Insects, especially blowflies, have become more widely used in the field of forensic science to determine the post mortem interval. This research contains twenty-four series of blowflies reared in a controlled environment, with daily recording and photography of their growth and development. The findings show that different the two different species, Phormia Regina and Phaenicia sericata emerge at different times in both their egg and pupae stage. Although maggot growth and development is temperature dependant, the observation of these twenty-four series allowed for the complete blowfly life cycle to be seen and recorded. This shows how different species of maggots' aid in identifying time elapsed since death.
A. Introduction
Although entomological evidence has been used for years, just recently is it becoming more commonly used and accepted as valid evidence in the crime laboratories as well as the court room. Forensic entomology in crime scene investigation has been used to analyze various aspects of a scene, such as, how the death occurred, whether a body has been moved, as well as the post mortem interval (PMI) [1]. Post mortem interval can be defined as, "the period of time between death and corpse discovery" [4]. Certain insects are extremely useful in PMI determination as well as forensic investigations because insects and other invertebrates sequentially colonize at a corpse during specific stages of decomposition, this is known as faunal succession. For example, blowflies (Calliphoridae) are invariably the first species to arrive to the corpse [3, 4]. Another common way that time is analyzed for blowfly larva is calculating the age of the insect through the use of accumulated degree hours (ADH) or accumulated degree day (ADD). ADH is the sum of the number of hours from egg to the current stage, which is then multiplied by the temperature (°C) after subtracting the temperature of the developmental threshold [ ]. However, it is very difficult to produce an accurate ADH because, first, the time of oviposition must be known, and second if the body temperature of the corpse is not known for the period of time it has been exposed, the ADH will generate an incorrect perception of time and accuracy [3, 4, 6]. ADH creates a more accurate value when the temperature is constant, such as the following experiment. Although the results not always accurate, the ADH values of this experiment will be listed for comparison purposes.
Decomposition of a body can take weeks to years depending on various factors including, but not limited to; weather, location, temperature and degree isolation. According to Goff, there are five stages of decomposition; fresh, bloated, decay, post-decay, and skeletal [6]. The succession of various insects can be related to the stages of decomposition. The first type of insects to arrive is the necrophagous species, which feed directly on the flesh and are primarily flies, followed by beetles. Blowflies and flesh flies continue to lay eggs and larve1 through the bloat stage. The bloat stage is physically noticeable because this is when the tissue of the body is broken down by bacteria through a process known as putrefaction. This 'bloat' occurs because the metabolic activities of the bacteria produce gases that ultimately inflate the abdomen [6]. As the amount of maggots increase, the next type of insects to arrive is the parasite and predators of the necrophagous species. Theses insects arrive more to consume the eggs and larvae then the corpse itself [6].
More beetle species arrive at the next stage of decomposition. Here, the skin breaks and the gases are released, resulting in drier skin, which is what most beetles prefer to feed on. During this stage ants and wasps also arrive to feed on both the body and other arthropods [6]. At the end of this stage about 15 to 20% of the corpse's weight remains and most fly species have either completely developed or have left the body. The post-decay stage consists mainly of rove and hister beetles and less than 10% of the corpse's weight remains [6]. The skeletal stage occurs when only the bones and hair remain, and the insects found at this stage are not insects pertaining to the body, but rather insects that are commonly found in the habitat, expanding their niche to the body [6]. Although this research focuses mainly upon blowflies, other insects were preserved and collected for analysis as well.
The developmental stages at which you find insects are vital to determine how long they have been present. Blowflies are holometabolous, or experience a complete metamorphosis and the life cycle of a blowfly consists of four main stages; egg, larva (maggot), pupa and the adult fly. The blowfly egg is small, approximately 1.0 - 1.5 mm in size, and looks like an off white piece of rice2 [3]. The larva stages consists of four discrete stages subsections consisting of the first instar, second instar, and the third instar which consists of actively separate parts, a third instar pre and post feeding stage. An instar is each of the successive incremental growth steps terminated by the larva molting [4]. The three instars can be determined and differentiated by the number of posterior spiracles present. Spiracles are external respiratory openings of an arthropod's tubular tracheal system3 [4]. The second and third instars indicate the amount of spiracles present. The first instar lacks an anterior spiracle. The anterior spiracles are located on the opposite end (the head region) of the maggots behind the mouth hooks4.The mouth hooks are sharp 'teeth' like objects that allow the maggot to tear through flesh, as well as secrete enzymes that breakdown tissue before consumption. Posterior spiracles are important because they allow a means for the maggot to constantly breathe while its fontal region is submerged in its food source. If larvae are found at a scene, some should be collected and preserved for observation; others should be collected and reared to insure species identification is correct. Pupae are also important to observe and collect as well.
Various flies emerge from their eggs as well as their pupa at different times depending on their species. The role of the following experiment was to determine the various lengths of the different stages for the two species identified. The length of time for the second and third instar was recorded for the green bottle fly (phaenicia sericata) and the black bottle fly (phormia regina) and their values were compared to values from previous research, in addition to providing information on growth for these species at a controlled temperature (22no °C). A classical way of analyzing decomposition is by observing the insect stage; most of the past research compares the developmental growth rate at various temperatures to one another. However, here we are making the constant (the life cycle) the variable by comparing each species within themselves and to each other.
B. Materials & Methods
Between June and August of 2009 twenty four experiments were carried out at the Suffolk County Crime Laboratory in Hauppauge, New York (N 40049'39.18", W 73014'7.08"). This research studied the growth development of two series while being held under a constant temperature to determine the average growth development and developmental stages among and between each species. Theses twenty four experiments were labeled series A-X, each contained what appeared to be a single egg deposition. A bucket containing a piece of rancid chicken meat was placed outside on June 15, 2009 and remained outside until July 7, 2009. The meat was checked hourly through the day (0800-1600), Monday through Friday. The bucket was closed and covered with a wired cage, with a heavy cement block placed on top to ensure that no scavengers would destroy the bucket. The bucket was reopened in the morning.
Once egg deposition occurred the eggs were than collected and placed in an individual 16oz Tupperware container. Each container had a 1" square removed from the top and covered with gauze to allow for ventilation. This 1" square was created with a scalpel and the gauze was then securely taped into position. Smaller pieces of the rancid chicken meat were placed in the Tupperware and the eggs were positioned on top. These containers were placed under the hood at a controlled temperature which was recorded using a Dickson 6" Temperature Chart. This recorded the temperature 24 hours a day 7 days a week with hourly increments labeled and was changed weekly. This ensured that each series growth development was unaffected by temperature difference. Having a constant temperature, the developmental stages of each series and species could be analyzed and compared. Although wild flies were not used in our analysis for PMI determination, wild flies were caught to observe the species that populated the area, as well as to count the amount of pregnant flies present. Other arthropods that were found around the meat were collected and analyzed to observe the faunal succession that occurred.
B.1 Collection and Larvae Measurement
Once each series was collected, a few eggs were removed and placed into a 50mL conical vial containing an 80% Ethanol solution to persevere the eggs. Each egg was measured using a 1/100" scale and photographed using a Nikon SMZ 1500 stereoscope. Three series among the twenty-two were placed under sequential imagining. A few eggs from each predetermined series were placed on a small amount of meat in a petri dish, which was lined with a wet paper towel for moisture. Sequential imagining entailed a photograph being taken every ten minutes for 100 minutes, to observe the emergence of the maggots from their eggs, this allowed for an exact time to be captured. When the maggots emerged from each series, a few were collected and placed in a 50mL conical vial possessing a 50:50 mixture of 95% Ethanol and Xylene. Larvae were collected and measured twice daily (in the morning and afternoon). Once the maggots began to migrate away from the meat, they were placed into a new 16 oz container to pupate, which consisted of the piece of meat from the original container placed on top of dirt (~1" deep).
B.2 Instar Determination
The instar was determined for each series at each measurement using various techniques. For the smallest of each series (usually the first three measurements) the larvae were placed in Potassium Hydroxide (KOH) for approximately 30 minutes. After 30 minutes, they were placed on a slide and compressed between the slide and a glass cover. They were observed then under a Nikon SMZ 1500 stereoscope; if the instars were still not determinable, they were observed under a compound light microscope. For the larger larva, one was removed from the preservation solution and was positioned with its posterior end facing up to be measured and photographed with the stereoscope.
B.3 Pupa Measurement
Once the maggots began to pupate, there was a daily measurement, using a 1/100" scale. The length and color was observed on average for the same three pupae. To ensure the continued use of the same pupa, once the maggot pupated, it was placed in a certain location within the Tupperware and was labeled with a number.
B.4 Fly Collection of Wild Flies and Reared Flies
Flies that were not reared in the laboratory were caught in two ways. The first method was with the use of a mesh wire cage measuring 12"x 12"x12". A rotten piece of chicken meat was placed underneath the wire mesh box which was resting on an angle on a 'y' shaped branch, which was connected to a string. When there appeared to be 15+ flies, the string was pulled and the box fell shut. A piece of paper towel soaked in chloroform was placed on top of the cage and a larger Tupperware box was placed over the smaller cage. After approximately 30 minutes the larger box was removed and the flies were placed into a 50mL conical vial. The vial was then labeled with the date and time and placed in the freezer. The second method to catching the wild flies was to submerge the 50mL conical vial into the bucket containing the meat when flies were in abundance. These flies were then killed by removing the cap, adding a drop of chloroform to gauze and placing the gauze into the cap of the tube, and replacing the cap. After, they were labeled with the time and date and placed into the freezer.
The collection of the reared flies occurred in one specific manner. Since the flies emerged into a closed 16oz Tupperware container, the containers were opened into a sealed box where five of the sides were solid, and one side consisted of mesh material where a hand/arm could be inserted. Once the flies flew or crawled out of the container, they were scooped up into the conical vial, and were labeled and killed the same method as the latter of the two previously mentioned.
B. 5 Other Insects
The other insects that were found on or around the meat were collected in a 50mL conical vial tube. They were labeled with the time and date of collection. The vials were placed into the freezer. After they remained frozen for a minimum of 2 hours, they were measured and photographed under the stereoscope. The species were identified based on unique characteristics.
A few problems were encountered during the span of this experiment. Firstly, two of the twenty four series (C and H) died prematurely. Series C died roughly within the first 24 hours of life, upon the realization that they were dead, the meat was analyzed and appeared to be dried out which would have led to a lack of nutrients. In Series H, only a few of the eggs hatched, the ones that did not hatch, upon analysis appeared to be more transparent and weak looking as opposed to the other series.
Another dilemma experienced was a holiday weekend. Fourth of July weekend led to the closure of the lab on Monday. Wanting to ensure the maggots remained at a constant temperature they were left at the lab; however six of the series began to pupate. Having a 72 hour window rather than a 48 hour window skewed the possibility of using those series for accurate hourly data. However, once the species were determined a rough estimate of when they pupated could be based upon when other series of the same species pupated.
The last issue observed during the experiment was the migration of Series W. This series was the largest series of them all, leading to the possibility that more than one fly oviposted in the same location. This series produced 200+ flies; however, once the maggots reached the post-feeding stage which is normally when they migrate away from the meat, they burrowed into the meat. They produced rows of pupae under the top layer of the meat, which resulted in the meat being sliced open with a scalpel for their removal. Unfortunately in this effort, approximately 30 pupae were destroyed.
D. Results
The twenty-two series of blowflies were continually analyzed during their entire development (egg stage to an adult fly). Through the use of various sources and unique characteristics it was determined that the twenty-two contained two different species, eight of them (Series A, D, I-M and Q) being the green bottle fly (phaenicia sericata) and fourteen of them being the black bottle fly (phormia regina). Before the species pupated it was concluded that there were at least two species present due to the fact that some series possessed buttons on their posterior spiracles while others did not. A button is a small circle located within the point of the spiracle connecting the outer spiracle line5. The actual species identification was determined when each species reached their adult stage.
Once the series were separated by species were we able to compare their development within species. Table one presents the emergence times for Phaenicia sericata and Phormia regina.
Table 1: Emergence from Egg Stage at a Constant Temperature of 23± 0.6 °C
Species
Mean Emergence Time
S.D. (hr)
Phaenicia sericata
19hrs 32 mins
±0.7
Phormia regina
23hrs 49 mins
±2.3
Since the size of the larvae were small in size, it was difficult to analyze the first instar, the data pertaining to the second and third instar are the main results analyzed. Therefore, the following table presents the average amount of time (hrs) required for each species to be in their second instar including the duration and standard deviation.
Table 2:
Species
Min (hrs)
Max (hrs)
Mean (hrs)
S.D. (hrs)
Phaenicia sericata
44
75
62
±5.3
Phormia regina
44.5
98.5
73.3
±4.3
The next table shows the average amount of time spent in each stage of development between the species.
Table 3:
Stage
Phaenicia sericata
Phormia regina
Egg
1stInstar
2ndInstar
Pupa
Total
TWO GRAPHS HERE
Table four below shows how each species was analyzed; the characteristics used are listed below.
Table 4: Characteristics Used in Determining Species Identification
Characteristic
Phaenicia Sericata
Phormia Regina
Presence of Button
Buttons Present in 3rdinstar
Buttons Absent in 3rdinstar
Average Length
Between 6.0 to 9.0 mm
Length between 7.0 to 9.0 mm
Common Color
Blue- green, yellow- green, green, golden bronze
Dark green to olive green thorax and abdomen
Unqiue Characteristic
Yellow Basicosta Present6
Anterior thoracic spiracles are surrounded by bright orange setae7
All of the characteristics in table 1 are observed once the blowfly is in its adult fly stage. Tables 2a and 2b show the average lengths obtained from four series of each species to observe and compare their length.
Table 2a: Average Fly Size of Phanecia Sericata
Table 2b: Average Fly Size of Phormia Regina
Series
Avg. Length
A
7.1 mm
D
6.8 mm
I
6.9 mm
L
7.8 mm
Series
Avg. Length
B
9.3 mm
P
8.8 mm
U
9.1 mm
W
8.3 mm
Figure 1: Complete Growth Development of Phaenicia Sericata at 23 ±0.6 °C
Figure 2: Complete Growth Development of Phormia Regina at 23 ±0.6 °C
Discussion
This entomological data collected was analyzed and compared between each other and to existing data. Table one shows that there approximately a four hour difference in emergence times between the species, such that Phaenicia sericata emerges earlier. Anderson listed the minimum and maximum time to reach the first instar in both Phaenicia sericata and Phormia regina at a constant temperature of 23.0±0.02°C and 23.3 ±0.02 °C. Listing the times until the first instar was reached we can conclude that they are the emergence times from the egg. Phormia regina took between 21.5±0.9 hrs and 22.5±0.5 hrs to reach the first instar, while Phaenicia sericata took between 21 hrs and 22 hrs. The latter of the two only contained two replicas, therefore the data can only produce an average based on two numbers, and not supply adequate data. However according to Greenburg, the average minimum duration for Phormia regina at 22 °C was 19.2hrs and Phaenicia sericata at 22 °C was 21.6 hrs. This data tends to contradict what my finding produced. Consulting a third source, Grassberger found that the average minimum duration for Phaenicia sericata at 22°C was17 hrs. Although there is no data obtained for Phormia regina for this study at least this study provided another average of minimum duration for Phaenicia sericata that was earlier that 21 hours.
The two charts show the minimum amount of time required by each species to reach the second and third instar. The axis provided are length of time (in hours) verse the percentage of the series in that specific instar. Looking at Phaenicia sericata, it is shown the by 70 hours 100% of the series are in their second instar, while the Phormia regina series, even by 80 hours, have not all reached their second instar. This data can be shown in table 3. The second chart shows the time verse percentage for the third instar. Here, the opposite occurs, Phaenicia sericata does not reach 100% even after170 hours, whereas, Phormia regina makes an interesting jump from approximately 40% of the series are in their third instar at the 165th hour and 100% of them are in their third instar by the 170th hour. In a mere five hours, the percentage more than doubles.
Complete growth development for each species is shown in figures 1 and 2. Comparing the two we can see that Phormia regina overall takes a shorter amount of time to develop, but a slightly longer time to emerge from their egg. Phaenicia sericata not only takes a longer time to develop, but takes a much longer time in their pupa phase (noted by when the line drops and levels off).
A few problems were encountered during the span of this experiment. Firstly, two of the twenty four series (C and H) died prematurely. Series C died roughly within the first 24 hours of life, upon the realization that they were dead, the meat was analyzed and appeared to be dried out which would have led to a lack of nutrients. In Series H, only a few of the eggs hatched, the ones that did not hatch, upon analysis appeared to be more transparent and weak looking as opposed to the other series.
Another dilemma experienced was a holiday weekend. Fourth of July weekend led to the closure of the lab on Monday. Wanting to ensure the maggots remained at a constant temperature they were left at the lab; however six of the series began to pupate. Having a 72 hour window rather than a 48 hour window skewed the possibility of using those series for accurate hourly data. However, once the species were determined a rough estimate of when they pupated could be based upon when other series of the same species pupated.
The last issue observed during the experiment was the migration of Series W. This series was the largest series of them all, leading to the possibility that more than one fly oviposted in the same location. This series produced 200+ flies; however, once the maggots reached the post-feeding stage which is normally when they migrate away from the meat, they burrowed into the meat. They produced rows of pupae under the top layer of the meat, which resulted in the meat being sliced open with a scalpel for their removal. Unfortunately in this effort, approximately 30 pupae were destroyed.
This analysis performed in identifying the developmental stages of the blowfly cycle to aid in PMI determination. These results were concluded with the use of a constant temperature which can only hold a value if a corpse was to be discovered in a location with a constant temperature such as in a building. This data is appropriate for the geographical region stated previously. Each location has different species have predators, insects and various animals. Since geographical regions can vary so much, this type of analysis must be continued to solidify the use of insects as indicators of PMI, and new techniques can eventually be developed to do so.
