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DNA typing for identification of some species of Calliphoridae
Forensic Science International 102 (1999) 111–119
DNA typing for identification of some species of Calliphoridae An interest in forensic entomology ¨ Coquoz b Yvan Malgorn a , *, Raphael a
´ Sueur, Institut de Recherche Criminelle de la Gendarmerie Nationale, 1 Bd Theophile ´ , France 93111 Rosny-Sous-Bois Cedex b Laboratory of the Institut de Police Scientifique et de Criminologie, UNIL–BCH, 1015 Lausanne–Dorigny, Switzerland
Received 15 February 1999; received in revised form 12 March 1999; accepted 31 March 1999
Abstract To determine precisely post mortem interval, larvae and puparium species found on a corpse have to be identified. Among more than 200 cases examined at the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale, two-thirds concerned corpses less than one month old. Therefore, insects from first and second screwworms are the most frequently found [1]. Some species commonly found in France, such as different Lucilia and Calliphora vicina Robineau–Desvoidy, are easily identifiable at an adult stage, but are almost impossible to differentiate at immature stages when only fragments of puparium or necrosed first instar larvae are available. For this reason, an easy and objective method of identification was thus searched by genetic analysis of these insects. Sequencing of partial gene of sub unit I of cytochrome oxydase has been used to predict restriction sites. Restriction enzyme cleavage of PCR products with Dde I allowed us to differentiate these species. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polymerase chain reaction; Mitochondrial DNA; Forensic entomology; Calliphoridae; Identification
1. Introduction The best method to obtain the most precise oviposition date is to raise larvae found on *Corresponding author. Tel.: 133-1-4935-5093; fax: 133-1-4935-5027. 0379-0738 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00039-0
112
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
a corpse [2,3], but this is not always possible and there are cases where only puparium (or fragments) or dead and necrosed larvae are available. Although these larvae or remains can easily be identified at the family level [4], it is much more difficult to identify species. It is a particularly tough challenge to differentiate Lucilia and Calliphora vicina Robineau–Desvoidy species among themselves [5,6]. These Calliphorids belong to families of the first screwworm described by ´ Megnin [7] and Leclercq [8]. But they have different biologies and then different growth and maturation rates [2,9–13]. Even if oviposition dates cannot be as precise as by raising, results obtained by the specific identification will be of a great interest for enquirers and judges and will narrow down the accuracy of the post mortem interval determination by a few days. Mitochondrial DNA is the logical target for a genetic analysis of puparium, necrosed larvae or small parts of insects. It is present in large numbers of copies. It has a high mutation rate leading to a rapid generation of sequence differences between sub-species. Its analysis can take advantage of all the modern and sensitive molecular biology methods. The only available sequences of Calliphorids in GenBank (The National Center for Biotechnology Information, Bethesda, MD, USA) were for the sub units I and II of cytochrome oxydase gene (access numbers L14945, L14946 and L14947). First, these sequences have been aligned. Then, highly conserved areas, with sufficient polymorphism between them, have been searched to design primers. Amplification and sequencing have then been carried out to predict informative restriction sites.
2. Materials and methods
2.1. Samples Insects were obtained either from colonies that are maintained in the forensic entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale or from collections. Depending upon availability, adults, puparium and larvae preserved in 708 alcohol were used. Four species of Lucilia and one of Calliphora were the subject of our study: Lucilia ´ Lucilia illustris (Meigen), Lucilia sericata ampullacea Villeneuve, Lucilia caesar Linne, (Meigen) and Calliphora vicina Robineau–Desvoidy. Three to seven individuals of each species were used for sequencing. For the restriction analysis experiments, the sample range was extended to necrosed larvae preserved for several months in 708 alcohol, first instar larvae, puparium fragments and adult fragments (wings, legs or parts of thoraces).
2.2. DNA extraction For all the samples, DNA extraction started by cutting them in small pieces, and crushing them in 1.5-ml microtubes with a piston pellet. A 500-ml volume of lysis buffer
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
113
[100 mM Tris, 50 mM ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), 0.5%, proteinase K 0.5 mg / ml] derived from Sambrook et al. [14] were added and the microtubes incubated over night at 568C. The resulted lysate was purified by extraction with phenol–chloroform–isoamylic alcohol (24:25:1) and chloroform. The final supernatant was then mixed with two volumes of ethanol, 8 ml of 5 M NaCl and 1 ml glycogen, 10 mg / ml at 2208C for 2 h to let the DNA precipitate. After centrifugation at 13 000 g the DNA pellet was dried at room temperature for 2 h, dissolved in 100 ml of sterile water and stored at 2208C. Total DNA was quantitated by reading the optical density with a spectrophotometer.
2.3. Primers Different sequences of Calliphoridae mitochondrial DNA (partial gene of subunit I of cytochrome oxidase) obtained from Genbank (access numbers L14945, L14946 and L14947) were aligned to design primers from conserved areas. The designed primer pairs are described in Table 1. Primers F1 and R1 allow the amplification of a 297 base pair (bp) fragment while primers F2 and R2 lead to a 304 bp fragment.
2.4. Polymerase chain reaction ( PCR) The PCRs were performed with reagents from the Perkin-Elmer Cetus GeneAmp PCR Core Reagents kit except for the primers which were synthesized by Eurobio (Les Ulis, France). Each 50 ml reaction contained 1X GeneAmp PCR buffer, 200 mM of each dNTP, 1.5 mM MgCl 2 , 0.5 mM of each primer, 100 ng of total DNA extracted from adults or larvae, respectively 10 ml of puparium DNA extract and 2.5 unit of AmpliTaq DNA polymerase. Amplifications with DNA amounts ranging from 1 to 500 ng were also performed. (1, 5, 10, 50, 100 and 500 ng). PCR was run for 30 cycles (958C for 20 s, 528C for 30 s, 728C for 30 s) with a final elongation of 10 min at 728C.
Table 1 Primers used for amplification a Primers
Sequence
Length
59 end
39 end
F1 R1 F2 R2
TTA TCA TTA CCA GTA TTA GC AGC TGA AGT AAA GTA AGC T CTA CTT TAT GAG CTT TAG G TTC AAG TTG TGT AAG CAT C
20 19 19 19
2056 2373 2477 2800
2075 2391 2495 2818
a
The nucleotide position numbering corresponds to that of the sequence of Drosophila yakouba published by Clary and Wolstenholme [15].
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Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
2.5. Sequencing Amplified products were first separated on agarose 1.5% gels (Pharmacia NA). The bands were then cut from the gel. The DNA was purified using Genelute Spin Column from Supelco (Supelco, Bellefonte, PA, USA), then precipitated by two volumes of ethanol and 0.1 volume of 1 M potassium acetate. The obtained pellet was resuspended in 20 ml of sterile water and then sequenced directly using ABI Prism TM dRhodamine Terminator Cycle sequencing Ready Reaction Kit according to Perkin-Elmer protocol. The PCR primers used above were used as sequencing primers to sequence each strand of the amplified products.
2.6. Prediction of restriction sites For each Calliphorid sequence of our study, cleavage sites for several restriction endonucleases were searched using DNASIS software: Hitachi Software, DNASIS for Windows , Sequence Analysis Software, Version 2.00.
2.7. Restriction analysis PCR products were first digested with restriction endonuclease Dde I according to manufacturer’s specifications (Boehringer Mannheim), then separated on 2% agarose Resophor Gel (Eurobio) containing 5 ml of GelStar (FMC Bioproducts, Rockland, ME, USA) for staining. The run was performed at 75 V during 1 h.
3. Results and discussion
3.1. Samples Sequences have been obtained from either adults, larvae or puparium. Every sample containing even a small amount of DNA (necrosed larvae, small parts of adults or puparium) allowed us to obtain an amplification. This is a good sign for forensic cases analysis but will have to be completed with the study of eggs and insects conserved by other means.
3.2. Polymerase chain reaction An amplification product has been obtained for the whole range of total DNA amounts tested (1, 5, 10, 50, 100 and 500 ng). Amounts from 10 to 100 ng of DNA gave the best results. After DNA has been resuspended in 100 ml of sterile water, the quantitation of about 30 adults and larvae allowed us to say that 10 ml of an extract diluted 100 times contains 50 to 100 ng of DNA. This dilution has been used successfully for all the samples who have not been quantitated and may be used for forensic cases. DNA obtained from small parts of adults or puparium could not be quantitated with the spectrophotometer because of insufficient sensitivity, but 10 ml of extracts gave us good results.
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
115
3.3. Sequencing A first study has been carried out for two individuals of each of the following species: Lucilia ampullacea Villeneuve, Lucilia illustris (Meigen) and Lucilia sericata (Meigen) for the PCR fragments of 297 and 304 bp (see, respectively, Appendices 1 and 2). As it could be predicted according to Sperling et al. [16] who studied Lucilia illustris (Meigen) and Phaenicia sericata (Meigen), which is the north-American equivalent of Lucilia sericata (Meigen), the polymorphism was sufficient to distinguish these three species. For the 297 bp fragment (see Appendix 1) there were 20 nucleotide differences between Lucilia ampullacea Villeneuve and Lucilia illustris (Meigen), 26 between Lucilia ampullacea Villeneuve and Lucilia sericata (Meigen) and 24 between Lucilia illustris (Meigen) and Lucilia sericata (Meigen). In comparison with sequences of Phaenicia sericata (Meigen) and Lucilia illustris (Meigen) published by Sperling et al. [16] we noticed, respectively, one and three different nucleotides. In the case of the 304 bp fragment (see Appendix 2), only this fragment of 304 bp has been used for further study because of the better yield at the amplification. In comparison with sequences of Phaenicia sericata (Meigen) and Lucilia illustris (Meigen) published by Sperling et al. we noticed respectively no difference and one nucleotide difference. The number of non conserved nucleotides and percentage of intra- and inter-specific polymorphisms for the five species of our study are given in Table 2. Among the five species tested, only two displayed intra-specific polymorphism, but with not more than one nucleotide difference. The inter-specific polymorphism is higher. It is in the range of 21 to 37 nucleotides differences, with an outlier at six nucleotides differences for the comparison of Lucilia caesar Linne´ and Lucilia illustris (Meigen). It is to be noted that this pair of insects is morphologically very similar and difficult to differentiate even at adult stage [17]. Calliphora vicina Robineau–Desvoidy which belongs to another genus shows logically the most important difference (higher than 33 nucleotides). The low intra-specific variation will have to be confirmed using a larger set of individuals from each species.
Table 2 Number of non-conserved nucleotides and percentage of intra- and inter-specific polymorphisms a
L. ampullacea (6) L. caesar (3) L. illustris (4) L. sericata (4) C. vicina (7) a
L. ampullacea
L. caesar
L. illustris
L. sericata
C. vicina
0/0 23 21 22 35
7.56% 1 / 0.32% 6 23 37
6.91% 1.97% 1 / 0.32% 25 33
7.24% 7.56% 8.22% 0/0 34
11.51% 12.17% 10.85% 11.18% 0/0
The number of sequenced individuals is given in parentheses.
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Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
Table 3 Size of restriction fragments Species
L. ampullacea
L. caesar
L. illustris
L. sericata
C. vicina
Fragments size (bp)
282–60
342
312–30
212–70–60
282–30–30
3.4. Restriction analysis Among all restriction enzymes available in DNASIS software, only Dde I alone enabled us to differentiate our five species. Sizes of the restriction fragments are given in Table 3. The restriction patterns obtained with this enzyme are shown in Fig. 1 and clearly allow us to differentiate our five species. In conclusion, an easy and fast method is now available to identify some Calliphoridae at immature stages. These results show again how powerful the analysis of mitochondrial DNA is. This study should be extended to other diptera which are difficult to differentiate at immature stages.
Acknowledgements Y.M. would like to thank Professor Pierre Margot, Director of the Institut de Police Scientifique et de Criminologie of Lausanne (Switzerland) who received him in his Institute where a large part of this work was carried out. He would also like to thank all the colleagues of the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale and Marie-Paule Carlotti, head of the biology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale who allowed him to use her sequencer without restriction.
Fig. 1. Restriction patterns obtained with Dde I. Lane 1: Lucilia illustris (Meigen); lane 2: Lucilia ampullacea Villeneuve; lane 3: Calliphora vicina Robineau–Desvoidy; lane 4: Lucilia sericata (Meigen); lane 5: Lucillia ´ lane 6: 100 base-pair ladder (Pharmacia Biotech). caesar Linne;
Sequences obtained for the 297 bp fragment
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
117
Sequences obtained for the 304 bp fragment
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Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
119
References ´ d’activite´ en entomologie [1] B. Ceccaldi, J. Salon, B. Chauvet, P. Masselin, Bilan de deux annees ´ ´ ´ Legale–Droit ´ ´ 39 (7 / 8) (1996) 517–519. medico–legale, J. Med. Med. [2] A.S. Kamal, Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera), Ann. Entomol. Soc. Am. 51 (1958) 261–271. [3] M.I. Marchenko, Medico–legal relevance of cadaver entomofauna for the determination of the time since death, Acta Med. Legalis Socialis 38 (1) (1988) 257–302. [4] K.G.V. Smith, A Manual of Forensic Entomology, Trustees of the British Museum of Natural History, 1986. [5] D. Liu, B. Greenberg, Immatures stages of some flies of forensic importance, Ann. Entomol. Soc. Am. 82 (1) (1989) 80–93. [6] Y.Z. Erzinc¸lioglu, Immatures stages of British Calliphora and Cynomya, with a re-evaluation of the taxonomic characters of larval Calliphoridae (Diptera), J. Natural History 19 (1985) 69–96. ´ ´ ´ [7] P. Megnin, La Faune des Cadavres – Encyclopedie Scientifique des Aide-Memoires, Masson, Gauthier, Villars, Paris, 1984. ´ ´ ´ ´ [8] M. Leclercq, Entomologie et medecine legale. Etude des insectes et acariens necrophages pour determiner la date de la mort, Spectrum 17 (6) (1975) 1–7. [9] B. Greenberg, Flies as forensic indicators, J. Med. Entomol. 28 (5) (1991) 565–577. [10] B. Greenberg, T.I. Tantawi, Different developmental strategies in two Boreal blowflies (Diptera: Calliphoridae), J. Med. Entomol. 30 (2) (1993) 481–484. [11] T. Spence, A taxonomic study of the females of the British Lucilia species (Diptera: Calliphoridae), Proc. Royal Entomol. Soc. London 23 (1954) 29–35. [12] C. Easton, D. Feir, Factors affecting the oviposition of Phaenicia sericata (Meigen) (Diptera: Calliphoridae), J. Kansas Entomol. Soc. 64 (3) (1991) 287–294. [13] R. Wall, N. French, K.L. Morgan, Effects, of temperature on the development and abundance of the sheep blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae), Bull. Entomol. Res. 82 (1992) 125–131. [14] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning – A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, 1989. [15] D.O. Clary, D.R. Wolstenholme, The mitochondrial DNA molecule of Drosophila yakuba: Nucleotide sequence, gene organization and genetic code, J. Molecular Evolution 22 (1985) 252–271. [16] F.A.H. Sperling, G.S. Anderson, D.A. Hickey, A DNA-based approach to the identification of insect species used for postmortem interval estimation, J. Forensic Sci. 39 (2) (1994) 418–427. [17] K. Rognes, in: E.J. Brill (Ed.), Blowflies (Diptera, Calliphoridae) of Fennoscandia and Denmark, Fauna Entomologica Scandinavia 24, Scandinavian Science Press, 1991.
DNA typing for identification of some species of Calliphoridae An interest in forensic entomology ¨ Coquoz b Yvan Malgorn a , *, Raphael a
´ Sueur, Institut de Recherche Criminelle de la Gendarmerie Nationale, 1 Bd Theophile ´ , France 93111 Rosny-Sous-Bois Cedex b Laboratory of the Institut de Police Scientifique et de Criminologie, UNIL–BCH, 1015 Lausanne–Dorigny, Switzerland
Received 15 February 1999; received in revised form 12 March 1999; accepted 31 March 1999
Abstract To determine precisely post mortem interval, larvae and puparium species found on a corpse have to be identified. Among more than 200 cases examined at the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale, two-thirds concerned corpses less than one month old. Therefore, insects from first and second screwworms are the most frequently found [1]. Some species commonly found in France, such as different Lucilia and Calliphora vicina Robineau–Desvoidy, are easily identifiable at an adult stage, but are almost impossible to differentiate at immature stages when only fragments of puparium or necrosed first instar larvae are available. For this reason, an easy and objective method of identification was thus searched by genetic analysis of these insects. Sequencing of partial gene of sub unit I of cytochrome oxydase has been used to predict restriction sites. Restriction enzyme cleavage of PCR products with Dde I allowed us to differentiate these species. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polymerase chain reaction; Mitochondrial DNA; Forensic entomology; Calliphoridae; Identification
1. Introduction The best method to obtain the most precise oviposition date is to raise larvae found on *Corresponding author. Tel.: 133-1-4935-5093; fax: 133-1-4935-5027. 0379-0738 / 99 / $ – see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00039-0
112
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
a corpse [2,3], but this is not always possible and there are cases where only puparium (or fragments) or dead and necrosed larvae are available. Although these larvae or remains can easily be identified at the family level [4], it is much more difficult to identify species. It is a particularly tough challenge to differentiate Lucilia and Calliphora vicina Robineau–Desvoidy species among themselves [5,6]. These Calliphorids belong to families of the first screwworm described by ´ Megnin [7] and Leclercq [8]. But they have different biologies and then different growth and maturation rates [2,9–13]. Even if oviposition dates cannot be as precise as by raising, results obtained by the specific identification will be of a great interest for enquirers and judges and will narrow down the accuracy of the post mortem interval determination by a few days. Mitochondrial DNA is the logical target for a genetic analysis of puparium, necrosed larvae or small parts of insects. It is present in large numbers of copies. It has a high mutation rate leading to a rapid generation of sequence differences between sub-species. Its analysis can take advantage of all the modern and sensitive molecular biology methods. The only available sequences of Calliphorids in GenBank (The National Center for Biotechnology Information, Bethesda, MD, USA) were for the sub units I and II of cytochrome oxydase gene (access numbers L14945, L14946 and L14947). First, these sequences have been aligned. Then, highly conserved areas, with sufficient polymorphism between them, have been searched to design primers. Amplification and sequencing have then been carried out to predict informative restriction sites.
2. Materials and methods
2.1. Samples Insects were obtained either from colonies that are maintained in the forensic entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale or from collections. Depending upon availability, adults, puparium and larvae preserved in 708 alcohol were used. Four species of Lucilia and one of Calliphora were the subject of our study: Lucilia ´ Lucilia illustris (Meigen), Lucilia sericata ampullacea Villeneuve, Lucilia caesar Linne, (Meigen) and Calliphora vicina Robineau–Desvoidy. Three to seven individuals of each species were used for sequencing. For the restriction analysis experiments, the sample range was extended to necrosed larvae preserved for several months in 708 alcohol, first instar larvae, puparium fragments and adult fragments (wings, legs or parts of thoraces).
2.2. DNA extraction For all the samples, DNA extraction started by cutting them in small pieces, and crushing them in 1.5-ml microtubes with a piston pellet. A 500-ml volume of lysis buffer
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
113
[100 mM Tris, 50 mM ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), 0.5%, proteinase K 0.5 mg / ml] derived from Sambrook et al. [14] were added and the microtubes incubated over night at 568C. The resulted lysate was purified by extraction with phenol–chloroform–isoamylic alcohol (24:25:1) and chloroform. The final supernatant was then mixed with two volumes of ethanol, 8 ml of 5 M NaCl and 1 ml glycogen, 10 mg / ml at 2208C for 2 h to let the DNA precipitate. After centrifugation at 13 000 g the DNA pellet was dried at room temperature for 2 h, dissolved in 100 ml of sterile water and stored at 2208C. Total DNA was quantitated by reading the optical density with a spectrophotometer.
2.3. Primers Different sequences of Calliphoridae mitochondrial DNA (partial gene of subunit I of cytochrome oxidase) obtained from Genbank (access numbers L14945, L14946 and L14947) were aligned to design primers from conserved areas. The designed primer pairs are described in Table 1. Primers F1 and R1 allow the amplification of a 297 base pair (bp) fragment while primers F2 and R2 lead to a 304 bp fragment.
2.4. Polymerase chain reaction ( PCR) The PCRs were performed with reagents from the Perkin-Elmer Cetus GeneAmp PCR Core Reagents kit except for the primers which were synthesized by Eurobio (Les Ulis, France). Each 50 ml reaction contained 1X GeneAmp PCR buffer, 200 mM of each dNTP, 1.5 mM MgCl 2 , 0.5 mM of each primer, 100 ng of total DNA extracted from adults or larvae, respectively 10 ml of puparium DNA extract and 2.5 unit of AmpliTaq DNA polymerase. Amplifications with DNA amounts ranging from 1 to 500 ng were also performed. (1, 5, 10, 50, 100 and 500 ng). PCR was run for 30 cycles (958C for 20 s, 528C for 30 s, 728C for 30 s) with a final elongation of 10 min at 728C.
Table 1 Primers used for amplification a Primers
Sequence
Length
59 end
39 end
F1 R1 F2 R2
TTA TCA TTA CCA GTA TTA GC AGC TGA AGT AAA GTA AGC T CTA CTT TAT GAG CTT TAG G TTC AAG TTG TGT AAG CAT C
20 19 19 19
2056 2373 2477 2800
2075 2391 2495 2818
a
The nucleotide position numbering corresponds to that of the sequence of Drosophila yakouba published by Clary and Wolstenholme [15].
114
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
2.5. Sequencing Amplified products were first separated on agarose 1.5% gels (Pharmacia NA). The bands were then cut from the gel. The DNA was purified using Genelute Spin Column from Supelco (Supelco, Bellefonte, PA, USA), then precipitated by two volumes of ethanol and 0.1 volume of 1 M potassium acetate. The obtained pellet was resuspended in 20 ml of sterile water and then sequenced directly using ABI Prism TM dRhodamine Terminator Cycle sequencing Ready Reaction Kit according to Perkin-Elmer protocol. The PCR primers used above were used as sequencing primers to sequence each strand of the amplified products.
2.6. Prediction of restriction sites For each Calliphorid sequence of our study, cleavage sites for several restriction endonucleases were searched using DNASIS software: Hitachi Software, DNASIS for Windows , Sequence Analysis Software, Version 2.00.
2.7. Restriction analysis PCR products were first digested with restriction endonuclease Dde I according to manufacturer’s specifications (Boehringer Mannheim), then separated on 2% agarose Resophor Gel (Eurobio) containing 5 ml of GelStar (FMC Bioproducts, Rockland, ME, USA) for staining. The run was performed at 75 V during 1 h.
3. Results and discussion
3.1. Samples Sequences have been obtained from either adults, larvae or puparium. Every sample containing even a small amount of DNA (necrosed larvae, small parts of adults or puparium) allowed us to obtain an amplification. This is a good sign for forensic cases analysis but will have to be completed with the study of eggs and insects conserved by other means.
3.2. Polymerase chain reaction An amplification product has been obtained for the whole range of total DNA amounts tested (1, 5, 10, 50, 100 and 500 ng). Amounts from 10 to 100 ng of DNA gave the best results. After DNA has been resuspended in 100 ml of sterile water, the quantitation of about 30 adults and larvae allowed us to say that 10 ml of an extract diluted 100 times contains 50 to 100 ng of DNA. This dilution has been used successfully for all the samples who have not been quantitated and may be used for forensic cases. DNA obtained from small parts of adults or puparium could not be quantitated with the spectrophotometer because of insufficient sensitivity, but 10 ml of extracts gave us good results.
Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
115
3.3. Sequencing A first study has been carried out for two individuals of each of the following species: Lucilia ampullacea Villeneuve, Lucilia illustris (Meigen) and Lucilia sericata (Meigen) for the PCR fragments of 297 and 304 bp (see, respectively, Appendices 1 and 2). As it could be predicted according to Sperling et al. [16] who studied Lucilia illustris (Meigen) and Phaenicia sericata (Meigen), which is the north-American equivalent of Lucilia sericata (Meigen), the polymorphism was sufficient to distinguish these three species. For the 297 bp fragment (see Appendix 1) there were 20 nucleotide differences between Lucilia ampullacea Villeneuve and Lucilia illustris (Meigen), 26 between Lucilia ampullacea Villeneuve and Lucilia sericata (Meigen) and 24 between Lucilia illustris (Meigen) and Lucilia sericata (Meigen). In comparison with sequences of Phaenicia sericata (Meigen) and Lucilia illustris (Meigen) published by Sperling et al. [16] we noticed, respectively, one and three different nucleotides. In the case of the 304 bp fragment (see Appendix 2), only this fragment of 304 bp has been used for further study because of the better yield at the amplification. In comparison with sequences of Phaenicia sericata (Meigen) and Lucilia illustris (Meigen) published by Sperling et al. we noticed respectively no difference and one nucleotide difference. The number of non conserved nucleotides and percentage of intra- and inter-specific polymorphisms for the five species of our study are given in Table 2. Among the five species tested, only two displayed intra-specific polymorphism, but with not more than one nucleotide difference. The inter-specific polymorphism is higher. It is in the range of 21 to 37 nucleotides differences, with an outlier at six nucleotides differences for the comparison of Lucilia caesar Linne´ and Lucilia illustris (Meigen). It is to be noted that this pair of insects is morphologically very similar and difficult to differentiate even at adult stage [17]. Calliphora vicina Robineau–Desvoidy which belongs to another genus shows logically the most important difference (higher than 33 nucleotides). The low intra-specific variation will have to be confirmed using a larger set of individuals from each species.
Table 2 Number of non-conserved nucleotides and percentage of intra- and inter-specific polymorphisms a
L. ampullacea (6) L. caesar (3) L. illustris (4) L. sericata (4) C. vicina (7) a
L. ampullacea
L. caesar
L. illustris
L. sericata
C. vicina
0/0 23 21 22 35
7.56% 1 / 0.32% 6 23 37
6.91% 1.97% 1 / 0.32% 25 33
7.24% 7.56% 8.22% 0/0 34
11.51% 12.17% 10.85% 11.18% 0/0
The number of sequenced individuals is given in parentheses.
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Y. Malgorn, R. Coquoz / Forensic Science International 102 (1999) 111 – 119
Table 3 Size of restriction fragments Species
L. ampullacea
L. caesar
L. illustris
L. sericata
C. vicina
Fragments size (bp)
282–60
342
312–30
212–70–60
282–30–30
3.4. Restriction analysis Among all restriction enzymes available in DNASIS software, only Dde I alone enabled us to differentiate our five species. Sizes of the restriction fragments are given in Table 3. The restriction patterns obtained with this enzyme are shown in Fig. 1 and clearly allow us to differentiate our five species. In conclusion, an easy and fast method is now available to identify some Calliphoridae at immature stages. These results show again how powerful the analysis of mitochondrial DNA is. This study should be extended to other diptera which are difficult to differentiate at immature stages.
Acknowledgements Y.M. would like to thank Professor Pierre Margot, Director of the Institut de Police Scientifique et de Criminologie of Lausanne (Switzerland) who received him in his Institute where a large part of this work was carried out. He would also like to thank all the colleagues of the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale and Marie-Paule Carlotti, head of the biology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale who allowed him to use her sequencer without restriction.
Fig. 1. Restriction patterns obtained with Dde I. Lane 1: Lucilia illustris (Meigen); lane 2: Lucilia ampullacea Villeneuve; lane 3: Calliphora vicina Robineau–Desvoidy; lane 4: Lucilia sericata (Meigen); lane 5: Lucillia ´ lane 6: 100 base-pair ladder (Pharmacia Biotech). caesar Linne;
Sequences obtained for the 297 bp fragment
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117
Sequences obtained for the 304 bp fragment
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