Molecular identification of the forensically important greenbottle fly species Lucilia caesar and L. illustris (family Calliphoridae)

Sonet, Gontran1, Desmyter, Stijn 2, Braet, Yves2, Jordaens, Kurt1

1. Joint Experimental Molecular Unit – JEMU, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B1000 Brussels and Royal Museum for Central Africa, Leuvensesteenweg 13, B3080 Tervuren, Belgium; gontran.sonet@naturalsciences.be

2. National Institute of Criminalistics and Criminology – NICC, Vilvoordsesteenweg 100, B1120 Brussels, Belgium

INTRODUCTION:

Some greenbottle flies (Calliphoridae: Lucilia; Fig. 1) colonise dead bodies and are useful in forensic

entomology to estimate the post-mortem interval (PMI). Two of them, Lucilia caesar and L. illustris, are

difficult to identify:-Only males can be identified with accuracy using the shape of the hypopygium (Fig. 2).-Sequence divergences between both species range from 0 to 2.9% for COI and from 0 to 5.1% for 16S. The

latter value is above the range of intra- and even interspecific p-distances observed in the genus Lucilia (1.2-

4.5%).

OBJECTIVES:-To discriminate between both species using DNA sequences.

MATERIAL & METHODS:-Sequencing 36 additional males of L. caesar and 24 of L. illustris for COI and, for a subset of samples, two

other mitochondrial (16S and COII) and two nuclear markers (28S and ITS-2).-Material originated from Europe, an area where both species co-occur.-95 homologous sequences from Canada, China, Europe, Korea, Singapore and USA were retrieved from

GenBank and used for network analyses.

PLUK DE SG

KR

PL

BE: NICC0067

UK:

UK DEPL FR

UK DEBE

UK: <

BE DEFR PLCN KR CA

DE:

DE BE

FR

KR

KR

KR

BEDE

BEDE KR

DE

BE

KR

KR

KRKR

BE

DE

FR

BEBE

NICC0250

AJ551445

KR

DQ453488AJ417703

BE:NICC0609

NICC0701 NICC0067NICC0725

DEUKPL

DE: NICC0250 NICC0251

UKBEDEFR BE:

UK:

UKDEBEPL

DE:CNDEUKBE

CN

CN: GU012425

DE

DE: NICC0254

DEUK

BE: NICC0609

BE: NICC0067UK: AJ551445

UK: NICC0729, NICC0731

DE: NICC0251

CN:GQ396697

UK: AY114186

1 sample of L. illustris

1 substitution

1 sample of L. caesar

COI

16S

COII ITS2

28S

CN

NICC0250

DE:BE:UK:

NICC0067NICC0725

UKCNDEBEFR

UKCNDEPL

1 indel of one bp

UKBEDEFR

UK : AJ417703

Fig. 1 Greenbottle fly, Lucilia caesar. © Krzysztof Szpila, Nicolaus Copernicus University, Torun

RESULTS AND DISCUSSION:

For each marker, both species shared at least one haplotype/genotype and

could therefore not be distinguished from each other (Fig. 3).

The apparent differentiation between both species that has been reported

previously is probably due to:-a too restricted sampling that did not include specimens with shared

haplotypes and-species misidentifications for 16S since one divergent (5.1%) sequence

(GQ396697) was identical to GenBank sequences of Chrysomya

megacephala and the others were highly divergent from any other

Lucilia species (>2.3%).

In the forensic practice, distinguishing between both species is only

useful if their biological characteristics are consistently different. The

accumulation of data about the life cycle and the ecology of these two

species will reveal the extent of phenotypic variation present in this

group of Lucilia.

Acknowledgments: The authors wish to thank the members of the team of DNA and Microtraces analysis of the NICC, J. Amendt, S. Swoboda, B. Vincent, J. Parrott, R. Wall, C. Richard, C. Rowlinson, K. Szpila, E. Pammeels and R. DeBry.

More info: Sonet et al., Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic? Forensic Science International, DOI: 10.1016/j.forsciint.2012.08.020

Fig. 3 Haplotype network for L. illustris and L. caesar based on COI, 16S, COII, ITS-2 and 28S. White = L. illustris, grey = L. caesar. Geographical origins are given as follows: BE: Belgium, CA: Canada, CN: China, DE: Germany, FR: France, KR: Korea, PL: Poland, SG: Singapore, UK: United-Kingdom. Voucher numbers or GenBank accession numbers of peculiar samples are indicated.

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Fig. 2 Detail of hypopygium. © Krzysztof Szpila, Nicolaus Copernicus University in Torun