LAB04: Forensic Entomology

REQUIRED READINGS (please read prior to attending lab section)

1. REVIEW Chapter 9: Fundamentals of Forensic Science 3rd ed.

Introduction
Forensic entomology is the study of insects in criminal investigations. Such investigations can include using the presence of
insect species and their life stages to gain insight into cases of abuse, ingestion of illicit substances, and the movement of
corpses, but most often post mortem interval (PMI). There are many important variables, however, that need to be
accounted for when using insect evidence such as: corpse temperature, ambient temperature, rain or snowfall, placement
of the corpse (e.g., indoors, outdoors, sun, shade, fully or partially buried), geographical location, among others. In this lab
you will be given a criminal case scenario that includes entomological evidence. You will process this evidence to assess PMI
of the corpse and while doing so, consider the ecological and environment variables that you can and can not account for in
creating your PMI estimate.

A crucial element in forensic entomology is species identification. Several species appear the same based on macroscopic
features but may have vastly different growth rates, behaviours and habitat preferences. Herein we will use a simplified
morphological key developed for species collected from campus. While many fly and beetle species are common colonizers
of corpses depending on the stage of decay: we will be focussing our investigation on the Calliphorid species found on a
corpse.

Faunal Succession
Insects arrive on a corpse in a somewhat predictable sequence depending on the stages of decomposition. This sequence of
colonization is called succession or faunal succession. These data can be used to provide a rough estimate of PMI as
different stages of decomposition are attractive to different species of insects. There are many environmental variables,
however, that influence succession: season, sun exposure, whether the body was found inside a building, immersed in
water or in urban vs. rural area. Bodies that are buried, left in vehicles, found in enclosed spaces, hanged or burnt are also
subject to varying insect succession patterns. All of the aforementioned variables can have an effect on the diversity of
species colonizing the corpse and also that have access to the corpse. Blow flies (Calliphoridae) and flesh flies
(Sarcophagidae) are usually the first two types of insects to arrive at a corpse. Blow flies are attracted first to the corpse
from a great distance by odour, generally within several hours, but sometimes within minutes after death. Blow flies are no
longer attracted when the remains have decomposed, are mummified or dry. Flesh flies arrive on human remains at the
same time or just after the blow flies. In some circumstances, the flesh flies may arrive first as they can fly under weather
conditions that would prevent most other types of flies from reaching the carrion. Successional patterns give a coarse
estimate of PMI and are often appropriate for later stages of decay in assessing PMI.

Species-Specific Life Cycle Development Rates

Once the flies have been collected and their species identified, lifecycle models of fly species are analyzed in relation to the
larvae collected from the corpse. Actual lifecycles of flies are variable between species depending on environmental
conditions and hence PMI estimates from these models must be assessed with some caution. From these life-cycles,
especially for early succession species, narrower estimates of PMI can be derived than from faunal succession estimates.
The effect of temperature on the rate of development is often the main variable that can be accounted for in a forensic
investigation where climatic data, such as high and low temperatures, can be accessed from meteorological stations in the
same vicinity as the corpse. Insects require a certain amount of heat to develop. For a fly to develop from an egg, the
temperature must remain between a minimum value, called the lower developmental threshold and a maximum value,
called the upper developmental threshold for a certain amount of time. On either side of this temperature range, the insect
will not develop or the rate of development will decrease. The total amount of heat required for an organism to develop
from one point in its lifecycle to another point is measured in units called degree-days.
FRSC2030H LAB04 Forensic Entomology

Accumulated Degree Days (ADD) & Accumulated Degree Hours (ADH)

One degree-day is the amount of development that occurs in one day (24 hours) when the temperature is one degree
above the lower development threshold. Degree-days accumulate over time. Each day the temperature is above the lower
developmental threshold, more degree-days are accumulated and referred to as accumulated degree days (ADD). For
example, if the temperature is 5°C above the lower development threshold for 2 days, the number of degree days is 10 (5°C
x 2 days). One degree above the lower development threshold for 10 days equals 10 Degree Days (or 240 accumulated
degree hours, ADH). Each insect species requires a certain total number of degree-days to complete their development. The
amount of heat required does not change and so ADD provides a common reference between species. Degree days are
calculated from actual temperature data.

For example, if the minimum threshold temperature for a fly is 9°C on a day when the average temperature is 10°C, one
degree day has accumulated. If the next day has a temperature of 11°C, another two degree days accumulate. The daily
accumulations are added together over time and used to predict insect development. When a certain number of degree
days have accumulated the adult insect will emerge. Constant temperatures are uncommon in the natural environment
where most insects live and grow. Insect development is affected differently by cyclic and constant temperature conditions,
even when the mean of the cyclic temperatures is equal to the constant temperature. Changes in environmental
temperature cause changes in development rate from day to day and even from hour to hour. A forensic entomologist must
consider both the internal and ambient temperatures when calculating the age of the fly larvae. Both temperatures
influence the amount of degree-days and the rate of larval development. To calculate ADD, calculate the mean (average)
temperature for that day. Compare the mean to the organism’s lower developmental threshold. If the mean is three
degrees higher than the lower developmental threshold, then there have been three degree-days.
Species Specific Growth Rates in Hours: depending on the life stage of maggot found, these data are used to estimate how
long the corpse has been present. For example, at an average temperature of 15°C, 2nd instar maggots from P. regina would
be 80-190 hours old (average 135 hours) or between 3-8 days old (avg 5.6 days). Since these flies arrive within minutes to
hours after death, these values would correspond to a PMI range.

Learning Goals/Objectives:
• Observation and documentation: continue to develop clear note-taking skills to permit you to accurately convey the
context and extent of entomological evidence presented within the lab.
• Protocols at a crime scene where entomological evidence is collected.
-Employ morphological keys in species determination and understand limitations of this technique.
-Morphological and size assessment of blowfly larvae and limitations of using these types of data
-Understand the use of accumulated degree days (ADD) in relation to species development and PMI estimations
and the limitations of using these types of data.

Schedule of Activities:
- Assess species composition of adult flies collected from crime scene using both macro and microscopic characters
relative to phenotype keys. Identify the two unknown species.
- Use these data to determine PMI using ADD/H for the species of interest.
- Determine the number of accumulated Degree-hours for a species to reach its observed larval instar stage
- Determine accumulated Degree-hours using weather data given for important days within your case scenario
- Estimate a PMI date.

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The Case
Gizelle was found dead in the woods at 11:23 on July 4 th. She was last seen at 13:00 on June 30th with her boyfriend
Francisco. He says they went for a walk in the woods but they got in an argument and he allegedly left at 15:00. His boss
confirmed that he was on time for his night shift at 19:00 that day until 07:00 July 1st. Upon Francisco’s arrival to their
shared apartment at 07:20 on July 1st Gizelle was nowhere to be found. He contacted the police and a missing persons was
issued. Francisco has an alibi from June 30th at 19:00 until the body of Gizelle was discovered but does not have one for June
30th between 13:00 – 19:00. In order to determine if Gizelle was killed on June 30th or later the post mortem interval (PMI)
must be calculated. Based on the maggots/adult blow flies collected from her body at 12pm on July 4 th and the previous
temperatures, can the possibility that Francisco killed Gizelle be excluded?

Gizelle’s father is a national political figure and this case has become extremely high profile. Two of the most revered
Forensic Entomologists have been called forth and asked to help give insight into the entomological evidence collected. One
will be testifying on the behalf of the prosecution and the other the defense. In groups of 2-4, use the entomological
evidence collected at the scene to determine the PMI estimates for the blow fly species found on the body.

Species ID
In order to distinguish blow fly species (Calliphoridae) from other two wing insects (Diptera), there are a number of physical
traits that can be examined (plumose arista, subscutellum weak/absent, and row of bristles on the meron). However, the
most common distinguishing feature is the sharply bent wing M vein. View your specimen’s wings to determine if the
species is a blow fly (see figure below). This can be accomplished with the naked eye.

If the specimens are found to be blow flies, continue your ID of

each using the Blow flies of eastern Canada – Subfamily key
(http://www.biology.ualberta.ca/bsc/ejournal/mwr_11/intro.ht
ml). You will have to use the discriminative phenotypic traits to
discern between groupings and species. The pictorial key will
guide you through which morphological features to examine (if
not shown, there are generally additional links) under the
compound microscope. Record the identified adult species name
in their corresponding blanks on the following page. You are only
required to identify the specimen to genus. Question 1 requires
you to record the step number and identifying feature(s) used to arrive at your ID (ex. Step 1 – Stem vein bare → Step 2 –
etc.). This identification will allow you to determine the lower developmental threshold for the species which is required to
calculate PMI.

Larval Developmental Stage

Upon examination the spiracles on the posterior end of each species of maggot found on the body, it was determined to be
in the 3rd instar and measuring roughly 12mm. Compare these findings to the table below, this may help in further
identifying the species in question.

Species egg 1st instar 2nd instar 3rd instar post-feeding pupae
Lucilia illustris 3mm 4-6mm 6-10mm 9-15mm 8-13mm ~8mm
Calliphora vicina 5mm 6-9mm 1-15mm 13-24mm 13-19mm ~12mm
Phormia regina 4mm 5-7mm 8-11mm 10-16mm 9-13mm ~9mm

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Thyssen, P. J. 2010. Keys for Identification of Immature Insects.

Thermal Unit Calculations (ADD/ADH)

Development data are given for three blowfly species in Tables 1, 2, and 5 below. Using the species and development stage
you determined, calculate the number of degree days required to assertain the identified stage. To do this you must convert
to thermal units, either accumulated degree days (ADD) or accumulated degree hours (ADH). To do this, you will require the
lower developmental threshold of the species.

Lower developmental thresholds (LDT) are reported in the literature as follows: Phormia regina 11˚C, Calliphora vicina
5˚C, and Lucilia illustris 9˚C.

ADD or ADH are calculated by multiplying the developmental duration (in days or hours) for the time to reach a given stage,
by the (rearing temperature – lower developmental threshold).

ADD/HRequired = Average development duration to given stage (in hours or days) × (Rearing temperature – lower
development threshold)

Sample calculation for Phormia regina 1st instar (Table 1 at 23 ˚C):

ADHreq = (21.5+22.5)/2 × (23.0 – 11)
ADH required to develop to 1st instar = 264

This gives the number of ADD or ADH required to reach a given stage of development. When calculating required ADH’s,
make sure you use development times closest to the average temperatures of the period of interest (in this case closest to
20˚C in boxes in Tables 1, 2, and 5). The average of the minimum and maximum time to reach a stage should be calculated
in order to give the degree days required for the certain stage (ex. Phormia regina 1st instar: min = 21.5; max = 22.5; avg =
22h).

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G.S. Anderson. 2000. Minimum and Maximum Development Rates of Some Forensically Important Calliphoridae (Diptera).
Journal of Forensic Sciences. 45(4):824-32.

The minimum and maximum temperatures are given in the table below for the time period when Gizelle was last seen and
when her corpse was discovered. Determine the PMI estimates using both blow fly species working back from July 4th. Are
the required ADH met for an estimated PMI on June 30th or are estimates later? (Required ADH are met when within ±200
ADH’s).
ADH = (mean temp – lower developmental threshold) × 24(hours)

ADH ΣADH
Date Min Temp ˚C Max Temp ˚C Mean Temp ˚C
Unknown Species Unknown Species

June 30th 10.7 24.6

July 1st 9.7 27.3

July 2nd 12.8 27.8

July 3rd 18.2 28.7

July 4th 14.3 28.5

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IN LAB QUESTIONS – Work together to understand the calculations.

Blowfly Identification

1. What genus did you assign to the adult blowfly?

2. Based on the online identification key, which morphological features did you observe that led you to this
determination for this blowfly?

PMI Calculations

3. How many ADH does the blowfly of this species require to reach the developmental stage you observed? Refer to
the tables on page 5 of your lab handout. Choose the correct table for your species and use the temperature
column closest to the average temperature in your case scenario.

4. Based on the chart on page 5 of the lab handout, what was the mean temperature in °C on July 1st?

5. Using the formula ADH=(mean temp-LDT) x 24 hours, how many ADH accumulated on July 1st for species 1?

6. How many total ADH (∑ADH) did this blowfly species experience between June 30th and July 4th?

7. Working backwards from July 4th, on what day are the required ADH reached for this blowfly? (Required ADH are
met when they’re within +/- 200ADHs of the calculated ∑ADH).

8. Based only on your post-mortem interval estimate, can Francisco be excluded as a suspect in Gizelle’s death?

Post-lab assignment is on BB9 you may begin during this lab, it will follow these same steps but with different values.