1. Maggots and Time of Death Estimation
Forensic Entomology
Maggots and Time of Death Estimation

3. Entomology is the Study of Insects
Images from:

4. Insect Biology
Insects are the most diverse and abundant forms of life on earth.
There are over a million described species- more than 2/3 of all known organisms
There is more total biomass of insects than of humans. of humans.
Insects undergo either incomplete or complete metamorphosis (Egg to larva to pupa to insect)
Larva have a soft tubular body and look like worms. Fly species larvae are “maggots”

5. What is Forensic Entomology?
Forensic Entomology is the use of the insects and other arthropods that feed on decaying remains to aid legal investigations. 
Medicolegal (criminal)
Urban (criminal and civil)
“legal proceedings involving insects and related animals that affect manmade structures and other aspects of the human environment”
Stored product pests (civil) 

6. Medicolegal Forensic Entomology
Often focuses on violent crimes
Determination of the time (postmortem interval or PMI) or site of human death based on identification of arthropods collected from or near corpses.
Cases involving possible sudden death
Traffic accidents with no immediately obvious cause
Possible criminal misuse of insects

7. Postmortem interval (PMI)
Forensic Entomology is used to determine time since death (the time between death and corpse discovery)
This is called postmortem interval or PMI).
Other uses include
movement of the corpse
manner and cause of death
association of suspects with the death scene
detection of toxins, drugs, or even the DNA of the victim through analysis of insect larvae.

8. Forensic Entomology is Applied Biology
If it weren’t for decomposition of all living things, our world would fill up with dead bodies.
When an animal dies, female insects will be attracted to the body. They enter exposed orifices or wounds and lay eggs or larvae.
A forensic entomologist:
identifies the immature insects
determines the size and development of the insects
calculates the growth of the insects and passage through stages of the life cycle in laboratory
compares the growth against weather conditions to estimate time of oviposition

9. Succession of Insects on the Corpse
Estimates of postmortem intervals based on insects present on the remains are based on:
The time required for a given species to reach a particular stage of development.
Comparisons of all insect species present on the remains at the time of examination.
Ecological succession occurs as an unexploited habitat (like a corpse) is invaded by a series of different organisms.
The first invasion is by insect species which will alter the habitat in some form by their activities. These changes make the habitat attractive to a second wave of organisms which, in turn, alter the habitat for use by yet another organisms.

10. Ecology of Decomposition
Necrophages - the first species feeding on corpse tissue. Includes rue flies (Diptera) and beetles (Coleoptera).
Omnivores - species such as ants, wasps, and some beetles that feed on both the corpse and associated maggots. Large populations of ominvores may slow the rate of corpse’s decomposition by reducing populations of necrophagous species.
Parasites and Predators - beetles, true flies and wasps that parasitize immature flies.
Incidentals – pill bugs, spiders, mites, centipedes that use the corpse as an extension of their normal habitat

11. Image: http://www.nlm.nih.gov/visibleproofs

12. Decay Rates Are Variable
Studies of decay rates of 150 human corpses at in the Anthropological Facility in Tennessee (The Body Farm)
Most important environment factors in corpse decay:
Temperature
Access by insects
Depth of burial
Other Factors
Chemical-- embalming agent, insecticides, lime, etc.
Animals disrupting the corpse

13. Time of Death can be broadly estimated up to about 36 hours
Temperature Stiffness Time of death
Warm Not stiff Dead less
than three hours
Warm Stiff Dead between 3
to 8 hours
Cold Stiff Dead between 8
to 36 hours
Cold Not stiff Dead in more
than 36 hours

14. Body Temperature
Stiffness/ Rigor Mortis
Time of Death
Warm

15. Temperature Stiffness Time of Death

16. Differentiate between PMI and Time of Death
These may not always equate.
Post mortem interval is restricted to the time that the corpse or body has been exposed to an environment which would allow insect activity to begin.
Closed windows
Body in box or bag
Cold temperatures
Deeper burial

17. Insect species arrive at a corpse in waves like clockwork
Calculate the heat/thermal energy (accumulated degree hour) required for each stage of the Green Bottle Fly’s life cycle.
Possibly the greatest potential source of error in using arthropod successional patterns lies in the collection of speciments.
Must only be done correctly to accurately sample the insects.

18. Image: http://www.nlm.nih.gov/visibleproofs

19. Calculating PMI from Accumulated Degree Hours (ADH)To
Temp
Hours
ADH
Cumulative ADH
Egg
1st Instar
70° F 23
23 x 70=
1610 ADH
1610
2nd Instar
70 ° F 27
27 x 70=
1890 ADH
1610+
1890
3rd Instar 22
22 x 70=
1540 ADH
1540
Pupa
130
130 x 70=
9100 ADH
Adult Fly
143
143 x 70=
10010 ADH
+10010
24100 ADH

20. Calculating ADH from Climate Data

21. Using the Data 3928 ADH in these three days (952+1488+1488).
How many ADH of 70º are there in these 3 days?
3928/70=56.11 hours
72 hours at 70º would have the insects passing to the 3rd instar. But 72 hours at colder temperatures and insects will only be at 2nd instar stage.

22. Five Stages of Decomposition Fueled by Insect Activity.
Fresh
Bloat
Decay
Post-decay
Dry (skeletal)

23. Fresh Begins at death Flies begin to arrive
Temperature falls to that of the ambient temperature.
Autolysis, the degradation of complex protein and carbohydrate molecules, occurs.
Gasses produced by the metabolic activities of the anaerobic bacteria first cause a slight inflation of the abdomen. The carcass may later assume a fully inflated, balloon-like appearance. Adult and larval blowflies in large numbers attracted to fluids seeping from body, normal soil dwelling fauna depart soil because of seepage of fluids; some muscid flies and ants which can feed on larvae and retard maggot activity.

24. Bloat Swells due to gases produced by bacteria
Temperature rise of the corpse
Flies still present

25. Decay Gases subside, decomposition fluids seep from body.
Bacteria and maggots break through the skin.
Large maggot masses and extreme amounts of fluid.
Unpleasant odor
Larvae beginning to pupate.
Corpse reduced to about 20% of it’s original mass.
Decay Stage - Black Putrefaction (Days 5-11) -- Decay stage begins when the abdominal wall is broken, allowing gasses to escape and carcass deflates. This process is facilitated by feeding activities of larval flies present on the exposed remains. Adult flies start to leave body, mainly larval mass. Carcass begins to assume a blackened, wet appearance, and most of the flesh will be removed by the maggots. Toward end of this period, carcass will begin to dry and beetles feed on drier tissue. Flies start to pupate. Predatory beetles such as rove beetles and histerids come to feed on other insects.

26. Post-Decay Carcass reduced to hair, skin, and bones.
Fly population reduced and replaced by other arthropods.
Hide beetles are dominant in dry environments.
Mite and predatory beetle populations increase.
Postdecay Stage - Butyric fermentation (Days 10-25) -- In dry habitats, remains consisted of dry skin, cartilage and bones. Site for dermestid beetles, histerids, fly pupae, immature and adult rove beetles. In wet habitats, a large quantity of wet, viscous material, termed byproducts of decomposition, was found in the soil under the remains. Site for immature and adult moth flies, sphaerocerid and muscid flies, rove beetles.

27. Dry (Skeletal)
Does not always occur especially if corpse is in a wet region. Maggots will stay longer and hide beetles will not appear.
In wet environments the hide beetles are replaced with nabid and reduviid insects.
The corpse is reduced to at least ten percent of the original mass.
In the last stage (Skeletal Stage), only bone and hair remain.
Dry Stage (Days 25 +) -- This stage is reached when mainly bones and hair remain. Odor is primarily that of normal soil and litter. Some dermestid beetles, histerids, fly pupae, immature and adult rove beetles, normal soil fauna (mites) start to return. Can last several months to even years.

28. Methods
This project took place at the Huntington landfill beginning on September 5, 2003.
Two different areas were chosen to deposit two pigs.
Pig 1 was laid in a sunlit area.
Pig 2 was laid in a shaded woodland area about 100 feet away at an elevation of approximately 20 feet.

29. Methods
Both pigs were placed in cages constructed of wood and one inch chicken wire that were staked to the ground to protect from predatory animals.
Prior to starting the project, great care was taken to prevent insect activity from taking place. After they died, the pigs were individually tied in two black garbage bags, placed in feed sacks, and secured.

30. Methods The pigs were kept at -80˚C in the laboratory.
They were placed in plastic bins in order to thaw for 48 hours prior to placement at the landfill.
Closed environment was maintained until they were deposited at the site.

31. Methods
Pigs with a genetic line of a minimum of fifty percent Yorkshire.
They were 8-10 weeks old and weighed approximately pounds.
Both died on July 11, 2003 approximately 12 hours apart. One died a natural death and the other was culled from the litter.
Both of the carcasses were in very similar condition; there were no breaks, tears or cuts in the skin.

32. Methods
Daily observations were made at both sites throughout the day at 7am, 1pm, 7pm, and 1am.
Air, ground, and maggot mass temperatures were taken at each visit and observations were recorded.
At 7am and 7pm they also collected maggot samples for analysis and photographed the scene.
Observations were noted and samples taken for a period of nine days.

33. Methods
Using insect tweezers, the investigators collected a number of maggots and dropped the samples immediately into boiling water, to kill the bacteria in the maggots and also to straighten their bodies for easier analysis.
The maggot samples were taken from different areas of the body in which there were large numbers present.

34. Methods
The maggots were then placed into a labeled jar and preserved with 70% EtOH.
They also collected interesting arthropods for analysis.
All of the samples were labeled and stored for later analysis in the laboratory.

35. Phormia regina
Spiracles are incomplete
Third-instar larvae

36. Phaenicia species
Spiracles are complete
Third-instar larvae

37. Results: Fresh Stage
Flies began to arrive within minutes of pig placement however, laying of eggs was delayed hours.
There was already some green discoloration on Pig 2 at the beginning of the fresh stage, possibly due to the fact that it was dead about 8 hrs before Pig 1.
72 hrs later, the first signs of bloating occurred, ending the Fresh Stage.

38. Results: Bloat stage
At about 72 hours, noticeable bloating began to occur in Pig 1.
However, Pig 2 did not show visible signs of bloating until about 92 hours.
The gap between the two pigs might have been even greater if they had both died at exactly the same time.

39. Results: Decay Stage Decay stage started around 102 hours.
At this point, the maggots had broken the skin and the pigs had begun to deflate.
Decompositional fluids began to seep from the carcass.
There was a green froth around the pig and also a dark fluid ring around the body of Pig 1.
Maggot activity increased tremendously, and maggot mass temperature reached its high during this stage.

40. Results: Post-decay Stage
When the experiment was terminated due to the fact that maggot activity had ceased, the pigs had reached the Post-Decay Stage.
They were mostly skin, bones, and hair, but there was some tissue remaining.

41. Temperature is a Factor: Pig 1
The graph shows an elevation for maggot mass temperatures over ambient
The fluctuation in ambient temperature induced elevated maggot activity which is consistent with other similar experiments.
Sunlit Pig

42. Temperature is a Factor: Pig 2
The ambient temperature for Pig 2 was more constant because it was in a shaded area.
The temperatures for Pig 1 fluctuated more than those of Pig 2.
Shaded Pig

43. Phormia Average Maggot Length vs. Time
Shows a gradual increase then decrease for the Phormia regina
The maggots feed and grow to a certain point when they begin to leave the carcass to find a safe place to pupate.

44. Phaenicia Average Maggot Length vs. Time
Two peaks for the Phaenicia
Infers two generations for Pig 1.

45. Two Different Maggot Generations
These are distinguishable by the length and obvious size difference.
This is why we believe there are two peaks in our graph data for the Sunlit Pig.
The photograph was taken at a time consistent with the influx at 132 hours.

46. Discussion
Two different species of maggots were collected over the nine day period.
These two species were analyzed at their third instar stages; they were able to determine the difference by comparing their spiracles.
The third instar was the only stage that they analyzed; species determination was more evident at this stage of development.
They also reared a sample of maggots from each pig for later species analysis.

47. Accumulated Degree Hours
ADH may be calculated using temperature and hours.
This works because there is direct correlation between temperature and maggot development.
These calculations were somewhat approximate but relatively accurate.

48. ADH and Pig Results
ADH for Pig 1 was calculated as after nine days.
ADH for Pig 2 was calculated as after nine days.
These can be used to determine PMI for carcasses found in this area in similar conditions.

50. The End