Use of PCRRFLP for differentiation of calliphorid larvae

(Diptera, Calliphoridae) on human corpses

H. Schroeder, H. Klotzbach
*
, S. Elias, C. Augustin, K. Pueschel
Institute for Forensic Medicine, University of Hamburg, Butenfeld 34, 22529 Hamburg, Germany
Received 23 July 2002; accepted 11 December 2002
Abstract
Blowy larvae found on human corpses are important for the estimation of the postmorteminterval (PMI) and other questions
of forensic relevance. Some of these species are difcult to differentiate morphologically, therefore a molecular method was
elaborated for species identication. Specic fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were
amplied followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identication of Lucilia sericata,
Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or
HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34
nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two
Calliphora species. These results aid in quick identication of species used for estimation of PMI.
# 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Postmortem interval; Blowy larvae; PCRRFLP; mtDNA; Cytochrome oxidase genes; Restriction enzymes
1. Introduction
In forensic entomology, necrophagous insects are used to
answer problems relating to estimation of the postmortem
interval (PMI) [17], postmortal transfer [8], diagnosis of
poisoning (in case of total absence of soft tissue) [912] and
neglect of living people [13,14].
Blowies (family Calliphoridae) are frequently found on
dead bodies shortly after death. Species within this family
differ in their developmental times, so an accurate identica-
tion of every species is necessary for the correct estimation of
the PMI. Most adult stages of species of this family can be
identied morphologically, but identication of the immature
stages is more difcult and sometimes even impossible. The
most important aspects for identication of immature stages
are the posterior spiracles and the cephalopharyngeal skeleton
[1517]. In criminal cases, rapid determination of PMI is
crucial for the investigation. Often, there is not enough time for
rearing larvae to adult ies. A second problem is the fact that
insect larvae may die before identication is possible.
PCRRFLP provides for rapid and accurate identication
of the forensically important y larvae. It is a modication of
the original restriction fragment length polymorphism(RFLP)
method [18] combined with the PCR technique [19]. For
PCRRFLP, regions of the mtDNA are amplied by use of
special primers and subsequently digested with restriction
enzymes. Using PCRRFLP, differentiation of forensically
important blowies in the USA [20] and France [21] was
possible. The species mentioned by Sperling et al. [20] and
Malgorn and Coquoz [21] are partly different from the for-
ensically important species found in Germany. We present an
elaboration of the PCRRFLPtechnique described by Sperling
et al. [20] suitable for three forensically important blowy
species (Calliphora vicina, Calliphora vomitoria and Lucilia
sericata) that are the most common in Germany.
2. Materials and methods
2.1. Samples
Fly larvae were collected alive in Hamburg, Germany,
from corpses in the Institute of Legal Medicine. They were
reared in the laboratory and identied as adults afterwards
Forensic Science International 132 (2003) 7681
*
Corresponding author. Tel.: 49-40-42803-2142;
fax: 49-40-42803-3934.
E-mail address: klotzbach@uke.uni-hamburg.de (H. Klotzbach).
0379-0738/03/$ see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0379-0738(02)00457-7
[2224]. The ies were then frozen at 30 8C until used for
DNA extraction. The following calliphorid species were
used: C. vicina, C. vomitoria and L. sericata. From each
of the species 1520 individuals were used for the research.
2.2. DNA extraction
Frozen ies were ground to powder using plastic pestles
in 1.5 ml microcentrifuge tubes. Total genomic DNA was
then extracted from these individual ies using the QIAmp
DNA MiniKit following the manufacturers protocol. This
system uses advanced silica-gel-membrane technology for
isolation of total cellular DNA without phenol, chloroform,
or ethanol precipitation. The buffer system allows selective
binding of DNA to a membrane.
2.3. PCR conditions
The PCR reactions contained 3060 ng template DNA,
50 mM KCl, 20 mM TrisHCl (pH 8.4), 1.5 mM MgCl
2
,
200 mM dNTPs, 1 unit of platinum Taq polymerase (Life)
and 0.4 mM primer in a total volume of 25 ml (for primer
sequences, see Fig. 1).
PCR was carried out in a Biometra Personal thermocycler
with a pre-denaturation step at 95 8C for 3 min, followed by
30 cycles at 94 8C for 1 min, 45 8C (47 8C for the primer
combination 2 4) for 1 min, and 72 8C for 1.5 min.
2.4. Restriction digestion conditions
Seven microliters of the amplicon was digested in a total
volume of 20 ml (restriction enzyme buffer) according to the
recommendations of the supplier of the restriction enzymes
(MBI Fermentas). The restriction enzymes were chosen
according to the work of Sperling et al. [20] (HinfI, DdeI,
EcoRV and DraI).
2.5. Gel electrophoresis
Separation was carried out with 7 ml of the digested DNA
on native polyacrylamide gels (8% T, 3% C, 500 mm,
piperazine diacrylamide as cross-linker, 60 mM formate,
20 cm separation distance, 2% agarose plugs in 2 Tris
borate (0.5 M Tris, 0.28 M borate, pH 9.0)) (according to
Allen et al. [25]). Bands were visualized by silver staining. A
25 bp ladder from GIBCO BRL (Life Technologies) was
used as size marker.
2.6. Sequencing
After purication of the PCR products with QIA-quick-
columns cycle sequencing was performed on both strands
using ABI PRISM Big Dye Terminator Cycle Sequencing
Ready Reaction Kit (Applied Biosystems). Primers used for
the sequencing of the PCR products were the same as for the
amplication. Removal of excess dye-deoxyterminators,
primers and buffer was accomplished by DYE-EX Spin-
columns (Qiagen).
3. Results
The mtDNA region used in this study includes a part of the
cytochrome oxidase b subunit I gene (COI), the cytochrome
Fig. 1. Scheme of the COI and COII region of the mtDNA, locations of the primers used and size of the fragments amplied with different
primer combinations. Primer 1: 5
0
-TACAATTTATCGCCTAAACTTCAGCC-3
0
; primer 2: 5
0
-CAGCTACTTTATGAGCTTTAGG-3
0
; primer 3:
5
0
-CATTTCAAGCTTGTAAGCATC-3
0
; primer 4: 5
0
-GAGACCATTACTTGCTTTCAGTCATCT-3
0
, modied after [20].
H. Schroeder et al. / Forensic Science International 132 (2003) 7681 77
oxidase subunit II gene (COII) and the tRNA leucine gene
(Fig. 1).
Different primer combinations were tested. Amplication
of the 2.4 kb fragment using the primer pair 1 4 (Fig. 1)
was not reproducible. Only DNA from ies frozen alive at
30 8C could be amplied. The primer combination 1 3
resulted in a 1.4 kb fragment, the combination 2 4 in a
1.3 kb fragment (Fig. 1). These fragments were amplied at
an annealing temperature of 47 8C. Amplication of the
smallest fragment (primers 2 3) was possible every time
with annealing temperatures of 45 and 47 8C, respectively.
For further experiments, the primer combinations 2 3 and
2 4 were used.
The PCR product of the primer pair 2 3 was a 349 bp
fragment (Figs. 1 and 4). In the respective publication of
[20], the fragment has a size of 348 bp. This fragment was
digested using the four restriction enzymes: DraI, EcoRV,
DdeI and HinfI (Fig. 2 and Table 1). Using DraI, the DNA of
all three species was cut, but resulted in identical fragments
(Table 1). With EcoRV, L. sericata showed a typical banding
pattern, whereas the DNA of C. vicina and C. vomitoria
remain uncut. L. sericata could as above be differentiated
from the other two species with DdeI, while C. vicina and C.
Fig. 2. PAA gel demonstrating the result of digestion of the 349 bp
fragment with four different restriction enzymes. VI C. vicina;
VO C. vomitoria; LS L. sericata. Marker 25 bp DNA ladder
(rst band consists of two bands: 500 and 475 bp).
Table 1
Fragments produced by digestion of the 349 bp COI gene region
(amplied by primer combination 2 3)
DraI (TTT#AAA)
L. sericata 208 141
C. vicina 208 141
C. vomitoria 208 141
DdeI (C#TNAG)
L. sericata 214 70 65
C. vicina 284 30 35
C. vomitoria 284 65
HinfI (G#ANTC)
L. sericata 349
C. vicina 169 180
C. vomitoria 349
EcoRV (GAT#ATC)
L. sericata 183 166
C. vicina 349
C. vomitoria 349
Fig. 3. PAA gels showing the result of digestion of the 1326 bp
fragment with four different restriction enzymes. VI C. vicina;
VO C. vomitoria; LS L. sericata. Marker 25 bp DNA
ladder.
78 H. Schroeder et al. / Forensic Science International 132 (2003) 7681
vomitoria showed identical fragments (Fig. 2). Though the
DNA of C. vicina has a second restriction side for DdeI
(Table 1), it is not useful for identication, because the
second cutting resulted in fragments too small (Table 1) to be
visible on a gel. HinfI allowed the identication of C. vicina,
the DNA of C. vomitoria and L. sericata was not digested
(Fig. 2 and Table 1).
The PCR product of primer pair 2 4 was a 1326 bp
fragment (Fig. 1) [20]. The same restriction enzymes were
used as for the smaller fragment described earlier. Digestion
with EcoRVand DdeI resulted not in differences between C.
vicina and C. vomitoria. Both were left uncut with EcoRV,
and did show identical fragment length with DdeI. L.
sericata had a typical banding pattern with these two
enzymes (Fig. 3). With both, DraI or HinfI, a differentiation
between all three species is possible (Fig. 3).
Because of the little differences found by the PCRRFLP
using the smallest fragment (349 bp), we sequenced this
fragment to get more information about the species. DNA of
ve individuals per species was sequenced at this region.
The comparison of these DNA sequences for the three
species is shown in Fig. 4. All three sequences were different
from one another. C. vicina and C. vomitoria showed a
divergence of 4.3%, for C. vicina and L. sericata the
divergence was 9.8% and the comparison of C. vomitoria
and L. sericata resulted in a divergence of 8.6% (Table 2).
Fig. 4. DNA sequence of L. sericata (LS), C. vicina (VI) and C. vomitoria (VO) across a part (349 bp) of mitochondrial cytochrome oxidase I
gene using primers 2 and 3 as shown in Fig. 1. The differences in the species sequences are shown using bold letters.
H. Schroeder et al. / Forensic Science International 132 (2003) 7681 79
4. Discussion
Differentiation of L. sericata and C. vicina was possible
with restriction of the small fragment (349 bp) using two
different restriction enzymes. C. vomitoria could not be
identied because the DNAremained uncut with two restric-
tion enzymes and had identical fragments with both other
species (C. vicina and L. sericata) (DraI) and C. vicina
(DdeI). Identication of a species by means of an uncut
fragment is not advisable, because in the case of unknown
larvae there could be another than one of the three species
investigated here present. Their DNA could also remain
uncut with the same enzymes. C. vomitoria could not be
identied by digestion with one enzyme, but only by com-
parison of the result of four enzymes (DraI, EcoRV, DdeI
and HinfI).
In contrast to Sperling et al. [20] who differentiated the
most common North American species (Phormia regina,
Phaenicia sericata and Lucilia illustris) using the 349 bp
fragment, it was not satisfactorily possible to differentiate
the three most common German blowy species using this
fragment. Therefore, we tried another primer combination.
The combination of primers 2 and 4 resulted in a 1326 bp
fragment and enabled a successful differentiation of the
three species. Each of the species showed different frag-
ments after digestion either with DraI or HinfI. Therefore,
the identication of each of the three species is possible
using only one restriction enzyme.
When using highly variable regions of the DNA for
identication of insect species, one must consider the pos-
sibility of intra-specic variation. Several parts of the COI
and COII regions of the mtDNAwere used to discover intra-
specic variation such as identifying the origin of a popula-
tion [26], uncovering geographic differences between popu-
lations [27] and separating morphological identical host
races [28]. For our investigation, 1520 individuals of each
species were used for the PCRRFLP technique, and 5
individuals for sequencing without observation of intra-
specic sequence variation. Further investigations will
include the analyses of more individuals from different cases
(and therefore from different locations) to establish whether
variation within one of the species is to be expected.
In accordance with the results of Sperling et al. [20],
sequencing of the 349 bp fragment throughout the part of the
COI gene region showed a high variability. Aclose degree of
relationship between insect species mostly is reected in low
genetic divergence. Hence, sequence divergence was lower
between the two most related species C. vicina and C.
vomitoria than between C. vicina or C. vomitoria and L.
sericata.
The presented results of sequencing and PCRRFLP
technique have shown that the divergence in the DNA
sequence of the three examined blowy species is suitable
for the identication of their larvae.
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