Egyptian Journal of Forensic Sciences (2016) 6, 203208

H O S T E D BY Contents lists available at ScienceDirect

Egyptian Journal of Forensic Sciences

journal homepage: http://www.journals.elsevier.com/egyptian-journal-of-forensic-sciences

REVIEW ARTICLE

Review of some recent techniques of age

determination of blow ies having forensic
implications
Madhu Bala *, Anika Sharma

Department of Zoology and Environmental Sciences, Punjabi University, Patiala 147002, India

Received 27 February 2015; revised 31 May 2015; accepted 22 June 2015

Available online 11 July 2015

KEYWORDS Abstract Forensic entomology can aid death investigation by using predictable developmental
Forensic entomology; changes to estimate the age of ies associated with a body. Forensic entomologists use size and
Postmortem Interval (PMI); developmental stages to estimate blowy age, and from those, a Postmortem Interval.
Age determination; Calliphorids are very interesting in forensic sciences from an applied point of view, because they
Pteridine accumulation; provide relevant evidence for estimating the Postmortem Interval. Since such estimates are generally
Cuticular banding; accurate but often lack precision, particularly in the older developmental stages, so there is a need of
Volatile organic compounds some alternative aging methods. The range of techniques available for age grading of adult insects is
reviewed, with particular emphasis on species of medical importance. The techniques described
include pteridine uorescence analysis, internal morphological analysis, cuticular hydrocarbon
analysis, gene expression analysis, cuticular banding pattern analysis, volatile organic compounds
analysis released by larvae and pupae.
2015 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by
Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
2. Pteridine uorescence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
3. Internal morphological analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
4. Cuticular hydrocarbon analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
5. Gene expression analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
6. Cuticular banding pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
7. Volatile organic compounds released by larvae and pupae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
8. Signicance of age grading techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

* Corresponding author.
E-mail address: madhubaladhakane@gmail.com (M. Bala).
Peer review under responsibility of The International Association of
Law and Forensic Sciences (IALFS).
http://dx.doi.org/10.1016/j.ejfs.2015.06.002
2090-536X 2015 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
204
9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The utilization of insects and other arthropods as forensic evi-
dence in both civil and criminal investigation is known as
Forensic entomology.1 Insects found on human remains are
useful in estimating a Postmortem Interval (PMI) during death
investigation. Accurate methods for determining the age of
adult insects in eld are of considerable importance, the age
of immature stages of insects found on the dead body can pro-
vide evidence for the estimation of minimum PMI ranging
from one day to more than one month. They are therefore
the primary and most accurate forensic indicators for time of
death (Postmortem Interval, PMI). Under eld conditions
however, when unidentied larvae are collected from a body,
reliable age determination is challenged by the fact that the lar-
vae of different species look extremely similar but grow to dif-
ferent body lengths, and that temperature has a variable effect
on development of some of the most common blow ies. Age
determination of postfeeding larvae (the period between the
cessation of feeding by third- instar larvae and the onset of
pupation) is still problematic, with no satisfactory age indica-
tor found in previous morphological studies.2
However, determining age of pupae is more difcult com-
pared to the age of larvae due to lack of morphological
changes; weight, length and even the color of the pupal case
does not change at all after the rst hour of pupation.
Metamorphosis is characterized by developmental process
such as cellular proliferation, tissue remodeling, cell migration
and programmed cell death. Many genes are involved in these
actions and changes in their gene expression function as a
modern tool in age determination. Age estimation methods
have most frequently focused on the larval stage, with little
published data for the age estimation of pupae.
Majority of age grading techniques are based on pre-
dictable changes in the reproductive system including the accu-
mulation of follicular relics3 and appearance of ovarian
tracheoles.4 Other methods rely on somatic changes, such as
deposition of growth bands in cuticle,5,6 the size of larval or
adult fat bodies,7 and the accumulation of uorescent pig-
ments in the eyes,8,4 The following age grading techniques were
reviewed in this article:
1. Pteridine uorescence analysis: Chronological age is calcu-
lated using the known relationship between age and pteri-
dine uorescence.
2. Internal morphological analysis: Provide additional inter-
nal development information to that of external morpho-
logical analysis, allowing a more accurate age and thus
PMI to be estimated.
3. Cuticular hydrocarbon analysis: Hydrocarbon prole dif-
fers at distinguishable ages, and this provides a reliable
method that complements the current methods used for
aging the immature life stages of the blowy life cycle.
M. Bala, A. Sharma
207
207
207
4. Gene expression analysis: Potentially serve as a molecular
tool to mirror the aging process of a pupae. Genetic assess-
ment of blowy age is more pronounced during the third
instar and pupal stages.
5. Cuticular banding pattern analysis: Used to give an accu-
rate estimation of the chronological age of young insects
of both sexes, and may be an appropriate technique for
use in studies where eld material needs to be stored.
6. Volatile organic compounds analysis: Increase the accuracy
of the estimated PMI, through improved estimation of the
age of blow ies present on the cadaver.
2. Pteridine uorescence analysis
Pteridine uorescence analysis method, based on the level of
uorescent pigments, i.e. pteridines, which are degradation
products of purine metabolism and accumulate over time in
the compound eyes, have the advantage not only of being
applicable to both male and female insects but also of easily
being assayable. Pteridine content in both laboratory and eld
captured ies is typically a level of magnitude higher than the
minimally detectable level and can be used to predict individ-
ual age in the laboratory population with high certainty.
Laboratory studies of individuals of known age indicate that
while pteridine levels increase linearly with age, they also
increase in a linear manner with rearing temperature and ambi-
ent light levels, but are independent of sex.
Insect pteridine has been discussed in detail by Ziegler and
Harmson9 and Peiderer.10 A number of biochemical
techniques have also been used to predict the age of individual
insects. Pteridines, a group of uorescent chemicals derived
from a pyrimidine-pyrazine ring structure9 increase with
chronological age in populations of various dipteran taxa pop-
ulations reared under laboratory conditions. After Mail et al.8
rstly develop the pteridine aging method for determining the
age of Stomoxy calcitrans adults, this method has been widely
employed to determine the adult age of many vector and
medicinal insects such as Glossina morsitans morsitans,11 S. cal-
citrans,12 Glossina pallidipes,13 Cochliomyia hominivorax,14
Chrysomya bezziana,15 Lucilia sericata,16 Bactrocera cucur-
bitae,17 Musca domestica,18 Musca autumnalis,4 Chrysomya
megacephala,19 Boettcherisca peregrine.20 Zhu et al.19 studied
the relationship between head pteridine uorescence (HPF)
levels and age in adult females and males of a common necro-
phagous y, C. megacephala. The effects of temperature and
y sex on the relationship were also studied by pteridine uo-
rescence spectrometry. Factors affecting HPF levels in ies
were found to include y age, temperature and sex, among
which the y age was the most dominate one. HPF level (P)
in adult ies grew with an increase of the adult chronological
age (d). A signicant linear relationship was found between
them. The linear regression equation is:
Recent techniques of age determination of blow ies
For females, P = 11.3256 (0.8710) + 0.6737 (0.0534) d
(r2 = 0.7452, P < 10 5) and
For males, P = 10.8509 (0.8978) + 1.1826 (0.0556) d
(r2 = 0.8975, P < 10 5).
Penilla et al.21 estimated total pteridine concentration
(TPC) in the head, Body parts (BP: abdomen, legs and wings)
and whole bodies of insectary- reared and eld collected
Anopheles albimanus mosquito by spectrouorometry, to
investigate whether they could be used for age determination.
Pteridine concentration diminishes with age in both mosquito
groups. TPC correlated with chronological age in insectary-
reared sugar fed-female (Head: r2 = 0.34, P < 0.001), but
lower correlation occurred in blood-fed female (Head:
r2 = 0.22, BP: r2 = 0.27).
In all cases, pteridines as measured by uorescence intensity
provided an index of age that was continuous, objective, and
seemingly unaffected by the nutrition of adults.
3. Internal morphological analysis
Metamorphosis comprises histolysis and histogenesis of tissues
and organs. During the pupal stage, the developing y rst
undergoes histolytic changes, then histogenetic changes and
nally differentiation. Relative development of external pupal
feature is useful, but there are also internal changes during
metamorphosis that may be indicator of age, utilizing histolog-
ical techniques. Histological analysis of blowy pupae pro-
vides additional internal development information to that of
external morphological analysis, allowing a more accurate
age determination. Robertson22 and Bainbridge and
Bownes23 constructed a timeline of morphological develop-
ment and use it to stage or estimate age of Drosophila melano-
gaster pupae. This species has a white translucent puparium
and unsclerotized pupal cuticle through which internal devel-
opment can be observed. The examination of pupal morphol-
ogy is readily conducted using light microscopy by removal of
puparium.
Methods such as scanning electron microscopy24 and micro
computed tomography (micro- CT)25 have been used to visual-
ize morphological development of preserved dipteran speci-
mens. Davis and Harvey26 have examined internal features
of blowy pupae, Calliphora vicina (Rob- Desvoidy) and L.
sericata (Meigen). The pupal stage comprises >40% of the
immature component of the blowy lifecycle during which vast
internal and external morphological development occurs.27
Hematoxylin and eosin stained pupal sections revealed differ-
ences in brain and thoracic muscle development throughout
the pupal stage with potential for age estimation. Brown et al.28
studied metamorphosis, focusing on the development of exter-
nal morphology (within the puparium), to provide a means of
age and PMI estimation for C. vicina (Rob-Desvoidy) pupae.
According to him age determination is based upon (1) The
combination of possible age ranges observed for each charac-
teristic and (2) Regression analysis to generate age estimation
equations. Morphological examination using stereomi-
croscopy would be the most suitable for forensic purposes.
Brown and Harvey29 use optical coherence tomography
(OCT) as a tool for invivo morphological observation and
pupal age estimation. Factors, such as temperature, humidity,
food, light, which are difcult to control, affect the duration of
metamorphosis in the blowy (Calliphora erythrocephala). To
205
avoid error of timing in development caused by these factors,
age of pupae is dened by using morphological critetia.30
4. Cuticular hydrocarbon analysis
To explore the potential of using cuticular hydrocarbons for
determining the ages of y larvae, changes of cuticular hydro-
carbons in developing larvae were investigated using gas chro-
matography with ame ionization detection and gas
chromatographymass spectrometry. Most of the hydrocar-
bons identied were alkanes with the carbon chain length of
2131, plus six kinds of alkenes. Cuticular hydrocarbon com-
position is a useful indicator for determining the age of larvae,
especially for postfeeding larvae, which are difcult to differen-
tiate morphologically. Composition of cuticular hydrocarbon
changes with age in some species of insects. Goodrich31 was
the rst to examine the cuticular lipids of puparia and adults
of sheep blowy Lucilia cuprina and found signicant qualita-
tive and quantitative differences between these two stages.
Quantitative and Qualitative changes were found throughout
the life cycle of Sarcophaga bullata.32
Cuticular hydrocarbons comprised of n-alkanes, alkenes,
terminally and internally branched monomethyl alkanes and
polymethyl alkanes. These function primarily to limit water
loss and to serve as pheromones or kairomones.3335
Cuticular hydrocarbons were also used to determine the age
of female Aedes aegypti.36 Tregenza et al.37 found that the
cuticular hydrocarbons showed signicant changes with age
and by the use of gas chromatography (GC) they estimate
the age of grasshoppers. Zhu et al.38 suggested that cuticular
hydrocarbon composition is a useful indicator for determining
the age of larvae of Chrysomya rufacies. Zhu et al.39 used gas
chromatography coupled with mass spectrometry to determine
the changes with the weathering time in cuticular hydrocar-
bons of puparial case of C. megacephala in the laboratory.
Moore et al.40 potentially use hydrocarbons for aging L. seri-
cata larvae to establish the Postmortem Interval. Hydrocarbon
prole shows distinguishing features over the period of the lar-
val life cycle, with signicant chemical changes occurring from
the younger larvae to the postfeeding larvae.
Gas chromatography (GC) and gas chromatographymass
spectrometry (GCMS) were used to characterize the age-
dependent, quantitative changes in cuticular hydrocarbons of
larval Aldrichina graham (Aldrich) (Diptera: Calliphoridae) at
24 C. The majority of low molecular weight alkenes decreased
in abundance with larval development. By contrast, the abun-
dance of high molecular weight alkanes of chain length greater
than C25 gradually increase with age.41 Moore et al.42 identied
rst instar larvae of three forensically important blowy species
L. sericata, C. vicina and Calliphora vomitoria using gas
chromatography- mass spectrometry (GCMS) and principal
component analysis (PCA). The result shows that each species
hold a distinct ngerprint hydrocarbon prole.
5. Gene expression analysis
The age of specimens can be calculated based on the measure-
ments of morphological characters and comparison to species-
specic growth curves. While outward characteristics such as
body size or instar have been used to estimate y age, other
traits that are developmentally regulated, including the
206
differential expression of genes, offer great potential for esti-
mating blowy age. Tarone et al.43 proled expression of three
genes bicoid, slalom, chitin synthase (bcd, sll, cs) throughout
the maturation of blow y eggs to determine the feasibility
of predicting age, identifying signicant linear trends in expres-
sion during their development. Zehner et al.44 monitored a dif-
ferential gene expression in C. vicina pupae at different ages by
using a specic ddRT PCR protocol. They have been worked
out for pupae at the beginning, the middle and the end of their
metamorphosis.
The analysis of differentially expressed genes (DEGs) dur-
ing blowy metamorphosis can lead to an understanding of
the process of formation of the adult y and may also repre-
sent a new approach to predict pupal age in death investiga-
tion. It has been demonstrated that metamorphosis is
induced and controlled by the hormone ecdysone which acti-
vates a hierarchy of response genes differentially expressed
throughout pupal development. Tarone and Foran45 age
forensically important L. sericata (Diptera: Calliphoridae) by
gene expression analysis throughout development.
Generalized additive models of development showed improved
statistical support compared to models that did not include
gene expression data, resulting in an increase in estimate preci-
sion, especially for postfeeding third instar larvae and pupae.
The models were then used to make blind estimates of develop-
ment for 86 immature L. sericata raised on rat carcasses.
Overall, inclusion of gene expression data resulted in increased
precision in aging blow ies.
Boehme et al.46 focuses on the differential expression of two
newly described, arbitrarily named genes (15_2, 2014192) and
two previously identied genes (actin, arylophorin receptor)
during C. vicina (Diptera: Calliphoridae) metamorphosis.
Quantication through real-time PCR revealed signicant
up- and down regulation of these transcripts found to be tem-
perature dependent and age specic, hence, a new possibility to
age forensically important blowy pupae. Among insects, gene
regulation during development has been most extensively stud-
ied in D. melanogaster (Diptera: Drosophilidae).47,48
6. Cuticular banding pattern
This method uses the diurnal differences in the deposition of
apodeme layers as a basis for counting the age in days since
eclosion. Neville5 rst described the presence of daily growth
layers in the rubber like cuticle resilin of locusts. Under ultra-
violet light the layers autouorescence brightly and dimly in an
alternating pattern. Neville49 report the presence of daily
banding on the apodemes of locusts. Schlein and Gratz50
determined the presence of daily growth rings in several
Diptera, including Sarcophaga falculata, C. erythrocephala,
Glossina austem, Culex pipiens and A. aegypti. The apodemes
are the internal cuticular projection of the exoskeleton on
which muscles attach. TyndaleBiscoe and Kitching6 used
cuticular growth rings to determine the age of L. cuprina.
They showed that clear growth bands occurred in response
to uctuating temperature in which the minimal temperature
must fall below 15.5 C and the maximum is at least 3.5 C
above this minimal threshold.
Cuticular banding appears to be the only effective way to
age males. Analysis of the reproductive state of the male would
M. Bala, A. Sharma
be of limited value since, according to Riemann51 mature
sperms are found in males as early as 1 day old. In
Drosophila, Johnston and Ellison52 have observed that the
bands seem to be due to the thickness of the cuticle. In
Drosophila and C. hominivorax the ability to detect these bands
using the scanning electron microscope conrms that thickness
of the cuticle is the major contributor to the detection of bands.
Cuticular banding offers two opportunities, the third furca and
the ejaculatory apodeme, for determining age in males.
The method is accurate to plus or minus one day, and can
be determined on material that has been dried, pinned, frozen
or preserved in ethanol or other xatives.53 Cuticular bands,
therefore may be used to give an accurate estimation of the
chronological age of young insects of both sexes, and may be
an appropriate technique for use in studies where eld material
needs to be stored.
7. Volatile organic compounds released by larvae and pupae
Volatile organic compounds (VOCs) are used to evaluate the
age of ies and their immature stages to establish a PMI in
medicolegal investigations. The composition of the volatile
compound released at different blowy developmental stages
is different. Changes in Volatile compounds are monitored
by headspace solid-phase micro extraction, followed by gas
chromatographymass spectrometry. Branched and
unbranched hydrocarbons, alcohols, esters and acids were
identied, and the volatile prole was shown to vary, in both
composition and quantity, with the age of the larvae/ pupae
under investigation.54 For larvae and pupae, analysis of
VOCs enables young pupae to be distinguished from older
ones. However two groups of factors could affect the chemical
composition39,55 and could also inuence the emission of
VOCs. The rst group comprises genetic factors, such as age
of the insect or the gender of a y.5658 The second group of
factors potentially affecting the emission of VOCs by blowy
larvae and pupae are those related to environmental factors,
such as diet, temperature or geoclimate.56,5861
The VOCs emitted by the larval stage arise from bacteria on
mouthparts, gut or skin of larvae. Bacteria from the families
Enterobacteriaceae, Pseudomonaceae and Bacillaceae have in
particular been shown to emit volatile compounds, such as
3-hydroxybutan-2-one, indole,3-methyl-1-butanol, 3-methylbu
tanal,2-methylbutanal, phenol, methyldisulphanylmethane
and limonene.6267 Cadaveric VOCs may be analyzed by gas
chromatography coupled with mass spectrometry (GCMS)
or more recently with comprehensive two dimensional gas
chromatography (GCxGC) coupled to time-of-ight mass
spectrometry (TOFMS).
8. Signicance of age grading techniques
The precise estimation of PMI is the most important goal of
forensic entomology by rening the techniques used.
Pteridine aging has the advantage of being applicable to both
male and female insects. Cuticular banding pattern method
reects chronological age directly, and thus lacks the intrinsic
inaccuracies of those techniques which require chronological
age to be calculated from physiological age. Internal morpho-
logical analysis produces a signicant preservation and
Recent techniques of age determination of blow ies
histology protocol for blowy pupae, suitable for routine
forensic application and useful for potential age and PMI
estimation.
Differential gene expression analysis is a promising technique
for discrimination of even small time intervals within pupal devel-
opment as expression patterns of genes seems to vary within a high
range during metamorphosis. Notably a combination of expres-
sion proles of several genes will be able to build a roster with a suf-
cient resolution for age determination. Cuticular hydrocarbons in
larvae and pupae change with age signicantly and serve as a useful
indicator for age determination, especially for post feeding larvae,
which are difcult to differentiate by morphology.
9. Conclusion
Age determination is the basis of determining the Postmortem
Interval using necrophagous y and their immature stages.
Some other measures like size and weight of larvae, changes in
intestinal content, length of larvae etc are also used for age deter-
mination, but there are some limitations of these techniques.
Weight of larvae cannot be used for Postmortem Interval esti-
mation in postfeeding stage. Scarcity of food or competition
between species may result in larval mortality or reduction in
size and weight of larvae. Age determination by changes in
intestinal content of maggots using radiological techniques is
difcult. Such techniques for aging blowy larvae have not been
reported for most of the species of calliphoridae. Thus the use of
some recent techniques for age determination of blowy, dis-
cussed above seems more reliable and can be used for
Postmortem Interval estimation. The aim of this article is to
use the internal features with potential for use in age estimation.
Age determination of adult y has forensic implications, espe-
cially in indoor death-scenes investigations. Given the informa-
tion of population age structure, it is then possible to draw a
conclusion on when the adult ies rstly arrive at the corpse
for laying eggs. The age of blowy larvae is closely related to
the time of death. This will make the criminal investigation team
aware of and familiar with forensic entomology, a step which
may initiate future studies and interest in the application of
insect evidence in legal investigations.
Funding
None.
Conict of interest
None.
Ethical approval
Necessary ethical approval was obtained from the institute
ethics committee.
Acknowledgement
The authors are thankful to the Department of Zoology and
Environmental Sciences, Punjabi University, Patiala for help-
ful suggestions and literature.
207
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