Volatile organic compounds released by blowfly larvae and pupae: New perspectives in forensic entomology

Forensic Science International 219 (2012) 215–220

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Volatile organic compounds released by blowfly larvae and pupae: New perspectives in forensic entomology C. Frederickx a,*, J. Dekeirsschieter a, Y. Brostaux b, J.-P. Wathelet c, F.J. Verheggen a, E. Haubruge a a

Department of Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liege, Passage des De´porte´s 2, 5030 Gembloux, Belgium Department of Applied Statistics, Computer Science and Mathematics, Gembloux Agro-Bio Tech, University of Liege, Passage des De´porte´s 2, 5030 Gembloux, Belgium c Department of General and Organic Chemistry, Gembloux Agro-Bio Tech, University of Liege, Passage des De´porte´s 2, 5030 Gembloux, Belgium b

A R T I C L E I N F O

S U M M A R Y

Article history: Received 10 February 2011 Received in revised form 14 December 2011 Accepted 3 January 2012 Available online 16 February 2012

To evaluate postmortem intervals (PMIs), one should take into account the determined age of necrophagous flies present on the cadaver. However, PMI determination needs further improvement, and rapid and accurate approaches have therefore to be developed. While previous studies have focussed on insect cuticular hydrocarbons, here we explore the volatile profile released by larvae and pupae of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). We monitored changes in volatile compounds daily, by headspace solid-phase microextraction, followed by gas chromatography–mass spectrometry. Branched and unbranched hydrocarbons, alcohols, esters and acids were identified, and the volatile profile was shown to vary, in both composition and quantity, with the age of the larva/pupa under investigation. We concluded, based on the analysis of the released volatile organic compounds, that it is possible to increase the accuracy of the estimated PMI, through improved estimation of the age of blowflies present on the cadaver. ß 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic entomology Calliphoridae Necrophagous fly Pupae Volatile organic compounds SPME

Forensic entomology is a branch of the forensic sciences which studies insects and other arthropods (e.g., mites) in a medico-legal context [1–5]. Insects are predominantly used in the discipline to determine timing of colonisation, which can be interpreted as time of death of a vertebrate organism, otherwise known as the postmortem interval (PMI). PMI is the period of time between death and corpse discovery [3,5–7]. In establishing the PMI, the forensic entomologist aims to establish the age of the oldest colonising species, for which various methods exist [3,8–10]. Currently, PMI is estimated on the basis of rearing of eggs, larvae and pupae collected at the crime scene. Blowflies are the most common invertebrates used to determine PMI, because this family is one of the first to invade a corpse [7,11,12]. Its age estimation depends on the rate of development of each species, which in turn depends on the temperature and humidity of the surroundings [7–9]. However, to avoid breeding efforts, forensic entomology would benefit from developing new rapid and accurate determination of PMI. One method could involve identifying hydrocarbons present on the cuticle of the insects, or even on their pupae and puparia [13,14]. Indeed, some recent studies [15–18] have shown that the profile of the composition of hydrocarbons found on the cuticle of larvae and pupae changes with time [14]. If these changes are integral to the development of larvae into

* Corresponding author. Tel.: +32 81622287; fax: +32 81622312. E-mail address: [email protected] (C. Frederickx). 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2012.01.007

adults, and if this can be incorporated into a model, these hydrocarbons could represent a very useful tool in estimating the age of a larvae or pupae and hence could increase the accuracy of the PMI. Many forensic entomological studies have been conducted on cadaveric volatile organic compounds (VOCs) released after death [19–23]. Numerous applications of VOCs are found in forensic sciences, including the aetiology of death [21], training of cadaver dogs [24,25], or the development of cadaveric material detection devices [22,23,26], but no information is available on the VOCs emitted by necrophagous Diptera throughout their life. These VOCs could be used to estimate the PMI more accurately. It is still not known or fully understood whether the VOCs emitted by larvae or pupae change over time during the life of an insect. However, the profile of hydrocarbons could change with genetic factors such as age [15] or gender of an insect [14], environmental factors such as diet [27–29] and temperature [30,31] or geoclimatic factors [30,32]. It therefore seems plausible to assume that the VOC profile of the cuticle will fluctuate with time, diet or temperature. Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) [1] is one of several species with the common name of blue bottle fly [33,34]. It has a holarctic distribution [35–37] and is known to produce myiasis [38–40]. C. vicina was selected because of its relative abundance on decaying corpses, and because it is one of the first species to arrive on a dead body, making them a highly useful forensic tool [7,11,41,42].

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Table 1 The 92 volatile organic compounds released by larval stages (L1, L2, L3) and pupal stages from one to ten days. 2 samples, : compound is present in 1 sample.

Retention time 1.25 1.55 1.60 1.64 1.69 1.79 2.29 2.31 2.68 2.79 2.83 3.16 3.21 3.64 3.78 4.10 4.18 4.19 4.31 5.93 6.24 7.30 7.54 8.15 8.56 9.01 9.12 9.39 9.65 9.76 10.16 10.39 10.50 11.20 11.21 11.83 12.27 12.52 13.09 13.17 13.38 13.39 13.39 13.46 13.70 13.85 13.95 14.09 14.43 14.59

Volatile chemicals N.N-dimethylmethanamine Methanethiol Ethanol Propan-2-amine Propan-2-one 2-methyl-propan-2-ol Ethyl acetate Acetic acid 3-methylbutanal 2-methylbutanal 2-methylpropan-1-ol Butan-2-one 1-methoxy-3-methylbutane 3-hydroxybutan-2-one Methylcyclohexane 3-methylbutan-1-ol 2-methylbutan-1-ol 4-methylpentan-2-one Methyldisulfanylmethane Octane Hexanal 2.4-dimethylheptane N-ethyl-1.3-dithioisoindoline 2-methylpropyl 3-methylbutanoate N-(2-aminoethyl)ethane-1.2-diamine Ethyl 2-methylbutanoate Ethyl 3-methylbutanoate Ethylbenzene 1.4-dimethylbenzene 1.3-dimethylbenzene 3-methylbutyl acetate 2-methylbutyl acetate 1.2-dimethylbenzene Heptanal Methoxybenzene 4-methyl-1-propan-2-ylbicyclo[3.1.0]hex-3-ene 4.7.7-trimethylbicyclo[3.1.1]hept-3-ene 6.6-dimethyl-5-methylidenebicyclo[2.2.1]heptane 2-methyl-5-propan-2-ylcyclohexa-1.3-diene Benzaldehyde (E)-4-methylidene-1-propan-2-ylbicyclo[3.1.0]hexane Methylsulfanyldisulfanylmethane (Z)-4-methylidene-1-propan-2-ylbicyclo[3.1.0]hexane 7.7-dimethyl-4-methylidenebicyclo[3.1.1]heptane 1-methyl-2-propan-2-ylbenzene Phenol 7-methyl-3-methylideneocta-1.6-diene 2.2.4.6.6-pentamethylheptane 3.7.7-trimethylbicyclo[4.1.0]hept-3-ene 1-methyl-4-propan-2-ylcyclohexa-1.3-diene

: compound is present in 3 or 4 samples,

: compound is present in

Developmental stage of C. vicina Aged of pupae Larvae L1 L2 L3 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10

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Table 1 (Continued ) 14.83 14.92

1-methyl-4-propan-2-ylbenzene 3-methylidene-6-propan-2-ylcyclohexene

15.14

1-methyl-4-prop-1-en-2-ylcyclohexene

15.33

1-methyl-4-propan-2-ylidenecyclohexene

15.47 15.71 15.78

(3E)-3.7-dimethylocta-1.3.6-triene 1-methyl-4-propan-2-ylcyclohexa-1.4-diene 2.2.5-trimethylhexane

16.17 16.96

2.4-dimethylundecane Nonanal

17.17 17.65 17.92

(1R.4S)-1-methyl-4-propan-2-ylcyclohex-2-en-1-ol 4.7.7-trimethylbicyclo[3.1.1]hept-3-en-2-ol 7.7-dimethyl-4-methylidenebicyclo[3.1.1]heptan-3-ol

18.09

4-methyl-1-propan-2-ylcyclohex-3-en-1-ol

18.25 18.30

2-(4-methylcyclohex-3-en-1-yl)propan-2-ol 2-(2-butoxyethoxy)ethanol

18.85

1H-indole

19.06 19.34 19.51

5-prop-2-enyl-1.3-benzodioxole Butyl butanoate (1S.3aR.4S.8aS)-decahydro-4.8.8-trimethyl-9-methylene-1.4-methanoazulene

19.53 19.57

3-hydroxy-2.4.4-trimethylpentyl) 2-methylpropanoate 8-Isopropyl-1.3-dimethyl-tricyclo[4.4.0.0(2.7)]dec-3-ene

19.65 19.78

1.2-dimethoxy-4-prop-2-enylbenzene (1R.4Z.9S)-4.11.11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene

19.78

2.4-ditert-butylphenol

20.12 20.37

4-methoxy-6-prop-2-enyl-1.3-benzodioxole 2.6-dimethoxy-4-prop-2-enylphenol

20.74 20.96 21.11(

Tetradecanoic acid Pentadecanoic acid 3-decanoyloxy-2-hydroxypropyl) decanoate

21.16

(E)-hexadec-9-enoic acid

21.22 21.25 21.42

Hexadecanoic acid Ethyl hexadecanoate Heptadecanoic acid

21.56 21.65 21.99

(Z)-octadec-9-enoic acid Heptadecene-8-carbonic acid Octadecanoic acid

22.56 22.66

Octadecane Nonadec-1-ene

22.75 22.93 24.37

Eicosane Z-tricos-9-ene (6E.10E.14E.18E)-2.6.10.15.19.23-hexamethyltetracosa-2.6.10.14.18.22-hexaene (3S.8S.9S.10R.13R.14S.17R)-10.13-dimethyl-17-[(2R)-6-methylheptan-2-yl]2.3.4.7.8.9.11.12.14.15.16.17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

27.52

The possibility of using VOCs emitted by larvae and pupae of C. vicina Robineau-Desvoidy (Diptera: Calliphoridae) as accurate components of PMI estimation was the main goal of this study. This study also included experiments to produce a daily identification of the VOCs emitted by C. vicina. 1. Methods and materials 1.1. Rearing of Insects C. vicina Robineau-Desvoidy were kept on a 16:8 light: dark photoperiod and at 22 8C  0.5 8C. Males and females were maintained together in a rearing cage (55  60  48 cm) supplied with sucrose, dried milk and water. The larvae were fed with pig meat chopped in a glass container (20  30  20 cm). After feeding, larvae were transferred to another container containing about 700 g of vermiculite for pupation. 1.2. SPME volatile collection and GC–MS analysis When the insects were at the stage of development for study, they were cleaned with distilled water and wiped. VOCs of larvae of L1, L2, L3 and pupae aged from 1 to 10 days were analysed. We selected 10 days as the appropriate period for study because the development period from puparium to imago emergence of C. vicina is 10 days at 22  0.5 8C [9]. Each developmental stage was placed into a glass vial of 20 ml to collect VOCs with solid-phase micro-extraction (SPME). The developmental stage sample filled half of the vial, corresponding to 7 mg of each developmental stage. Preliminary experiments were carried out with different types of SPME fibre, to select the most adequate. The SPME volatile collection of larvae was conducted using a 50/30 mm divinylbenzene–carboxen–polydimethylosiloxane (DVB–CAR–PDMS,

stableflex) (Supelco, State College, PA, USA). The SPME volatile collection of pupae was conducted using a 75-mm carboxen–polydimethylosiloxane (CAR–PDMS, fused silica) (Supelco, State College, PA, USA) coating fibre. Before each use, the fibre was conditioned at 270 8C (DVB–CAR–PDMS) or 300 8C (CAR–PDMS) for 1 h in a split– splitless GC injector. After conditioning, the fibre cleanliness was checked by analysing it using gas chromatography–mass spectrometry (GC–MS), as the control run. Volatile collections were performed at 25  1 8C during 1 h on a hot plate, in a 20ml vial. After each volatile collection, the SPME fibre was withdrawn from the vial and analysed by GC–MS. GC–MS analyses were carried out on an Agilent 6890N Network GC System coupled with an Agilent 5973 Network mass selective detector, equipped with an HP-5 (Agilent) capillary column (30 m  0.25 mm ID, 0.25 mm film thickness). The oven temperature programme was initiated at 40 8C, held for 8 min, then raised first at 8 8C min 1 to 115 8C and raised in the second ramp at 50 8C min 1 to 290 8C and held for 8 min. Other operating conditions were as follows: carrier gas, helium, with a constant flow rate of 1 ml min 1; injector temperature, 270 8C, splitless mode. Mass spectra were taken at 70 eV. Mass range was from m/z 35 to 350 amu. The larvae and pupae volatile components were identified by comparing their mass spectra fragmentation patterns with those stored in the Wiley275.L computer library and confirmed, when possible, by injection of pure standards. Experiments were replicated four times for each developmental stage and for each age of pupae, for a total of 52 samples plus 52 used as control runs.

1.3. Statistical analyses The data used in the analysis were the presence/absence of the VOCs. However, only VOCs which were present in three or four samples were considered, and any pre-treatment of the data was carried out. The VOCs emitted by L1, L2 and L3 larvae, and the pupae aged 1–10 days, were analysed by an ascending hierarchical clustering (AHC) using Ward’s method. AHC consists in gradually incorporating the individuals according to their resemblance as measured using an index of

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Fig. 1. Chromatogram resulting from 6 days old pupae headspace sampling 1.55: methanethiol; 1.60: ethanol; 1.69: propan-2-one; 2.29: ethyl acetate; 2.68: 3methylbutanal; 2.79: 2-methylbutanal; 3.21: 1-methoxy-3-methylbutane; 3.64: 3-hydroxybutan-2-one; 4.10: 3-methylbutan-1-ol; 4.18: 2-methylbutan-1-ol; 4.31: methyldisulphanylmethane; 13.39: methylsulphanyldisulphanylmethane; 15.14: 1-methyl-4-prop-1-en-2-ylcyclohexene; 19.51: (1S,3aR,4S,8aS)-decahydro-4.8.8trimethyl-9-methylene-1.4-methanoazulene.

dissimilarity, based here on the Jaccard’s index [43]. The initialisation of this method consists in calculating a table of Jaccard’s dissimilarities between the individuals to be classified. The algorithm then begins by grouping more similar individuals into couples, and then gradually to incorporate the other individuals or groups of individuals, according to their resemblance. At each stage, the clusters that are amalgamated are those whose dissimilarity is the weakest. Analysis was conducted with R statistical software v2.10.0 [44], using vegan package v1.17-4 to compute the dissimilarities’ table.

2. Results 2.1. Volatile collection Table 1 presents the VOCs identified in one, two, three or four of the samples according to the developmental stage. However, statistical analysis was carried out on compounds emitted by three or four of the samples. Independently of the developmental stage, 92 VOCs were identified by GC–MC (Fig. 1). Many chemical families were represented (in parentheses, the number of chemical compounds identified): cyclic hydrocarbons (32), non-cyclic hydrocarbons (13), alcohols (11), esters (10), acids (9), aldehydes (6), nitrogen compounds (4), ketones (4) and sulphur compounds (3). There are some differences between the life stages in terms of sampled chemical molecules. Indeed, a total of 54 VOCs was identified in L1, L2 and L3 larvae, whereas a total of 52 VOCs was identified in pupae. The larval stages and the pupal stage emit 14 common VOCs. Eight cyclic hydrocarbons were emitted at each stage of larval development: safrole (IUPAC name: 5-prop-2-enyl1,3-benzodioxole), myristicin (IUPAC name: 4-methoxy-6-prop-2enyl-1,3-benzodioxole), terpinen-4-ol (IUPAC name: 4-methyl-1propan-2-ylcyclohex-3-en-1-ol), gamma-terpinene (IUPAC name: 1-methyl-4-propan-2-ylcyclohexa-1,4-diene), alpha-thujene (IUPAC name: 4-methyl-1-propan-2-ylbicyclo[3.1.0]hex-3-ene), sabinene (IUPAC name: (E)-4-methylidene-1-propan-2-ylbicyclo[3.1.0]hexane), 3-carene (IUPAC name: 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene) and para-cymene (IUPAC name: 1-methyl-4propan-2-ylbenzene). However, only one VOC was emitted during the entire pupal stage: dimethyldisulphide (IUPAC name: methyldisulphanylmethane).

All the acidic compounds, except for acetic acid, were emitted exclusively during the larval stage. This is also true for nitrogen compounds, cyclic alcohols, non-cyclic and cyclic alkenes except 4,7,7-trimethylbicyclo[3.1.1]hept-3-ene, 1-methyl-4-prop-1-en2-ylcyclohexene and (1S,3aR,4S,8aS)-decahydro-4,8,8-trimethyl9-methylene-1,4-methanoazulene. However, the non-cyclic and cyclic alkanes were mainly emitted by the pupae. All the sulphur compounds, ketones, aldehydes and alcohols were emitted by pupae. Moreover, ester compounds were detected during the last 3 days of the pupal stage. The compounds containing a benzene ring were emitted by larvae, as well as by pupae. 2.2. Ascending hierarchical clustering The AHC dendrogram shows the cluster of larvae and pupae and the evolution of the volatile profile over time (Fig. 2). The dendrogram shows four groups. A first group is represented by the

Fig. 2. Ascending hierarchical clustering dendrogram.

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three larvae instars, with a volatile profile significantly different from that of pupae. Pupae volatile profiles allow three other groups to be distinguished: pupae of 1–3 days old; pupae of 4–7 days old and pupae of 8–10 days old. The VOCs which made it possible to group the larval stages were: 5-prop-2-enyl-1,3-benzodioxole, 4methoxy-6-prop-2-enyl-1,3-benzodioxole, 4-methyl-1-propan-2ylcyclohex-3-en-1-ol, 1-methyl-4-propan-2-ylcyclohexa-1,4-diene, 4-methyl-1-propan-2-ylbicyclo[3.1.0]hex-3-ene, (E)-4methylidene-1-propan-2-ylbicyclo[3.1.0]hexane, 3,7,7-trimethylbicyclo [4.1.0] hept-3-ene and 1-methyl-4-propan-2-ylbenzene. The older pupae, from 1 to 3 days old, were grouped in a cluster because they emitted methyldisulphanylmethane and 4,7,7trimethylbicyclo[3.1.1]hept-3-ene (alpha-pinene). The pupae from 4 to 7 days old were grouped because they emitted 3methylbutanal, ethanol, methyldisulphanylmethane (dimethyldisulphide), methylsulphanyldisulphanylmethane (dimethyltrisulphide) and alpha-pinene. Moreover, pupae of 4 days old were closer to the pupae of 5 days old because both emitted 3methylbutan-1-ol. Acetic acid allowed 6-day and 7-day-old pupae to be clustered. The last cluster, grouping 8- to 10-day-old pupae, included 2-methylpropan-1-ol, 1-methoxy-3-methylbutane, ethyl 3-methylbutanoate, 3-methylbutyl acetate and methyldisulphanylmethane. The pupae aged 8 days resembled more closely those aged 9 days, because both emit methanthiol, ethyl acetate and phenol. These three VOCs were not emitted during any other period. 3. Discussion The first aim of this study was to determine whether VOCs can be used to evaluate the age of flies, to establish a PMI in medicolegal investigations. Our study showed that the composition of the volatile compound blend released at different blowfly developmental stages is different. For larvae and pupae, analysis of VOCs enables young pupae to be distinguished from older ones. However, two groups of factors could affect the chemical composition [14,16,45] and could also influence the emission of VOCs. The first group comprises genetic factors, such as age of the insect or the gender of a fly [14,46–48]. In general, the age or gender of an insect can influence the chemical profile. The developmental stage of an insect is a factor relating to genetics. In Chrysomya rufifacies Macquart larvae, it has been shown that the hydrocarbon profile can change over time [15]. Previous studies report similar age-related changes in cuticular hydrocarbons in many other insect species [45,47,49–51]. In this study, modifications observed among the GC–MS patterns of the VOCs are clearly dependent on the developmental stage, because we standardised the environmental conditions. The second group of factors potentially affecting the emission of VOCs by blowfly larvae and pupae are those related to environmental factors, such as diet, temperature or geoclimate [30,32,46,48,52]. Before such an analysis can be translated into a new approach in forensic entomology for the estimation of the PMI, the effects of genetic factors and environmental factors on the profile of VOCs of the developmental stage should be investigated. The second aim was the characterisation of VOCs released by the juvenile stages of C. vicina. Previous studies of cadaveric VOCs released by the decaying process collected compounds from a global cadaver micro-ecosystem, without knowing whether the volatile compounds collected were released from the corpse itself or associated insects [20,22,23,26]. However, maggot masses can be very abundant during the ‘active decay’ stage. Dekeirsschieter et al. [19] identified the main cyclic compounds of this decaying stage as being dimethyldisulphide, dimethyltrisulphide, indole and phenol. In this study, indole and phenol were collected from larvae. These two compounds, identified as being cadaveric

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compounds, can be emitted to a large degree by the larval masses and not by the corpse itself. However, there are not published studies concerning phenol and indole emitted by the corpse itself, or by the maggot mass. The VOCs emitted by the larval stage could arise from bacteria on mouthparts, gut or skin of larvae. Bacteria from the families Enterobacteriaceae, Pseudomonaceae and Bacillaceae have in particular been shown to emit volatile compounds, such as 3-hydroxybutan-2-one, indole, 3-methyl-1butanol, 3-methylbutanal, 2-methylbutanal, phenol, methyldisulphanylmethane and limonene (IUPAC name: (1S,3aR,4S,8aS)decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene) [53–61]. In this study, larvae emit 3-methylbutanal, phenol, indole and limonene, but more research is necessary to identify microorganisms present at the larval stage and their production of VOCs. In the late pupal stage, we observed a large number of new VOCs. During this late pupal stage, the eyes were pigmented and the setae of head, thorax and abdomen were tanned [62], which could explain the new compounds. However, it is not yet possible to determine whether this is in fact the case, because it would be necessary to study the hormonal and morphological development inside the pupae. Twenty VOCs emitted by pupae aged 1–10 days were also identified as cadaveric VOCs [19–23,26,63]. Among these VOCs, 10 were recognised as important markers of the cadaveric decomposition: dimethyldisulphide, dimethyltrisulphide, butan-2-one, propan-2-one, ethylbenzene, 1,3-dimethylbenzene, benzaldehyde, nonanal, hexanal and ethanol [20–23]. Further studies on VOCs released during the developmental stages of necrophagous Diptera are currently being conducted at the Department of Functional and Evolutionary Entomology (AgroBio Tech, Gembloux, Belgium). Different environmental factors are also being investigated. Acknowledgement Christine Frederickx and Jessica Dekeirsschieter are financially supported by a Ph.D. grant from the Fonds pour la Formation a` la Recherche dans l’Industrie et l’Agriculture (F.R.I.A.), Belgium. References [1] R.D. Hall, Medicocriminal entomology, in: E.P. Catts, N.H. Haskells (Eds.), Entomology and Death, A Procedural Guide, Forensic Entomology Associates, 1990 , pp. 1–8. [2] R.D. Hall, Introduction: perceptions and status of forensic, in: J.H. Castner, J.L. Byrds (Eds.), Forensic Entomology: The Utility of Arthropods in Legal Investigations, CRC Press, Boston, 2001, pp. 1–16. [3] J. Amendt, R. Krettek, R. Zehner, Forensic entomology, Naturwissenschaften 91 (2004) 51–65. [4] J. Wallman, A key to the adults of species of blowflies in southern Australia known or suspected to breed in carrion, Med. Vet. Entomol. 15 (2001) 433–437. [5] D.E. Gennard, Forensic Entomology: An Introduction, John Wiley & Sons, Ltd., 2007. [6] H. Klotzbach, R. Krettek, H. Bratzke, K. Puschel, R. Zehner, J. Amendt, The history of forensic entomology in German-speaking countries, Forensic Sci. Int. 144 (2004) 259–263. [7] C. Wyss, D. Cherix, Traite´ d’Entomologie Forensique: Les insectes sur la sce`ne de crime, Presses Polytechniques et Universitaires romandes, Lausanne, 2006. [8] M. Marchenko, Medico-legal relevance of cadaver entomofauna for the determination of the time of death, Acta Med. Leg. Soc. 38 (1988) 257–302. [9] M. Marchenko, Medicolegal relevance of cadaver entomofauna for the determination of the time of death, Forensic Sci. Int. 120 (2001) 89–109. [10] C. Henssge, B. Madea, Estimation of the time since death in the early post-mortem period, Forensic Sci. Int. 144 (2004) 167–175. [11] A. Gunn, Essential Forensic Biology, John Wiley & Sons, Ltd., Liverpool, 2006. [12] B. Greenberg, J.C. Kunich, Entomology and the Law. Flies as Forensic Indicators, Cambridge University Press, Cambridge, 2005. [13] A.R. Gilby, J.W. McKellar, Composition of empty puparia of a blowfly, J. Insect Physiol. 16 (1970) 1517–1529. [14] F.P. Drijfhout, Cuticular, Hydrocarbons: a new tool in forensic entomology? in: J. Amendt, C.P. Campobasso, M. Lee Goff, M. Grassbergers (Eds.), Current Concepts in Forensic Entomology, Springer, Dordrecht, Heidelberg, London, New York, 2010 , pp. 179–203.

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