Entomology Chapter 9 ©2010 Elsevier, Inc..

INTRODUCTION Forensic entomology is the application of the study of arthropods to criminal or legal cases Entomology is divided into three topics: urban entomology, stored products entomology, and medicolegal entomology Insects and their life cycles may help to establish postmortem interval (PMI) Forensic entomology is closely linked with medical entomology, pathology and taxonomy Taxonomy is the classification of living things ©2010 Elsevier, Inc.

INTRODUCTION Forensically important conclusions may be drawn by analyzing the phase of insect invasion of a corpse or by identifying the life stage of the necrophagous insects found in, on or around the body Necrophagous means dead-flesh eating Knowledge of insect biology and habitats may provide information for accurate estimates of how much time a body has been exposed to insect activity Taphonomy is the study of an organism from the time it dies until the time it reaches the laboratory Investigate how different processes affect organisms Distinguish between natural phenomena and human intervention ©2010 Elsevier, Inc.

INSECTS AND THEIR BIOLOGY
Insects are the largest group of arthropods Six legs and three-segment body Most numerous and diverse group of organisms known Possess an external skeleton, or exoskeleton Composed of chitin and protein Protects animal’s internal organs, conserves fluids and acts as the structure for muscle attachment ©2010 Elsevier, Inc.

INSECTS AND THEIR BIOLOGY
Three body segments are joined by flexible joints Head, thorax, and abdomen Head contains insect’s eyes, sensory organs, including antennae, and mouth parts Thorax contains prothorax, mesothorax, and metathorax Each subsegment has a pair of legs and depending on the insect the meso- and meta-thorax have wing attachment sites Abdomen carries internal organs and is also segmented Segments have a pair of holes, called spiracles, which the insect uses for breathing ©2010 Elsevier, Inc.

LIFE CYCLES OF INSECTS Arthropods pass through a series of maturation phases In ametabolous, meaning “without change,” or metamorphosis, eggs yield immature forms that look like smaller forms of adults In paurometabolous, or gradual, metamorphosis, hatchlings emerge in a form called a nymph Nymphs resemble wingless version of adults of the species Grow by molting; each successive molt produces a new instar or growth phase ©2010 Elsevier, Inc.

LIFE CYCLES OF INSECTS In holometabolous, or complete, metamorphosis, an adult lays an egg or depositis larva onto a food source Larvae eat or hatch from the egg and begin eating Increase in size by molting through instars Larvae form is very different than adult form At end of instars, the larvae transition into pupal stage Pupa is hardened outer shell or skin that protects larva while it undergoes its final growth stage to the adult form Frequently encountered type of pupa is puparium, which is hardened skin of the last larval instar Seen in flies ©2010 Elsevier, Inc.

LIFE CYCLES OF INSECTS Time it takes to go from egg to adult varies greatly Many factors affect timing of insect reproduction: weather environment, season, food supply, rainfall, humidity, etc. Many other factors affect number and timing of successive generations of necrophilious (“dead loving”) insects Location, shade, slope, where the body lies, etc. Necrophilious insects are very sensitive to chemical changes in a dead body Chemicals are by-products of decomposition Habitat of a decomposing body is a finely tuned environment ©2010 Elsevier, Inc.

COLLECTING INSECTS AT A CRIME SCENE
Number, type, and distribution of insects drawn to a dead body will vary by the environmental conditions, time since death, location, geography, weather, etc. Forensic entomology is complicated and specialized discipline Coordination with all those involved in evidence collection is important Suggested sequence of stages for a forensic entomological investigation: Visually observing and taking notes of the scene Recording notes Approximating the number and kinds of insects Recording locations of major insect infestations Noting immature stages Identifying the precise location of the body Observing any other phenomena of note (trauma, coverings, etc.) ©2010 Elsevier, Inc.

Collecting of climatological data from the scene Recording ambient air temperature Measuring ambient humidity Taking ground surface temperature Taking body surface temperatures Taking below-body temperatures Taking maggot mass temperatures Taking post-body removal sub-soil temperature Collecting of specimens from the body before its removal from the scene, the area surrounding the body (up to 20′) before its removal, and the area directly under the body after the body has been removed ©2010 Elsevier, Inc.

Necrophilious insects are attracted to dark, moist areas: face, open wounds, etc. Insects can be collected in a variety of ways Sweeping a net back and forth over the body End of net, with insects in it, can then be placed in a wide-mouth killing jar A glass jar containing cotton balls soaked in ethyl acetate Preserve insects in 75% ethanol Label vials Crawling insects can be collected with forceps or fingers Care must be taken to avoid disturbing other evidence Eggs, a mixture of larvae of various sizes, and any adults should be collected Soil under body should be sampled ©2010 Elsevier, Inc.

Buried or enclosed remains present problem due to insects’ limited access Alters the estimation of time since death because the “clock” of insect succession rate has been altered Environmental changes, natural or artificial, need to be measured and noted Wide-angle and close-up photographs should be taken ©2010 Elsevier, Inc.

THE POSTMORTEM INTERVAL
Forensic entomologist’s main contribution to death investigation is an estimate of the postmortem interval (PMI). A minimum and maximum time since death based on the insect evidence collected and developed ©2010 Elsevier, Inc.

THE CLASSIFICATION OF INSECTS
Classification of insects on body can only be performed by an experienced forensic entomologist with the proper reference collections Requires differentiation between closely related insects Science of identifying and classifying organisms in called taxonomy Categorized by relatedness through the recognition of significant evolutionary traits ©2010 Elsevier, Inc.

THE CLASSIFICATION OF INSECTS
Order of relatedness is broken into taxa, or related groups Kingdom, Phylum, Class, Order, Family, Genus, and Species Genus and Species are typically used to describe an organism Capitalize Genus and italicize both Genus and Species Some species have well-defined variants or subspecies Taxonomic key is method for classifying organisms whereby each trait identified separates otherwise similar groups of organisms ©2010 Elsevier, Inc.

REARING INSECTS Significant step in identifying immature insects is the rearing of larvae into adult insects Growth environment must be closely controlled and monitored Requires vent hood Laboratory space equipped to keep samples and insects separate Work space that is easily cleaned after case work is completed ©2010 Elsevier, Inc.

DNA and Insects DNA analysis is used to identify insect species
Extraction of the gut contents of maggots may reveal nuclear or mitochondrial DNA from the deceased Indicates if maggot was on a particular corpse ©2010 Elsevier, Inc.

CALCULATING A PMI Basis of method is to study which insects, or their young, inhabit a dead body and in what sequence they do so Recognition of each species in all stages and knowledge of the time occupied in each stage allow for estimate of time since death Four ecological stages exist in cadaver community Necrophagous species feed on carrion Predatory and parasitic species prey on other insects, which inhabit the cadaver Omnivorous species eat material from body, other insects, or whatever food source presents itself Incidental species use cadaver as extension of their normal habitat ©2010 Elsevier, Inc.

CALCULATING A PMI Faunal succession on carrion is linked to the natural changes that take place in a body following death Temperature falls Gases are released Putrefaction follows Estimate may be accurate from less than one day to just over one month depending on conditions and species reared PMI may be determined by estimate age of immature insect Estimate of PMI using a model of insect development or succession is a more complete method ©2010 Elsevier, Inc.

CALCULATING A PMI If no data are available that take into account the parameters that the forensic entomologist faces, experimentation is required Decomposition studies of small pigs Temperature is most influential factor in determining PMI Living, feeding, moving maggots, which are larvae of necrophagous flies, can raise the temperature of an area by many degrees Maggot mass effect ©2010 Elsevier, Inc.

OTHER FORENSIC USES FOR INSECTS
Ingestion of drugs, toxins, or other substances from body DNA may be obtained from guts of insects May give information about the location, travel, or geography associated with items ©2010 Elsevier, Inc.

T. Trimpe 2009 http://sciencespot.net/
Forensic Entomology Insects as Evidence Warning: Some material in this presentation and related videos may be too graphic for some people. T. Trimpe

Cool Jobs: Forensic Entomology
What do they do? Forensic entomologists apply their knowledge of entomology to provide information for criminal investigations. A forensic entomologist’s job may include: Identification of insects at various stages of their life cycle, such as eggs, larva, and adults. Collection and preservation of insects as evidence. Determining an estimate for the postmortem interval or PMI (the time between death and the discovery of the body) using factors such as insect evidence, weather conditions, location and condition of the body, etc. Testifying in court to explain insect-related evidence found at a crime scene. Did you know? Maggots can be used to test a corpse for the presence of poisons or drugs. Some drugs can speed up or slow down the insect’s development. Cool Jobs: Forensic Entomology Discovery Video

Insects as Evidence Forensic entomologists use their knowledge of insects and their life cycles and behaviors to give them clues about a crime. Most insects used in investigations are in two major orders: 1 – Flies (Diptera) and 2 – Beetles (Coleoptera) Blow Fly Carrion Beetle Species succession may also provide clues for investigators. Some species may to feed on a fresh corpse, while another species may prefer to feed on one that has been dead for two weeks. Investigators will also find other insect species that prey on the insects feeding on the corpse. Images: Top Right - Chart -

Click the image to view a video about the Body Farm!
Weather data is also an important tool in analyzing insect evidence from a corpse. Investigators will make note of the temperature of the air, ground surface, the interface area between the body and the ground, and the soil under the body as well as the temperature inside any maggot masses. They will also collect weather data related to daily temperature (highs/lows) and precipitation for a period of time before the body was discovered to the time the insect evidence was collected. Other factors that might affect their PMI estimates: Was the body enclosed in an area or wrapped in a material that would have prevented flies from finding the corpse and laying eggs? Were other insect species present that may have affected the development of the collected species? Were there drugs or other poisons in or on the body that might have affected the larvae’s development? Did you know… The “Body Farm” in Knoxville, Tennessee is a university research facility to investigate human decomposition under various conditions in order to understand the factors which affect its rate. Click the image to view a video about the Body Farm!

Blow Fly Metamorphosis
Blow flies are attracted to dead bodies and often arrive within minutes of the death of an animal. They have a complete life cycle that consists of egg, larva, pupa, and adult stages. 1st – Adult flies lay eggs on the carcass especially at wound areas or around the openings in the body such as the nose, eyes, ears, anus, etc. 2nd – Eggs hatch into larva (maggots) in hours. 3rd– Larvae continue to grow and molt (shed their exoskeletons) as they pass through the various instar stages. 1st Instar - 5 mm long after 1.8 days 2nd Instar - 10 mm long after 2.5 days 3rd Instar – mm long after 4-5 days 4th – The larvae (17 mm) develop into pupa after burrowing in surrounding soil. 5th – Adult flies emerge from pupa cases after 6-8 days. Adult Pupa Eggs 3rd Instar Larva 1st Instar Larva 2nd Instar Larva It takes approximately days from egg to adult depending on the temperatures and humidity levels at the location of the body. Image: Information: and

Examples of Diptera (Flies)
Early Stage Decomposition Blow & Greenbottle Flies (Calliphoridae) Metallic thorax and abdomen Flesh Fly (Sarcophagidae) Striped thorax Life Cycle of a Calliphoridae Fly Late Stage Decomposition House Fly (Muscidae) Cheese Skipper (Piophilidae) Informational Source: Images: Top Left - Middle-Left: Top Right - Bottom -

Examples of Coleoptera (Beetles)
Early Stage Decomposition Early to Late Stage Decomposition Rove Beetles (Staphylinidae) Predator of fly eggs Clown Beetles (Histeridae) Predator of fly eggs Carrion Beetles (Silphidae) Adults & larvae feed on fly larvae Late Stage Decomposition Ham & Checkered Beetles (Cleridae) Predator of flies & beetles; also feed on dead tissue Hide Beetles (Scarabidae) Usually the last to arrive Skin Beetles (Dermestidae) Feed on dried skin & tissues Informational Source: Images: &

Let’s give it a try … Click the image above or click here to visit the website at

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

Entomology is the Study of Insects
Images from:

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”

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)

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

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.

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

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.

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

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

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

Time of Death can be broadly estimated up to about 36 hours
Temperature Stiffness Time of death Warm Not stiff Not dead more 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

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

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.

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

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

Calculating ADH from Climate Data

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.

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

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.

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

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.

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.

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.

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.

Methods Both pigs were housed 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. Subsequent to death, the pigs were individually tied in two black garbage bags, placed in feed sacks, and secured.

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.

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.

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.

Methods Using insect tweezers, they 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.

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.

Phormia regina Spiracles are incomplete Third-instar larvae

Phaenicia sp Spiracles are complete Third-instar larvae

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.

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.

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.

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.

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

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

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.

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

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.

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.

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

ADH and Our 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.

The End

Entomology Chapter 9 ©2010 Elsevier, Inc..

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