Entomology-based methods for estimation of postmortem interval

Abstract

Forensic entomology involves the use of insects and other arthropods to estimate the minimum time elapsed since death, referred to as minimum postmortem interval (min PMI). This is based on the assemblage of insects found in association with remains, and most often, the time required for development of the first colonizing insects to develop to their size/ life stage at time of collection. This process involves the accumulation of appropriate data for the development of the species of insect at a variety of relevant temperatures and consideration of the other biotic and abiotic factors that may affect developmental rate. This review considers the approaches to the estimation of min PMI, focusing largely on the age estimation of specimens collected from remains and the limitations that accompany entomology-based PMI estimations. Recent advances and newly developed techniques in the field are reviewed in regard to future potential.

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http://dx.doi.org/10.2147/RRFMS.S68867
Entomology-based methods for estimation of
postmortem interval
Michelle L Harvey1
Natalie E Gasz1
Sasha C Voss2
1School of Life and Environmental
Sciences, Centre for Chemistry
and Biotechnology (Waurn Ponds
Campus), Faculty of Science,
Engineering and Built Environment,
Deakin University, Geelong, VIC,
2Cen tre for Forensic Anatomy
and Biological Sciences, School of
Ana tomy, Physiology and Human
Biology, The University of Western
Australia, Crawley, WA, Australia
Correspondence: Michelle L Harvey
School of Life and Environmental
Sciences, Centre for Chemistry and
Biotechnology (Waurn Ponds Campus),
Faculty of Science, Engineering and
Built Environment, Deakin University,
75 Pigdons Road, Locked Bag 20000,
Geelong, VIC 3217, Australia
Email michelle.harvey@deakin.edu.au
Abstract: Forensic entomology involves the use of insects and other arthropods to estimate
the minimum time elapsed since death, referred to as minimum postmortem interval (minPMI).
This is based on the assemblage of insects found in association with remains, and most
often, the time required for development of the first colonizing insects to develop to their size/
life stage at time of collection. This process involves the accumulation of appropriate data for
the development of the species of insect at a variety of relevant temperatures and consider-
ation of the other biotic and abiotic factors that may affect developmental rate. This review
considers the approaches to the estimation of minPMI, focusing largely on the age estimation
of specimens collected from remains and the limitations that accompany entomology-based
PMI estimations. Recent advances and newly developed techniques in the field are reviewed
in regard to future potential.
Keywords: forensic entomology, PMI, blowfly, decomposition, death investigation
Introduction
Forensic entomology, the use of arthropods as tools in legal investigations, primar-
ily focuses on the estimation of the time length between death and the discovery of
decomposing remains in cases of homicide, suicide, or accidental death.1–3 Termed
“minimum postmortem interval” (minPMI), the entomological estimation of this time
period is based on the assumption that insects, commonly found in association with
decomposing remains, arrive at a carcass shortly after death.4 Decomposing remains
present a transient habitat and food resource opportunity for numerous insect species.5,6
Within hours of death, insect groups such as blowflies (Diptera: Calliphoridae) are
olfactorily attracted to decomposing remains which are both a source of protein for
egg development and a site for oviposition.7,8 The colonization time, development time,
and departure time of the different insect species inhabiting remains are closely linked
to the progression of carcass decomposition.9 As such, the age of the oldest immature
insect specimen collected from remains, in the context of expected arrival time of adult
females, provides an indication of the minimum time that the decomposing remains
were available for insect colonization and thus minPMI.10–12
As insect development is primarily governed by temperature, where this relationship
has been quantified for a species, the age of a specimen can be determined based on the
level of development and the thermal history at which that specimen developed.13 Where
immature insect specimens are not present or have already completed development,
as is often the case in advanced stages of decomposition, minPMI is estimated based on
the assemblage of insects associated with the remains, referred to as the predictable
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Harvey et al
process of insect succession.14 The insect species present
are compared to known patterns of insect colonization and
the time frames associated with each phase of colonization.
Such estimates are not as precise as age-based estimates,
but they do provide a broad time frame within which death
occurred.15 Forensic entomology is most commonly used to
provide minPMI estimates where early colonizers are still in
association with remains, and thus, the fundamental issue
in such cases is to estimate the age of a specimen collected
from remains. This review concentrates on the methods used
to achieve this, considering the strengths and weaknesses of
such approaches and scope for further research and technique
development.
Entomological approaches for age
determination
Current approaches to age estimation are based around the
species-specific time required for an immature insect to
progress through developmental landmarks such as length,
weight, and life cycle stage in relation to temperature. While
measures of developmental duration based on length and
weight are valuable, life cycle stage is a preferred land-
mark for age estimation due to the confounding issues of
diet, competition, and application of different preservation
methods for forensic specimens (shrinkage) on weight and
length.16,17 Thus, determination of specimen age is predomi-
nantly based on predetermined development data detailing
the predictable relationship between temperature and insect
growth for the onset and completion of each life stage of
insect development.18–20 Applicable reference data details the
duration of development of specific life stages of immature
insects encompassing egg, larval instars, pupation, and eclo-
sion under a range of constant or fluctuating temperatures.21
The application of data detailing the relationship between
insect development and temperature is then used in several
modeling approaches to predict insect specimen age based on
the thermal history of the collected forensic specimens.
Isomorphen and isomegalen diagrams
The simplest approach is termed an “isomorphen diagram”
which is essentially a scatter plot of the time from eggs hatch-
ing until eclosion plotted against constant temperature.1,22
Associated error bars provide a 95% confidence interval for
each developmental event. A slight variant, termed “isomega-
len diagram”, plots larval size since hatching (length, weight,
or width) rather than life stages against temperature.22 Use
of size as a component within the isomegalen diagram has
the advantage of greater time point resolution compared to
life stage event landmarks for age estimation; however, size
measures have been reported as poor indicators of age.23,24
Estimation of specimen age is achieved with considerable
accuracy where the thermal history of the specimens examined
is consistent with the constant temperatures used to generate
the reference data of the diagram. Considerable error occurs,
however, in the derived age estimation using this approach
when the ambient temperatures under which specimens are
developing on decomposing remains fluctuate over time.22
As the majority of crime scenes within which decomposing
remains are found experience fluctuating temperature condi-
tions, a series of mathematical models have been developed
which are generally more applicable and widely used.
Thermal summation model
The most commonly applied method for modeling insect
development rates in a forensic context is the thermal sum-
mation model which applies a linear regression analysis to the
positive relationship between temperature and development.1
Insect development can be measured at close intervals over
a range of temperatures, and where the rate of development
(measured as reciprocals of development time, 1/D) is plot-
ted against temperature, a sigmoid-shaped curve results.25 At
temperature extremes, insect development is either slowed or
completely halted corresponding to an upper and lower devel-
opmental threshold.26 A large proportion of the relationship
between temperature and development is linear between the
upper and lower developmental threshold (species specific).
Linear regression can thus be used to determine an x-intercept
(lower developmental threshold, TL) and inverse of the slope
of the linear regression (thermal summation constant, K)
which allow prediction of development time from the thermal
history of a specimen.1 Under this linear regression model,
development is measured as physiological time with units
of “degree days” or “degree hours”, where one degree day is
equal to one degree above the lower developmental threshold
over either 24 hours or 1 hour, respectively. Each life stage
(egg, first instar, or pupation) requires a certain amount of
accumulated degree hours to develop to the next life stage
and complete development equating to K.26 Standard practice,
under this method, would be to rear insects collected from
a scene at a constant, controlled temperature, record time
elapsed at point of eclosion, and subtract the physiological
time required for laboratory development from the total
physiological time required for development in this species.
When used in conjunction with crime scene temperatures,
the period of time elapsed between oviposition and insect
collection at the scene may then be calculated.

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Problematically, while linear models have the advantage
of simplicity and allow estimation of lower developmental
thresholds and thermal summation constants, they do not
incorporate the nonlinearity observed in insect development
at low and high temperatures.27 Several alternative models
have been proposed including a revised linear model28 that
calculates an improved fit for TL and K by accounting for the
high variability at extremes of the linear range and multiple
nonlinear approaches29–31 encompassing a recent “new simu-
lation model” termed “ExLAC”.32,33
Curvilinear models/ExLAC
Nonlinear or curvilinear models can more accurately describe
the relationship between development and temperature
for insect populations by incorporating the curvilinearity
observed at the upper and lower temperature extremes of
the plotted relationship between development rate and tem-
perature.27,34 While offering improved parameters for estima-
tion, the complexity of such models, however, reduces the
practicality of application to forensic estimation of minPMI.
Additionally, no one curvilinear model, above others, has
been identified that consistently outperforms linear models
across relevant species data.35
A recently proposed curvilinear model known as ExLAC
has been demonstrated to offer an alternative to linear thermal
summation modeling.32,33 While the ExLAC model shows
minimal performance improvement over linear modeling, it
has the advantage of generating error rates associated with
the age estimate derived by the model. Again, the devel-
opment model is based on the duration of each life stage
during development as a function of temperature.33 An indi-
vidual exponential function is, however, applied to each life
stage, and additional parameters are included in the model
that account for variation in measurement of input values
such as thermal history data and the strength of the relation-
ship between life stage duration and temperature.32,33 The
included measurement of error for the inputted temperature
data offers a distinct advantage over the currently applied
thermal summation model. Standard practice involves the
use of temperature data gathered at the weather station near-
est to the crime scene as a measure of the insect specimen’s
thermal history prior to collection.36 This data is corrected
for potential variation between locations using regression
modeling incorporating data acquired at the crime scene
following the discovery of decomposing remains. While this
aspect of the proposed model offers a measure of potential
error in estimates of minPMI, the performance of the model
is only marginally better than the linear thermal summation
method for establishing specimen age.32 To date, the ExLAC
model has yet to be thoroughly evaluated and assessed prior
to implementation in forensic practice, and the linear thermal
summation model is still the preferred method of estimating
specimen age and thus minPMI.
Current issues
Regardless of the developmental modeling approach taken
to determine age, several problems arise in regard to the
available development data used in such models. Available
reference data for use in forensic practice is typically focused
on the development of early colonizing dipteran species.
A considerable body of reference literature exists document-
ing the development of forensically relevant fly species under
varied constant temperatures within the laboratory.11,37–41
Problematically, reference data is not available for all spe-
cies reported in association with crime scenes, particularly
in the case of alterative indicators of minPMI such as beetles
and parasitic wasps. Aspects of life history and development
in relation to temperature are often unknown for specimens
collected off remains or of limited scope for application in
development models.
Additionally, research has indicated that populations of
the same species can differ physiologically depending on
their geographic origin.2,42,43 For instance, there are often
considerable discrepancies in the reported development time
of blowfly populations from different geographic origins
when reared at the same developmental temperature.44,45
Such differences have been attributed to regional genetic
variation between populations, although the effect of dif-
fering environmental factors between regions cannot be
discounted. The possibility that inherent biogeographical
variation exists between populations of species in relation to
development time is of considerable consequence in respect
to the accuracy of minPMI estimation. At present, development
data for a species from a single source location are applied to
the same species from different geographic locations, even
though there is little evidence supporting the validity of such
procedures and considerable evidence to the contrary.17,44–46 At
present, the applicability of extrapolating development data
to a population from a different geographic origin to that of
the source data is likely to be called into question under cross
examination during court proceedings. Thus, further work is
urgently needed to address and quantify this issue.
Further error in the accuracy of minPMI can arise in regard
to a species physiological response to fluctuating temperature
regimes, a common occurrence at crime scenes. Thermal
summation assumes that a species development rate at a given

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Harvey et al
constant temperature is independent of the overall thermal
regime.47 A number of studies, however, have indicated that
development rate under fluctuating temperatures does not
correspond to development under the resulting mean constant
temperature.24,48,49 As reference data detailing the relationship
between temperature and insect development is typically
generated using constant temperatures, the use of this data
to model specimen age for forensic estimation of minPMI can
lead to erroneous estimates where daily ambient temperatures
at crime scenes fluctuate. As such, there exists a need for
expanded research of species-specific development under
both variable and constant temperatures to provide compre-
hensive reference data for use in forensic case work.
Moreover, temperature is considered the primary fac-
tor influencing insect development; however, a variety of
abiotic and biotic factors additionally influence the rate
of development including, humidity,50 photoperiod,51 and
diet (nutrition).52–54 These factors are essentially ignored in
the majority of studies documenting developmental time-
frames for use in forensic practice, yet are likely to have
an impact on development rate and hence the accuracy
of any associated minPMI estimation. Other limitations in
the currently available reference data include the lack of a
standardized rearing methodology between research studies.
Where development data is generated using different food
substrates, photoperiods, sampling protocols (ie, sampling
interval, larval density), and rearing conditions unrelated to
temperature, reported development rates for a particular spe-
cies often vary.55,56 Thus, inconsistency in the methodology
behind the generation of development data for use in estimat-
ing forensic specimen age can contribute to inaccuracies in
the associated minPMI estimate.
Estimates of minPMI based on modeling of the relation-
ship between insect development and temperature are widely
accepted in criminal courts throughout the world; however,
there are substantial issues regarding the appropriateness of
the reference data available in the literature and used to for-
mulate such estimates. As such, further research on aspects
of forensically relevant insect growth and life history are
still needed to establish a comprehensive, global, reference
database of applicable developmental data for use in both
linear and nonlinear models.
Recent advances in age estimation
methodology
Current approaches to age estimation are generally based
on measurement of the timeframes associated with the start
and end of the life stage collected, such as the onset of an
instar or pupation. The duration of a life stage such as the
pupal stage can be considerably long, and thus, the use of life
stage landmarks alone (ie, onset of pupation and eclosion) as
indicators of specimen age can introduce considerable error
into associated minPMI estimates.57–59
Several approaches have been proposed to address this
issue, and provide refined approaches to limiting the possible
age range of a specimen. These range from observations
of insect morphological features as an indication of age to
quantitative approaches based on gene expression or hormone
levels. These approaches vary in their utility, objectivity, and
reliability.
Morphology
Calliphorids are the primary colonizers of carrion, and as
such generally represent the oldest insects present on a corpse
and perceived best indicators of minPMI. They exhibit holom-
etabolous development, meaning a complete metamorphosis
through egg, larval, pupal, and adult stages occurs. These life
stages are all morphologically distinct from each other. Within
each life stage, relative degrees of development based on size
or the relative level of development of certain features may be
useful in characterizing the insects as having experienced a
certain amount of physiological time. In the egg stage, which is
relatively short, physical markers refining age estimates are less
important than in the longer persisting larval or pupal stages.
The pupal phase alone may comprise .50% of the total life
cycle of the insect;57–59 thus, the ability to refine an age estimate
from a 2-week window, down to perhaps a 24- to 48-hour
window, is highly desirable. Current approaches to pupal aging
rely frequently on rearing to adult stage,60 at times a lengthy
process and sometimes compromised by insect death due to the
presence of hymenopteran parasitoids or other factors.
External morphology
Size measures are often proffered as useful methods to
refine insect age within the larval stage, utilizing width,
length, or weight of larvae as an indicator of relative period
of development.61 These measures are subject to numerous
influences, summarized suitably by Villet and Amendt,60
including drugs, maggot mass-generated heat, competition
for food, substrate fed on, preservation, and measurement
errors. These factors make the use of size measures, as used
in isomegalen- and isomorphen-based methods, prone to
significant error. An alternative is to concentrate on discern-
ible developmental changes within a life stage.
Eggs, pupae, and adults display no obvious measurable
size-related changes that may be utilized for age refinement.

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Eggs are generally reared to hatching, and then age is derived
via back-calculation of physiological time required for
development. Discernible morphological features are gener-
ally lacking unless the specimen can be observed to be in
the process of emerging from the chorion via the plastron. In
larvae, non-size-related changes are largely confined to the
posterior spiracle morphology, with instar determined based
on the number of slit-like openings to the trachea in each
posterior spiracle.62 These instars are clear intra-life stage
events that may be used to further refine larval age.
Adult age estimation is generally confined to the period
immediately following eclosion, where wings are gradu-
ally unfurled as hemolymph moves into veins, and general
body coloration of the fly develops.62 There is little call
for determining age of adult flies, given that their mobility
makes connecting them to development on a specific source
of carrion problematic. The puparium left behind following
eclosion is likely to provide as much information as the
adult fly itself.
It is the pupal phase that necessitates the greatest age
estimation refinement, given the extended duration of the
stage and thus large timeframes provided for minPMI based
on the landmarks of life stage commencement and end. In
the pupal stage, there are few obvious changes on the exterior
of the puparium itself, with the exception of the initial tan-
ning and sclerotization process.62 Pupae are sedentary, and
their extended close association with remains makes them
useful targets for provision of temporal information regard-
ing remains. It is for this reason that most age refinement
methods concentrate on this extended stage.
The calliphorid pupa is coarctate, with the puparium
formed following “loosening” of the third instar cuticle. The
casing darkens with time,63 but coloration is obviously altered
with soil type/moisture level and/or preservation. Removal
of the puparium, however, reveals significant morphological
remodeling as the larva metamorphoses to adult form, with
the development of key features such as tagmosis, append-
ages, setae, and coloration.57 Examination of these features, in
consultation with species-specific morphological timelines,57
may be utilized to refine an age estimate for a specimen.
Brown et al57 utilized 23 morphological features of Cal-
liphora vicina pupae to refine an age estimate to within 5%
of a pupa’s actual age with 95% reliability. This illustrates
the utility of the technique for intra-pupal stage aging but is
heavily reliant on appropriate datasets for numerous features
in all species of relevance, and confirmation that between
population variation is minimal, before data may be applied
in other localities.
Internal morphology
Internal changes have been studied in the pupal phase of
C. vicina for their use in age estimation of specimens.64
Internal metamorphosis during this phase involves histolysis
and histogenesis of tissues and organs during the transition to
adult form, with changes such as the utilization of larval fat
bodies and glycogen stores. Thoracic flight muscles become
recognizable, and digestive, reproductive, and nervous
systems are modified and developed.64 These changes can
be used to create a chronology of development relative to
physiological time, and utilized, either alone or in conjunc-
tion with external morphological data, to provide a minPMI
estimate. Other life stages have not been considered, and
future studies are needed to further develop this approach
across relevant life stages.
Optical tomography
Optical coherence tomography utilizes benchtop-sized
instrumentation to provide high-resolution images of samples
without the need for destructive analysis. Specimens may be
analyzed while still alive, remaining in the puparium and suc-
cessfully continuing development and emerging in expected
time frames, allowing rearing-based confirmation of age, and
species identification from adult morphology. Morphological
features such as brain, mouthpart, and leg development have
been successfully visualized in calliphorid pupae;65 however,
the puparium limits resolution due to absorption of light
and thus limits penetration of light into the insect itself. The
method holds promise but requires refinement for extensive
use in casework.
Hyperspectral imaging
The ability to analyze specimens nondestructively is also
provided using hyperspectral imaging, a technique used in
other areas of forensics66–68 and in agriculture.69 This method
allows specimens to be analyzed either live or preserved in a
noninvasive manner, utilizing the technique to provide spatial
and spectral information regarding a specimen. Preliminary
work assessing the validity of this approach to the analysis
of entomological specimens has been conducted using pupae
of Chrysomya rufifacies and Calliphora dubia. Specimens
reared at 24°C and 30°C were imaged daily to provide data for
predicting age, in conjunction with morphological changes.
Hyperspectral imaging allowed determination of pupal
age with .82% accuracy and also reliably distinguished
between the two species based only on spectral data.70 The
technique has distinct advantages including the portability
of the equipment required which, once developed, will allow

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Harvey et al
analysis of specimens at the crime scene. Additionally, the
nondestructive and noninvasive advantages of the technique
allow alternative and/or later reanalysis of evidence. Such
advantages place hyperspectral imaging at the forefront of
future advances in the forensic field, and there is consider-
able potential for the approach to be developed as a regular
tool in forensic case work.68 Problematically, the technique
requires the development of a reference database for relevant
species prior to implementation in case analysis. It is antici-
pated, however, that upon further development, hyperspectral
imaging will provide a valuable approach to age estimation
of all life stages of insect development.
Molecular methods
Molecular methods form a useful basis for estimation of
insect age, as they are based on objective, quantitative data,
not subject to the interobserver error associated with mor-
phological methods. In theory, these should be more reliable
indicators of insect age. As with morphological methods, the
focus is largely on the long-lasting pupal phase.
Steroidogenesis
Steroidogenesis examines levels of ecdysteroids (polyhy-
droxylated steroid hormones) produced throughout insect
development. Moulting and metamorphosis are triggered by
such hormones, and therefore, developmental change can be
measured relative to quantitative levels of specific steroids.
Ecdysteroid levels were examined in pupae of Protophormia
terraenovae based on enzyme immunoassay, and it was shown
that with suitable preservation, ecdysteroid peaks could be
determined between 36 and 96 hours after pupariation.71
This may provide valuable information regarding time of
pupariation, to correlate with morphological development
observations; however, utility may be limited by state of
preservation, for example, when samples have been frozen
following collection. Furthermore, conclusions may only be
narrowed to quite wide time frames. A more precise method
of estimation would be useful, given that the pupal stage may
be considerably long.
Gene expression
During insect development, genes are switched on and off at
various times, triggering the synthesis of products including
proteins. For a gene to be switched on, the DNA within a
cell must first be transcribed as mRNA, called a transcript.
Gene expression studies measure the level of transcription at
a given point in time.72 Measurement of the level of expres-
sion of a particular gene over time can provide a useful
indication of age of a specimen, as specimens of known age
can be examined to build reference data regarding levels of
expression expected at various times in development.
Age estimation of Lucilia sericata eggs has been shown
to be possible,73 successfully aging within 2 hours based
on transcript levels of three genes. The use of three genes,
rather than one gene alone, allows three expression levels to
be compared, providing corroboration and refining age esti-
mations to smaller windows. Other studies59,72 have further
extended the approach to larval and pupal stages of the same
species with success, providing good support for traditional
morphology-based methods of age estimation. Pupae, in
particular, are useful targets for gene expression-based aging,
given the extensive tissue remodeling and cellular prolifera-
tion that take place during this stage.
Cuticular hydrocarbons
On the surface of the insect cuticle is a lipid wax layer of
hydrocarbons that function to protect the insect from drying
out, provide defense against attack by microorganisms, and
may play important roles in behavioral events such as mate
selection by acting as pheromones or kairomones.74 These
hydrocarbons may be either saturated or unsaturated com-
pounds, and have been suggested to be useful in forensic
entomology for a number of applications, including deter-
mining species identity, determining geographical origin of
specimens, and estimating age of individual insects for the
purpose of refining minPMI estimates.75–77
Hydrocarbon analysis has been proven useful with
forensically significant calliphorids, separating adults of
Phormia regina according to population origin and sex,75 and
identifying empty puparia to species level.77 The utility of
cuticular hydrocarbon composition to pinpoint larval age in
C. rufifacies determined that the method is particularly useful
for post-feeding larval age estimation,77 mirrored in a study
of L. sericata showing distinct difference between young and
post-feeding larvae.78 This is usually limited with respect to
morphological changes, so it represents a useful application
for the technique. Cuticular hydrocarbons could also be used
to help estimate PMI based on weathering of puparia, but this
is obviously complicated by the numerous factors that may
affect rate and degree of weathering.79 Other studies have
suggested that insect cuticular hydrocarbons can be used
for PMI estimation for egg to 8-day-old adults;80 however,
large databases of expected hydrocarbon compositions are
required for species of different ages at different locations,
given that conspecific populations have been shown to be
distinguishable based on hydrocarbon composition.

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The possibility of extending analyses to additionally
measure volatile organic compounds released by insects
to estimate their age has been proposed.81 Volatile organic
compounds were collected daily throughout larval and pupal
phase and analyzed using headspace solid-phase microex-
traction and gas chromatography–mass spectrometry, and
profiles were shown to carry in quantity and composition.
While promising initial results were reported for C. vicina,
the effect of age and genetic factors, as well as environmen-
tal factors (temperature, diet, geoclimate), requires further
investigation. In addition, some compounds are produced by
the insects, some by the corpse itself, and some by bacteria,
so the source of emission needs to be clarified.81
The future
Forensic entomology has become an integral technique for
the forensic sciences, providing important information for the
investigator regarding time of death. This is dependent initially
on examination of the assemblage of insects present, followed
by accurate identification, and then refinement of minimum
age of insects based on knowledge of developmental rates
and ambient temperatures to calculate physiological time.
This entire process is limited greatly by lack of data regarding
developmental rates in the plethora of decomposition environ-
ments that may be encountered, including burials, wrapping,
enclosure in vehicles, and aquatic submersion, plus the numer-
ous other complicating variables including drug presence in
remains, clothing, rainfall, and humidity. Of all the variables
requiring consideration in minPMI estimation, temperature is
fundamental and is the best studied factor, but there remains a
need for significant study into the effect of other variables.
The current methods utilized for minPMI estimation such
as thermal summation and isomegalen/isomorphen diagrams
suffer limitations, and as such, several new approaches
have been suggested and preliminary work undertaken. But
ultimately, entomology is a locality-specific science, and
techniques must be thoroughly examined for reliability and
utility in all geographic regions of use with all potentially
encountered species. Currently, work has focused largely on
calliphorids as the first colonizers, but even data gathered for
these species requires consideration of numerous biotic and
abiotic factors to ensure it is reliable and repeatable and thus
suitable for use in legal proceedings. Ultimately, the most
reliable methods for estimation will likely be corroborated
using multidisciplinary approaches, for example, the use of
gene expression, and external and internal morphology to
estimate the age of a pupa in conjunction with ExLAC or
thermal summation.
In recent times, a number of approaches to age estimation
of insect evidence have been proposed and assessed in pre-
liminary validation studies that appear to offer considerable
advances in the accuracy of age determination. However, all
require substantial development prior to application in stan-
dard investigative practice of forensic cases. Further research
and establishment of relevant reference data for application
is required and warranted in the case of techniques such as
hyperspectral imaging, gene expression, and ExLAC.
There are numerous factors that will induce error
into minPMI calculations, and fundamental to all conclu-
sions is an awareness, and acknowledgment, that biological
data, and organisms themselves, are subject to variation. It
is unlikely that a one-size, easy-answer approach will be
applicable, as each life stage has different features, and each
case introduces a new range of challenges, whether they are
factors affecting the development of the insect at the crime
scene, altered succession patterns, or human-induced chal-
lenges such as preservation errors or damage of specimens.
A flexible, but scientifically sound and well-tested approach,
that encompasses variables as much as possible, but acknowl-
edges shortfalls, is the model that forensic entomologists
are ultimately working toward in the provision of minPMI
estimates for forensic purposes.
Disclosure
The authors report no conflicts of interest in this work.
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