Identification of forensically important Diptera from...

Identification of forensically important Diptera from

Belgium and France using the Barcode of Life Data

Systems (BOLD) and GenBank

Gontran Sonet1, Kurt Jordaens2,3, Yves Braet4, Luc Bourguignon4, Eréna Dupont4,

Thierry Backeljau1,3, Marc De Meyer2 & Stijn Desmyter4

4 National Institute of

Criminalistics and Criminology,

Belgium

1 & 2 JEMU – Joint Experimental Molecular Unit,

Royal Belgian Institute of Natural Sciences &

Royal Museum for Central Africa, Belgium

3 University of Antwerp,

Belgium

Lucilia caesar (Linné 1758)

© Krzysztof Szpila, Nicolaus Copernicus University, Torun

INTRODUCTION Forensic entomology

© Crime Scene Unit of South-Western Finland, Turku Police Department

Pohjoismäki et al. Forensic Science International 2010

INTRODUCTION Dipteran species of forensic interest in Western Europe

Calliphoridae

Calliphora vicina Robineau-desvoidy, 1830

© jorgemotalmeida Viseu Portugal Calliphora vomitoria (Linnaeus, 1758)

© Roger Thomason Mossbank UK

Lucilia sericata

(Meigen 1826)

© Alvesg Lisbon Portugal

Chrysomya albiceps

(Wiedemann, 1819)

© Nikita Vikhrev Moscow Russia

Protophormia terraenovae

(Robineau-desvoidy, 1830)

© jari.flinck Helsinki Finland

Muscidae

Muscina levida (Harris 1780)

© Tim worfolk Devon UK

Musca autumnalis De Geer, 1776

© Nick Upton Wiltshire UK


Sarcophagidae

Sarcophaga spp.


Reference dataset

INTRODUCTION DNA-based specimen identification

sp1

sp2

sp3

Unknown species

Query

sequence

INTRODUCTION Validation of a reference dataset

Lucilia sericata

Protophormia

terraenovae

Reference sequences

available in BOLD

New in

this

study

Reference dataset

INTRODUCTION Validation of a reference dataset

sp1

sp2

sp3

sp4

New reference

sequence

Before applying the DNA barcoding identification method to

samples of a certain location, it is necessary to validate the method

with samples from that specific location.

Material & Methods Specimens

85 specimens from 27 localities in Belgium and France

Adults identified using morphology 16 species of forensic interest

Calliphoridae 14 Calliphora vicina Robineau-desvoidy, 1830

16 Calliphora vomitoria (Linnaeus, 1758)

7 Chrysomya albiceps (Wiedemann, 1819)

1 Cynomya mortuorum (Linnaeus, 1761)

2 Lucilia ampullacea Villeneuve, 1922

16 Lucilia sericata (Meigen 1826)

12 Protophormia terraenovae (Robineau-desvoidy, 1830)

Muscidae 1 Eudasyphora cyanella (Meigen, 1826)

1 Musca autumnalis De Geer, 1776

5 Muscina levida (Harris 1780)

5 Muscina prolapsa (Harris, 1780)

1 Neomyia cornicina (Fabricius, 1781)

1 Polietes lardarius (Fabricius, 1781)

Fanniidae 1 Fannia sp1

1 Fannia sp2

1 Fannia sp3

Material & Methods Searches in BOLD and GenBank

Barcode fragment (642-658 bp):

85 sequences,

42 haplotypes

GenBank

BOLD: Public Records

341 580 sequences;

45 368 nominal species

11 732 interim species 24 May 2013

BOLD: Species Level Records

(including early releases)

1 367 662 sequences

127 679 species

53 394 interim species 24 May 2013

+ keyword

“barcode”

1 2 3 4

Material & Methods Searches in BOLD and GenBank

Best matches

>99% similarity

with query

(max. 100)

Correct match

Incorrect match

Query

Ambiguous

identification

Unambiguous

identification

Results Local reference dataset

1st COI records

Muscina prolapsa (Harris, 1780)

© cgm crex Sweden

Cynomya mortuorum (Linnaeus, 1761)

© brundlefly


2nd COI record

Neomyia cornicina (Fabricius, 1781)

© Hakon Cyprinoid Norway

Polietes lardarius (Fabricius, 1781)

© Nick Upton Wiltshire UK

Eudasyphora cyanella (Meigen, 1826)

© Tim Worfolk Devon UK

Potential forensic interest


Search procedure: 1 & 2 3 4

Matches > 99% similarity

(% of queries):

86%

95%

62%

(% of species): 69% 88% 31%

Results and Discussion Searches in BOLD and GenBank

References considered in the search procedures:

1&2 Public records (GenBank & BOLD)

3 Public + early releases (BOLD)

4 Records labelled as BARCODE (GenBank)

Search procedure: 1 & 2 3 4

Presence of correct best

match(es) when > 99%

similarity :

100% 100% 100%

Ambiguous identifications

(% of queries):

50%

62%

27%

(% of species): 27% 43% 20%


References considered in the search procedures:




6

64

3 1 0

10

20

30

40

50

60

70

incorrect

correct 1&2 3 4


Number of best matches > 99%

obtained for Calliphora vomitoria Search procedure:




10

55

5 2

2

2

0

10

20

30

40

50

60

incorrect

correct 1&2 3 4


Muscidae ??

=


obtained for Calliphora vicina Search procedure:




2

16

2 4

5

0

5

10

15

20

25

incorrect

correct

1&2 3 4


Lucilia caesar

& L. illustris

=


obtained for Lucilia ampullacea Search procedure:





15

4

3

0

20

40

60

80

100

120

incorrect

correct

1&2 3 4

Lucilia

cuprina

=


obtained for Lucilia sericata Search procedure:




Conclusion

Validating public reference libraries using reference sequences of the

local entomofauna allowed us to:

1. make sure that the local entomofauna is well represented in the

reference library

2. “red flag” cases of unreliable identifications and

3. determine ways to address each problematic identification:

• correction of mislabelled reference sequences (dynamic libraries)

• adaptation of the identification method (distance threshold)

• usage of alternative markers (hybridisation, introgression, etc.)

Value of the Species Level records of BOLD

Acknowledgements

The authors would like to thank:

• you for your attention,

• the organisers of the conference and

• the Belgian Science Policy Office (Belspo)

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