to be flattened, most likely caused
by drying of the specimen. To conclude, another
species of fly (Phaenicia) was examined in order
to compare the spiracle morphology between
related species. Figure 9 presents the results.
The peritreme is seen completelely surrounding
each spiracle and the button is easier to
distinguish. These features that are clearly
seen with the SEM are much harder to discern
using a light microscope. We therefore believe
that the SEM will be a valuable tool for forensic
entomologists as the field develops and precision
techniques become more refined.
Figure 3. (1)Peritreme and (2) Button
Figure 5. Low magnification image of a second
instar (4 day) larvae.
Introduction Forensic entomologists are
concerned with the many insects that appear at a
carcass. Realizing that these insects have their
own stories to tell, they can piece together the
events that occurred in the mysteries that they
are attempting to solve (Schrof 1991). Insects
are relatively predictable due to their
persistent nature of remaining in the same
environment and eating the same food (Fernandez
2001). By examining the crime scene, a forensic
entomologist can determine facts that would be
otherwise unknown. For example, by examining if
the certain species of fly will appear indoors or
out, in warm or cool weather, in the shade or the
sun, or in daylight or night, a forensic
entomologist will be able to recreate the events
that occurred prior to the discovery of the body
(Schrof 1991). Many insects bombard a body after
a death, but the most common post-mortem insect
is the blowfly. To distinguish between species
of blowflies, forensic entomologists will examine
such features like hooks that surround the mouth
and the spiracles at the rear of the developing
larvae. The spiracles are the breathing organs
of the larvae and allow the larvae to place their
heads directly into the flesh of the body that
they are feeding on (Sachs 1998). A common
blowfly that is encountered around fresh corpses
is known as Phormia regina (Figure 1) (Joy et al.
2002). These flies will go through three larval
developmental stages (instars), a pupa stage and
will emerge as adults. In the development of
Phormia regina, one oval opening is apparent in
each spiracle of the first instar of development.
As the larvae mature into the second instar,
another opening is developed and each spiracle
will contain two oval openings (Figure 2a). When
the larvae reach the third instar state of
development, a third slit (Figure 2b) will become
apparent in each spiracle. As the larvae develop
through each stage, an incomplete peritreme and a
feature known as a button (Figure 3) will be
seen surrounding each of the spiracles that are
present (Hall 1948). By determining the number
of openings in each spiracle, a forensic
entomologist is able to accurately determine time
of death through the specific larval stage that
is present (Sachs 1998).

• Objectives
• Examine the morphological characteristics of the
  spiracles of the larvae of Phormia regina.
• Distinguish between the differences in spiracular
  formation of second (4 day) and third (6 day)
  instar larval stages of Phormia regina.
• To compare spiracle morphology in third instar
  larvae of Phaenicia sp. with that of Phormia
  regina.

Figure 7. High magnification image of a second
instar (4 day) larvae.
Materials and Methods Sample The larvae of
Phormia regina were collected from raccoon
carcasses (Joy et al. 2002). Ten four day and
ten six day larvae were prepared for use in the
SEM. All the larvae were collected during an
experiment preformed by Dr. Joy of the Marshall
University, Department of Biological Sciences.
The single larva of Phaenicia sp. was collected
from a hawk carcass in the spring of 2002.
Specimen Preparation Due to the delicate nature
of the samples, three different preparation
techniques were compared to observe which
resulted in less deformation of the sample.
These included Procedure 1 Dehydration of the
four day and six day larvae in an ethanol series.
This involved soaking the larvae in 30, 50, 70,
90 and 100 percent ethanol for intervals of 10
minutes. Procedure 2 Fixing the larvae in four
percent glutaraldehyde. Followed by dehydration
in an ethanol series (See Procedure
1). Procedure 3 The process with Karnovskys
fixative began by placing the larvae in the
fixative for an hour. Then the larvae were
washed with Cocadylate buffer, pH of 7.5.
Finally, the larvae where post-fixed in 0.47
OsO4 for an hour and a half. Once this process
was completed the larvae were processed through
an ethanol series (See Procedure
1). Imaging All the test samples were sputter
coated and imaged under the Jeol 5310 SEM that
was provided by the staff at Marshall
University. Electron energies ranged from 15 to
20kV. Working Distance was relatively long (20-30
mm) to improve depth of field. Magnification
ranged from 75x to 500x.
Figure 6. Low magnification image of a second
instar (4 day) larvae.
Figure 8. High magnification image of a third
instar (6 day) larvae.
Figure 1.Adult Phormia regina
Figure 2. Montages of (a) second instar and (b)
third instar phormia larvae