Forensic Science International 206 (2011) e96–e98
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Simple clearing technique as species determination tool in blowfly larvae Senta Niederegger a,*, Nelly Wartenberg a, Roland Spieß b, Gita Mall a a b
Department of Forensic Entomology, Institute of Legal Medicine, University Hospital Jena, Germany Department of Neurobiology, Institute of Zoology, University Bonn, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2010 Received in revised form 17 December 2010 Accepted 12 January 2011
A simple clearing technique is presented by which species specific structures and organs of blowfly larvae can easily be visualized and displayed without any danger of mechanical damages or dislocations of delicate formations and without fixation of the object. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Blowfly larvae Clearing technique Methyl salicylate Species determination
Forensic investigations of human corpses at death scenes involve an estimation of the post mortem interval (PMI) which indicates the time elapsed between death and the discovery of the body. Long term PMI estimations are best performed calculating the age of blow fly larvae developing on the corpse [1–4]. It has to be accounted for however, that different blow fly species develop at different rates under the same conditions [5]. An exact species determination is therefore crucial for any further calculations and analyses. Species determination in adult flies is a tedious, but accomplishable task by the use of a dissecting microscope. In larvae however, species specific morphological features are considerably smaller and often internal organs or structures need to be consulted. The analysis of external features such as spiracles and spine bands can be performed by the use of a stereomicroscope [6–8] or scanning electron microscopy [9–12]. Analysis of internal organs however asks for challenging dissections and elaborate histological treatments [13]. An important internal structure used for species determination in blowfly larvae is the cephalopharyngeal skeleton (CPS) [13–17]. It is embedded in the anterior end of the larvae and comprises the mouth hooks which are used for feeding and locomotion [18]. The CPS furthermore provides attachment sites for the pharyngeal
muscles [19,20]. The integument which covers the body of the larvae consisting of a two-layered epicuticle [21] as well as the above mentioned pharyngeal muscles need to be removed in order to have a clear view of the CPS. This preparation is elaborate and the risk of damaging the structure considerable. Szpila and Pape [22] used Hoyer’s medium (gum arabic, glycerol, chloral hydrate, distilled water) to clear these structures. The technique eliminates removing the structure or tissues from the larval body. An other clearing technique using potassium hydroxide (KOH) was proposed by Sukontason et al. [23]. Both techniques however demand the mounting of the specimen onto slides. In order to reduce time and operating expenses as well as the usage of above mentioned hazardous chemicals a far simpler procedure is required. An established method to visualise morphological details of previously stained neurons embedded in the insect nervous system uses methyl salicylate (e.g. [24]). This method was modified in order to obtain a simple and reliable tool for the visualisation of the CPS as well as intersegmental spine bands and spiracles in maggots. No potentially structure damaging or dislocating preparation is needed and species determination can be performed by the use of a simple dissecting microscope. Methyl salicylate, also known as wintergreen oil, is a natural product of many plants and is used as a rubefacient in deep heating liniments and in small amounts as a flavoring agent [25]. Methyl salicylate has a refraction index (n20 D) of 1.535–1.538 [Merck data sheet].
* Corresponding author at: Institut fu¨r Rechtsmedizin, Fu¨rstengraben 23, 07743 Jena, Germany. Tel.: +49 3641 937927; fax: +49 3641 937902. E-mail address:
[email protected] (S. Niederegger).
‘‘Objects can be seen because they differ from their surroundings in colour or refractive index. The latter causes light to bend
1. Scientific note
0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.01.012
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S. Niederegger et al. / Forensic Science International 206 (2011) e96–e98
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Fig. 1. Before (a) and after (b) clearing in methyl salicylate. Lateral (top) and ventral (bottom) views of a third instar Calliphora vomitoria maggot.
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Fig. 2. Lateral (a) and ventral (b) view of the cephalopharyngeal skeleton. Third instar Calliphora vomitoria maggot in methyl salicylate. AOS, accessory oral sclerite; MH, mouth hook; PPS, parapharyngeal sclerite; HPS, hypopharyngeal sclerite; IPS, infrapharyngeal sclerite; TPS, tentropharyngeal sclerite; PDP, posterodorsal process; VC, ventral cornua; DC, dorsal cornua; as, anterior spiracle; is, intersegmental spine band; cl, cephalic lobe; do, dorsal organ; to, terminal organ.
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at the surface of the object. Clearing media are designed to closely, but not perfectly, match the refractive index of cell walls or organelles. An object becomes invisible when immersed in a medium of equal refractive index. Variation in refractive index among cellular components assures that some aspect of the specimen will always be visible when the refractive index of the medium is close to that of the bulk specimen.’’ [Cited from 26, p. 35] Feeding third instar Calliphora vomitoria larvae were used in our analyses. Larvae were doused with boiling water for about 30 s to prevent shrinkage and browning and stored in 70% ethyl alcohol for 2 h. Larvae were then kept in 96% ethyl alcohol for 2 h for dehydration before transfer in methyl salicylate. First effects could be seen after 1 h or less, full clearance was reached after 3 h (Fig. 1). Pictures were taken using a Zeiss Stemi 2000-C stereomicroscope with a Zeiss AxioCam ICc 1 1.4-megapixel CCD camera and corresponding software. Terminology was adapted from various authors [13,27–29]. Upper-case letters indicate parts of the CPS, lower-case letters organs and other structures located on the surface of the maggot (Fig. 2). By the use of methyl salicylate species specific structures and organs, specifically the distinct features of the CPS, can easily be visualized and displayed without any danger of mechanical damages or dislocations of the delicate formations. Large numbers of larvae can be handled simultaneously. If larvae are not needed for further examinations the posterior part can be truncated in order to accelerate penetration of methyl salicylate into the body. Dehydration time can be prolonged if needed, larvae will not suffer any damages. Furthermore larvae stored in 70% EtOH for a long time can be analyzed without difficulty as long as no browning of the larvae occurred so far. References [1] E.P. Catts, M.L. Goff, Forensic entomology in criminal investigations, Annual Reviews Entomology (1992) 253–272. [2] M.L. Goff, A Fly for the Prosecution, Harvard University Press, Cambridge, 2000. [3] D.E. Gennard, Forensic Entomology. An Introduction, Wiley, Chichester, 2007. [4] K.G.V. Smith, A Manual of Forensic Entomology, The Trustees of the British Museum (Natural History), London, 1986. [5] S. Niederegger, J. Pastuschek, G. Mall, Preliminary studies of the influence of fluctuating temperatures on the development of various forensically relevant flies, Forensic Science International (2010). [6] A.J. Prins, Morphological and biological notes on six South African blow-flies (Diptera, Calliphoridae) and their immature stages, Annals of the South African Museum 90 (1982) 201–217. [7] Y.Z. Erzinclioglu, Immature stages of British Calliphora and Cynomya, with a reevaluation of the taxonomic characters of larval Calliphoridae (Diptera), Journal of Natural History 19 (1985) 69–96.
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