The effect of cold temperatures on Dipterian development and its implication for current
forensic entomological practices
E. H.
5/25/2004
Abstract
Forensic entomology is utilized in criminal investigations by exploiting the close
association of insects and corpses. Species of Diptera, especially blowflies, are particularly
useful in this field because they are some of the first insects to invade a corpse, and can therefore
be useful in estimations of time of death, also known as post-mortem interval (PMI). However,
previous methods of both insect evidence handling and estimation of PMI don’t adequately
account for aspects of the effect of cold temperatures on blowfly development. Here I examine
two experiments, Myskowiak and Doums (2002) and Ames and Turner (2003), which sought to
explore the effects of low temperature on blow fly development with regard to how these effects
would impact current forensic techniques. They found that revision of current practices is called
for in order to accurately utilize insect evidence to determine PMI. These findings highlight the
importance of experimental work along with case work for the development of the field of
forensic entomology.
Introduction
Corpses attract a vast myriad of arthropod species, most prominently including Diptera,
Coleoptera, and their larvae; this abundance lends to their application for forensic entomology,
the study of insects and other arthropods recovered from crime scenes and corpses (2). Insects
provide an important source of forensic information, since after the temperature of a dead body
has equilibrated with its surrounding environment and initial putrification has occurred, a reliable
estimation of time of death cannot be made (1). Forensic entomology was first documented by a
Chinese lawyer and death investigator in the 13th century, but insects were not first used to
estimate postmortem interval until the mid-nineteenth century, and forensic entomology was not
used to determine postmortem interval in forensic cases until the mid-twentieth century (2).
Three major concentrations in the still somewhat burgeoning field of forensic entomology have
been recognized: the identification of species, the study of larval growth and the changes
associated with different developmental conditions and the determination of how differences in
how thermal differences affect insect invasion of the cadaver (4).
Entomology is used in estimating time of death by the examination of blowfly maggots
(seasonally), and by examining the temporal succession of species found on a decomposing body
(3). There are four identified ecological categories that can be identified in the carrion
community (Smith, 1986): necrophagous species; predators and parasites of those (which include
schizophagous species which initially feed on carrion but may be predaceous at later larval
stages); omnivorous species which feed on both carrion and colonizers; and species which use
the corpse as an extension of their environment. The first two groups, which are considered to
be the most important for forensic entomology, include mostly species from the orders Diptera
and Coleoptera, with blowflies usually being the first colonizers of a corpse (2).
Forensically important blowflies of temperate regions are generally the Calliphora
species, including Calliphora vomitoria (Linnaeus) and Calliphora vincina (RobineauDesvoidy), which are widely distributed through Europe (6). These species lay their eggs around
natural orifices or wounds on a fresh corpse shortly after death (1, 6). The larvae which hatch
from these go through 3 molts before pupation (3, 6). By utilizing temperature data and the stage
of larval development, an estimation of minimum time since death (MTD), also known as
minimum postmortem interval (PMI), can be made (3, 6).
There are several methods for the determination of minimum PMI, which include
measuring the length or dry weight of the oldest larvae and comparing that information with
reference data, or an approach known as thermal summation (1, 6). Thermal summation is the
calculation of accumulated degree days or hours (ADD or ADH) needed to reach a particular
stage of development; this calculation is a linear model which applies well to a thermal range
where temperature is directly proportional to developmental rate; this is also examined in
conjunction with Developmental Threshold Temperature (DTT), which is estimated by
measuring developmental rates at a range of temperatures and extrapolating to the x-axis where
development is zero (6).
An important consideration in the determination of temperature, though, is the effect of
microclimates on the corpse and larval development, as well as the constantly fluctuating
temperatures present in the natural environment, as these can cause developmental times to differ
greatly (3, 7). The effects of low temperature on insect development are well known, with
diapause and quiescence being the main physiological responses (8). Since diapause is a period
during which growth and development of the insect is suspended due to seasonal weather
changes, it is an obstacle to the forensic entomologists’ accurate estimation of PMI, especially in
temperate climates where it is commonly observed in the spring and autumn months (1).
Conversely quiescence is a drop in insect metabolism in response to a drop in temperature and
only stops development for a short time (8).
Myskowiak and Doums (2002) and Ames and Turner (2003) studied the effects of low
temperature and low temperature episodes, respectively, on the development of blowfly maggots
through a series of experiments. Myskowiak and Doums (2002) studied the impact of the
common forensic practice of refrigeration of insect evidential material to halt temporarily halt
development for later analysis using Protophomia terraenovae (Robineau-Desvoidy). That
study was then complemented by Ames and Turner (2003) who studied the effects of short
periods of low temperature on the development of Protophomia terraenovae (Robineau-
Desvoidy) and Calliphora vomitoria L. with reference to ADD/ADH and DTT estimations and
calculations. These studies both call into question the accuracy of common forensic
entomological practices based upon a better understanding of Dipteran developmental biology.
Results
Myskowiak and Doums (2003) observed significant effects on biometry and
developmental rate for all developmental stages (egg, L1, L2, L3, prepupa and pupa) of blowfly
after applying refrigeration at 4 degrees Celsius to different stages and then allowing
development to proceed. The refrigeration significantly decreased the weight of the two-day-old
pupae, but this decrease was not a function of time spent in the refrigerator. The total
development time was significantly affected by the treatment for all stages as well, and
significantly varied linearly with time in the refrigerator, except in the L3 stage. The direction of
the linearly relation depended on the developmental stage, though; development time decreased
for L1 and prepupae but increased for L2 and pupae refrigeration. This data suggests that
placing immature stages of blowfly at low temperatures could cause significant error in PMI
estimates, especially since the effect on total development time was primarily due to its affect on
larval development time rather than on metamorphosis.
Ames and Turner (2003) ran two series of experiments: the first series had different
developmental stages experiencing a five day period at 20 degrees Celsius replaced by a five day
cold episode of 5 degrees Celsius, whereas the second series were reared for 3 days at 20 degrees
Celsius and then cohorts of each stage were placed at four different low temperatures for 5 days.
The first experimental series did not experience a linear delay in development due to the cold
exposure period as would be predicted by the linear hour-degree model, but instead showed
significant differences between the treatment groups for both species, with the development
stage at which the cold episode occurs being statistically important for C. vicina, with a
significant difference between the 1st stage larvae and pupal treatments. This effect is not
observed in the C. vomitoria, however.
The second experimental series found that with increasing temperature of the cold
episode the development time to reach the adult stage for both species decreased linearly. The
results for this series also depart from classical linear expectations since a significant difference
between development times for each low temperature in the series was observed. Adult ADH
values also followed a decreasing linear relationship, again departing from expectations which
would predict that they be constant across the range of temperatures. However, the ADH values
for the three highest temperatures were not significantly different, suggesting that the linear
model was accurate at these temperatures. Ames and Turner (2003) also found that the level
DTT is set at on the ADH estimate affects the controls because it affects the calculation of ADH,
though this only has a marked effect on the 20 degree Celsius controls.
Conclusions
Myskowiak and Doums’ (2003) finding that refrigeration of P. terraenovae can affect the
development and biometry of larval specimens after only one day of refrigeration suggests that a
common forensic practice could be introducing significant error in to PMI estimations.
Furthermore, the different relationships between duration of refrigeration, the stage refrigerated,
and the time to development indicate that only L3 larvae return to their standard development
rate after quiescence, indicating that a period of refrigeration has a lasting impact on the
development of the specimen. The developmental sensitivity and increased mortality of
pupating larvae to refrigeration is explained by the pupaes’ increased metabolic demands due to
extensive tissue remodeling (8), further proving that the insect developmental biology must be
accounted for in forensic entomological evidential practices.
Ames and Turner (2003) add to this conclusion with their finding that only third larval
stage ADH values are statistically similar between blowfly species, with no difference in the
effects of temperature on developmental rate, suggesting that the use of generic linear ADH
values is questionable and could lead to underestimation of PMI. The basic foundation of the
ADH concept is that there is a fixed quantity of metabolic activity, which is correlated with time
and temperature, necessary to complete development, which is not reflective of the true
developmental patterns found experimentally.
While Myskowiak and Doums (2002) briefly address the difference between diapause
and quiescence, they never return to the concept or really explain its significance in context with
the relationship of refrigeration and development. Ames and Turner (2003) revisit this point in
their concluding remarks, suggesting that diapause, since it requires specific triggers for its
activation and deactivation, is unlikely to be the physiological mechanism at work here, and
instead it is likely that the flies are experiencing quiescence, indicating that it can not be assumed
that no growth has occurred during cold periods.
The first true application of forensic entomology was in a French courtroom in 1850, and
while this instance was a breakthrough for forensic entomology, the forensic examiner in the
case believed that the development of adult flies took a full year (2). This case clearly stresses
the importance of a clear understanding of insect biology in forensic entomology. By further
studying the biology of carrion insects and combining that experimental work with case work, as
Amendt et al. (2004) suggest, the field of forensic entomology could become one of the most
valuable scientific tools available for criminal investigation.
Citations and References
(1) Amendt J., Krettek R., Zehner R. 2004. Forensic Entomology. Naturwissenschaften 91:5165.
(2) Benecke, M,. 2001. A brief history of forensic entomology. Forensic Science International.
120: 2-14.
(3) Erzinçlioglu, Z. 2003. Forensic Entomology. Clinical Medicine 3: 74-76.
(4) Marchenko, M.I. 2001. Medicolegal relevance of cadaver entomofauna for the determination
of the time of death. Forensic Science International 120: 89-109.
(5) *Smith, K.G.V. 1986. A Manual of Forensic Entomology. British Museum, London.
(6) Ames C., Turner B. 2003. Low temperature episodes in development of blowflies:
implications for postmortem interval estimation. Medical and Veterinary Entomology 17: 178186.
(7) Grassberger, M., Reiter, C. 2002. Effect of temperature on development of the forensically
important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera:
Calliphoridae). Forensic Science International 128: 177-182.
(8) Myskowiak, J., Doums, C. 2002. Effects of refrigeration on the biometry and development of
Protophormia terranovae (Robineau-Desvoidy) (Diptera: Calliphoridae) and its consequences in
estimating post-mortem interval in forensic investigations. Forensic Science International 125:
254-261.