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Forensic Science International: Genetics Supplement Series 1 (2008) 584–585

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Research article
MtDNA analysis for genetic identification of forensically
important insects
F. Alessandrini, M. Mazzanti, V. Onofri, C. Turchi, A. Tagliabracci *
Institute of Legal Medicine, Università Politecnica delle Marche, Via Conca, 60126 Ancona, Italy
Received 13 August 2007; accepted 1 October 2007

Abstract
Background and purpose: Unequivocal identification of insect specimens is a crucial step in forensic entomology and could be difficult using only
morphological features, especially for immature stages. The majority of published studies consider the cytochrome oxidase subunits one (COI)
analysis and the obtained sequences are representative for insect population of specific geographic regions. This work describes a different
approach for genetic identification of insect specimens.
Method: The immature specimens found in human corpses recovered in Italy were bred to obtain adult flies. DNA was extracted and analyzed by a
900 bp fragment of COI and COII genes, using four degenerate primers. The sequences were aligned and compared with the Diptera sequences in
GenBank for species-specific identification.
Results: The sequenced fragment allowed an insect species-specific identification; intra-specific and geographic variations were found. COI and
COII sequences were compared with GenBank sequences independently and sometimes species-specific identification was ambiguous.
Conclusion: The combined analysis of COI and COII fragments is a more accurate approach for Diptera species identification than single COI
fragment analysis. The geographic and intra-specific variations are important for insect identification and local database set up are strongly
recommended.
# 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic entomology; Genetic identification; COI; COII

1. Introduction 2. Materials and methods

Correct identification of an insect specimen is usually a
crucial early step in a forensic entomological analysis [1].
Although traditionally accomplished using morphological
characters, this can be difficult or impossible for the
immature stages of many species [2]. DNA analysis can be
used to overcome these problems. This technique of
analyzing forensically relevant insects focuses mainly on
the mitochondrial encoded subunit I of the cytochrome
oxidase gene (COI) [3–8] and the obtained sequences are
representative for insect population of specific geographic
regions. The present study explores the utility of DNA
sequences from the mitochondrial genes cytochrome oxidase
subunits one and two (COI and COII) to the identification of
forensically important flies.
* Corresponding author. Tel.: +39 0715964716; fax: +39 0715964723.
E-mail address: a.tagliabracci@univpm.it (A. Tagliabracci).
1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2007.10.128
100 insect samples at different life stage were recovered
from human corpses deceased at different time in our region
(central Italy) and bred to obtain adult flies. DNA was extracted
by the QiAmp DNA mini kit (Qiagen) from empty puparia,
puparia, maggots and adult flies. Genetic identification of
insects was performed by amplifying a segment of 900 bp of
subunit I and II of the cytochrome oxidase gene (COI and COII)
of MtDNA, using degenerate primers, modified from Sperling
et al. [3]. Quantitation of amplification products was performed
by agarose gel electrophoresis. 5 ng of amplified product were
submitted to sequencing reaction using the Big Dye Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems).
Extension products were purified with the DyeEx Spin kit
(Qiagen); capillary electrophoresis was carried out on an ABI
Prism3130 Genetic Analyzer (Applied Biosystems). Data were
analyzed with SeqScape software v2.5. The resulting COI and
COII sequences were compared with the Diptera sequences in
the NCBI web site by Blastn function to identify species.
 F. Alessandrini et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 584–585
Morphological analysis on adult flies was performed to confirm
genetic identification.
3. Results
For all samples representative nucleotide sequences were
obtained. MtDNA analysis identified seven Diptera species:
Calliphora vicina (47), Lucilia sericata (35), Sarcophaga
crassipalpis (3), Sarcophaga argyrostoma (5), Chrysomya
albiceps (5), Phormia regina (2), Musca domestica (3) with
sequence identity varying between 97 and 100%, because of
intra-specific and geographic variations. Genetic identification
was confirmed by morphological analysis. When COI and COII
sequences were compared with GenBank sequences indepen-
dently, sometimes species-specific identification was ambig-
uous: for a sample COI sequence identified Calliphora vicina
voucher with 100% of identity, while COII sequence identified
Calliphora vicina (99% identity); the combined COI + COII
sequence identified Calliphora vicina. In one case, when
aligning the two single fragments with Blastn, a Calliphora
vomitoria was recognized by each of them, while the
comparison with the combined fragments was statistically
significant for Calliphora vicina. Morphological identification
confirmed the species Calliphora vicina.
4. Conclusion
The combined analysis of COI and COII fragments is a more
accurate approach for Diptera species identification than single
COI fragment analysis. Due to intra-specific and geographic
variations, a genetic difference up to 3% in the sequences of the
same species can be found. The set up of local databases to
reduce the incidence of geographic variations on the species
identification is recommended.
585
Funding source
Grants from ‘‘Università Politecnica delle Marche’’ and
‘‘Ministero per Istruzione, Università e Ricerca’’. The funding
sources had no involvement in the development of the paper or
decisions related to the paper.
Conflict of interest
None.
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