Microscopy and forensic entomology

Transcription

1 A. Microscopy and forensic entomology N. Ubero-Pascal, I. Arnaldos, R. López-Esclapez and M.D. García Department of Zoology and Physical Anthropology, University of Murcia, Campus Espinardo s/n Murcia, Spaim Forensic Entomology is a speciality contributing more frequently to Forensic Sciences. It can be critical for solving forensic cases such as criminal cases through estimation of the postmortem interval, whether the corpse has been moved... or civil cases such as infestation of food or structures, urban pests... In any case, a clear identification of evidence is necessary since a proper expert report depends on the precise knowledge of the species that were involved in each case. It is known that the specific identification of the insects is a complex task, even if they are large, since certain parts of the body, usually of small size, are used. Therefore, from the beginning of Entomology it has been necessary to use microscopical techniques. Most of the studies use optical microscopy, although lately scanning electron microscopy (SEM) is becoming a very popular technique. One of the main goals of Forensic Entomology, the estimation of postmortem interval, deals with the larval stages of Diptera, which need to be identified. Morphological studies are being conducted using optical and electronic microscopy to establish consistent characters of larval stages that will be useful for quick identification, and detemine whether the distinctive characters set can be properly observed at optical microscopy, since this would be a faster way to yield timely results for forensic purposes. Keywords SEM; Light Microscopy; Entomology; Forensic Sciences 1. Introduction Forensic Sciences, defined as the application of scientific methods to legal issues, are an exciting field of study, complex and multidisciplinary. They use scientific arguments (chemical, physical, biological...), as well as social and legal, to the resolution of legal cases. In fact, the term "forensic" defines any science applied to law [1]. The importance that Forensic Sciences have in the administration of justice resides in its potential ability to provide vital information about how a criminal action has been committed and who has committed it [2]. Nonetheless, Forensic Science may be involved in many other activities which, while having legal implications, do not fall within the criminal field. There are many ways in which Forensic Sciences can be applied, from the estimation of postmortem interval on the basis of pollen or algae in the corpse to the technology for vocal identification or analysis. The application of Entomology to legal cases is called Forensic Entomology [1]. Following Hall and Huntington [3],...is the broad field where arthropod science and the judicial system interact. Therefore, Forensic Entomology deals with very diverse applications, the most well-known being, probably, the medico-legal practice. Although the estimation of postmortem interval on the basis of entomological evidence is, even, fashionable, Forensic Entomology can also be applied to the evaluation of the place where death took place, the perimortem circumstances, a guilty or suspicious identification, neglect cases, sudden deaths, myiasis therapy, detection of drugs or toxins, wild fauna cases, stored products cases, urban and house pests... All these applications are based on the best possible correct identification of the evidence. Thus, Taxonomy becomes an essential tool for Forensic Entomology, and Taxonomy is based, in most cases, in morphological characters. Almost any arthropod can be involved in a Forensic Entomology case. However, the most frequent group consists of flies (Insecta, Diptera). They are typically related to corpses in the first moments after death, and stay in the corpse for a variable time depending on the species, the season, the geographical area, etc. Adults, mainly the females, arrive very early to the corpse to lay eggs (or larvae, in Sarcophagids). After hatching, larvae begin to feed on the corpse tissues. The development of arthropods is strongly influenced by environmental conditions, mainly temperature. Each species of fly has different environmental requirements which contribute to its development. The larval age, expressed in hours or days, is one of the criteria that is used for estimating the postmortem interval (PMI); the estimated age of an immature insect that has fed on a body provides a minimum PMI [4]. Accurate identification of specimens is usually the first priority in a forensic analysis of the entomological evidence. Identification of the genus or species allows the forensic entomologist to access the correct developmental data and distribution ranges to be applied to the case [5]. An incorrect species determination can lead to a potential error in the estimation of PMI since the carrion species differ in terms of growth rate, arrival time and position within the order of succession [4]. As mentioned above, the morphological characters are the most important ones for quickly identifying a taxon. Despite the large number of molecular identification techniques, their cost hinders its implementation. A drawback is the lost of the specimen studied of which, at best, only fragments can be preserved. On the contrary, morphological examinations can be done quickly and are extremely cost-effective [5]. For forensic purposes Diptera is the most interesting group. Among them, three families are most relevant to forensic practice: Calliphoridae (blow flies), Sarcophagidae (flesh flies) and Muscidae (house and muscid flies) and their larvae are very interesting to estimate the PMI. These larval stages are not always easily identified and even the morphology of the immature stages of some species is still unknown. Because of the size, especially in the early stages of development, and the fine details to be 1548

2 A. considered, it is necessary to do a microscopic study. In fact, only few species can be easily identified without the aid of microscopic study. Scanning electron microscopies (SEM) provides excellent and detailed morphological overview, but has the disadvantage of being rather expensive and require preparation of the material to be studied. Therefore, we try to determine the utility of optical microscopy studies by comparing the characters observed with this method and with SEM in order to determine their applicability to forensic practice. Fig. 1 Macroscopical morphology of immature stages of C. vicina. a) Comparative size and general morphology of egg and larva instars. b) General morphology of pupa. c) Pseudocephalon and first thoracic segment of larval instar III. d) General morphology of anal division of instar III. Magnification: 8x a) and b); 55x c) and d). 2. Materials and methods The preimaginal stages studied came from colonies bred in laboratory under controlled environmental conditions (25 C and 60% relative humidity), using pig liver as substrates for egg laying and larval food. Pupation substrate was sand surrounding the liver. Adults of Calliphoridae were collected in the Campus of Murcia University (Southeastern Spain) using a modified Schoenly trap [6], and those of Sarcophagidae in Sierra Espuña (Murcia), using the same trap. Muscid adults were collected in the city of Murcia using pet s food as bait. All studied specimens, as well as adults, are kept at the Área de Zoología in the Departamento de Zoología of Universidad de Murcia. For the stereomicroscope study, larvae were killed in water near to boil and then preserved in 70% ethanol. Observations were made by a Leica stereomicroscope MZ 9,5 attached to a Nikon digital camera DS-Fi1. For light microscopy analysis, larvae were killed and preserved in the same way. Afterwards, they were cleared so that the detailed structure of the mouthparts and spiracles (anterior and posterior) coud be studied. This was achieved by macerating the specimen in a 10% aqueous solution of KOH [7]. Maceration time varies depending on the larval size, from a few hours for the smallest one to almost one day for to the most developed larvae. Once cleared, larvae were rinsed and transferred to glacial acetic acid for at least 15 minutes to neutralise any residual potassium [7]. Then, specimens were mounted on slides to be studied using Hoyer medium. Observations were made using a Nikon 80i microscope with differential interference contrast (DIC) and pictures were taken using the same camera mentioned above. For SEM, selected specimens were rinsed and killed in the usual way (except pupae), and fixed in McDowell fixative solution, ph 7.4, at 4 C for 24 h. Specimens were rinsed again twice in sodyum cacodylate-sacarose solution; dehydrated until absolute acetone in a gradient of increasing concentration of ethanol and ethanol-acetone solution (50% of each reagent), and either dried using critical point method or air-dried by hexamethyldisilizane treatment [8]. Dried specimens were mounted on stub with conductive adhesive tape, trying to lay them in different position (dorsal, lateral and ventral views). Because the third instar larvae are the largest ones, they were cut in order to study efficiently the fore and hind parts of the body. Specimens were coated with Au-Pd in a Polaron Bio Rad Sputter Coat and observed in a JEOL JSM 6100 SEM located in the Microscopy Service of Murcia University. Pictures were directly digitized from the SEM. 1549

3 A. The considered morphological features of eggs, larvae and pupae, the body divisions and the terminology follow those of [9, 10, 11, 12, 13]. Abbreviations used in figures: a, antenna; ae, anterior spiracle; an or ap, anal protuberance; b, button; bm, bubble membrane; c, chorion; cl, cephalic lobe; eb, spines band; h, respiratory horn; la, labial lobe; m, micropyle; ma, median area; mp, maxillary palpus; og, oral groove; p, papillae; pa, anterior pole; pe, posterior spiracle; pl, respiratory plastron; pp, posterior pole; pr, peritreme; ps, pseudocephalon; psc, pseudocephalon collapsed; pt, peristigmatic tuft; rs, respiratory slit; sk, cephaloskeleton; vo, ventral organ. Fig. 2 General morphology and ultrastructure of eggs in C. vicina (a-b, d, f) and S. nudiseta (c, e, f-g). a) General morphology by light microscopy. b and c) General morphology by SEM. d and e) Ultrastructure of anterior pole and micropyle. f and g) Ultrastructure of plastron area. Magnification light microscopy: 30x a). 3. Results and Discussion Eggs, three larval instars and pupae are the immature stages in a typical Diptera life cycle (Fig. 1 a-b). The morphology of all these stages provides many data of great interest for taxonomic purposes. However, many Diptera species of forensic importance are still not well known from a morphological point of view, or the main study available for a specific identification was only performed with light microscopy. This kind of microscopy can be useful for a general overview of immature stages (Figs. 1 and 2a), the study of big structures like band of spines or anterior and posterior 1550

4 A. spiracles (Fig. 4c-d) in specimens of great size (instar III of larvae and pupae) and, even, for studying internal structures such as mouthparts or cephalopharingeal skeleton (Fig. 3a-b). However, the morphology of stages whose specimens are small, such as eggs (Fig. 2a) and larval instars I and II, or tiny structures in the other stages (Fig. 3e), are poorly described. This implies two taxonomical problems. One relates to the fact that general description by light microscopy only allows to distinguish species very different morphologically and only in the last stages of life cycle (instar III and pupae). Second, light microscopy does not allow connecting first and late larval stages if they are not bred in the lab, because this technique doesn t allow a good discrimination of morphological characters of taxonomic importance. So, SEM is now being used for this purpose, providing fine details of morphological features that allow distinguishing species regardless of stage and connecting the stages without having to breed them in the lab. Nevertheless, the morphological knowledge of immature stages of Diptera species of forensic importance using SEM is currently insufficient to establish the best characters to accomplish this task. Below, we will detail the usefulness of microscopy for the study of Dipteran immature stages, illustrating what each technique affords and how they can complement each other. The purpose is to define the key features at SEM that could also be identified using light microscopy, since this technique is easier to use, and results can be obtained more quickly. The fast processing feature is crucial for solving forensic cases in a brief period of time. Fig. 3 Internal and external morphological features of pseudocephalon in Phaenicia sericata (Meigen, 1826) (a), S. nudiseta (b, d, e), Sarcophaga cf. cultellata Pandelle, 1896 (c) and C. vicina (f). a and b) Cephaloskeleton morphology by light microscopy. c and d) Ultratructure of pseudocephalon. e) Morphology of antenna and maxillary palpus by light microscopy. f) Ultrastructure of antenna and maxillary palpus. Magnification light microscopy: 40x a and b); 400x e). 3.1 Morphology of eggs According to Smith [14], eggs of Diptera of forensic importance are very simple morphologically although some main or typical structures can be distinguished [10, 15]: micropilar plate, median area, respiratory plastron, hatching lines, and chorion sculpturing. Diptera eggs are larger than those already studied in other groups, such as Ephemeroptera [16], 1551

5 A. however SEM is necessary for a detailed morphological analysis [10]. Light microscopy doesn t provide the accurate observation necessary to describe the main chorionic structures (Fig. 2a). In fact, all previous papers that use eggs to distinguish Diptera species are performed by SEM [10, 17, 18, 19]. Nevertheless, not all Diptera especies of forensic importance can be distinguished by the egg morphology, since they are very similar, and specific features have not been yet described. This is the case of species belonging to the same genus, such as Calliphora [10]. Despite it, egg morphology allows to distinguish many species of Diptera. An example is showed in Fig. 2b-g, where it can be seen that eggs of Calliphora vicina Robineau-Desvoidy, 1830 and Synthesiomya nudiseta van der Wulp, 1883 are very similar in their general shape or in the location of micopyles, but they also show clear differences. For instance: 1) plastron runs along the complete length of egg in S. nudiseta, while in C. vicina doesn t arrive to the posterior pole (Fig. 2 b-c), having clear morphological differences (Fig. 2f-g); 2) boundaries of median area are expanded in S. nudiseta, while in C. vicina are very short (Fig. 2b-e); and c) chorionic sculpturing is clearly different, while S. nudiseta shows longitudinal ribs and a great magnification is necessary to observe the hexagonal chorionic reticulation (Fig. 2e), C. vicina has not ribs and the hexagonal reticulation is observed at very low magnification (Fig. 2d). In this respect, many structural variations have been described among eggs of other species, mainly in the plastron structure and the boundaries of median area [10, 15, 17, 18, 19]. Fig. 4 Morphological features of anal division in S. nudiseta (a, c, e), S. cf. cultellata (b), P sericata (d) and C. vicina (f). a and b) Ultrastructure of anal division. c and d) Morphology of posterior spiracle by light microscopy. e and f) Ultrastructure of posterior spiracle by SEM. Magnification: 200x c and d). It is noteworthy that in some Diptera of forensic importance eggs can not be studied because the species are ovoviviparous and lay first stage larva directly, as normally happens with Sarcophagidae and, also, with some Calliphoridae when food is scarce [14]. 1552

6 A. Fig. 5 Morphological comparison of pseudocephalon and anal division in the three larval instars of C. vicina. a and b) Instar I. c and d) Instar II. e and f) Instar III. 3.2 Morphology of larvae Larvae are the main source of morphological useful data to identify Diptera of forensic importance in immature stages, specially the instar III (Figs. 3 and 4). Although the size of larvae changes drastically from one instar to other, usually instar III may measure five or six times the length of instar I (Fig. 1a), the morphology of the body is basically maintained in all of them (Fig. 1a). Following Courtney et al. [11], larvae of cyclorraphan Diptera are composed of twelve segments. Three regions can be differentiated in the body: cephalic region or pseudocephalon (first segment), thorax (from second to fourth segments) and abdomen (from fifth to twelfth segments), although the twelfth segment is also known as anal division. Every region and even each segment shows several unique structures that usually appear in all instars, although they may appear more complex in the last instar (Fig. 5). These structures allow to distinguish between species and to relate larval instars in the same species (Figs. 3-5). External larval morphology provides the main number of structures of taxonomical value, although the mouthparts or cephalopharingeal skeleton, an internal structure, is widely used for this purpose, especially in instar III [14]. Light microscopy is the appropriate technique to study the cephalopharingeal skeleton without dissecting the specimen since all sclerites that form it do not move and maintain their position (Fig. 3a-b). However, to get good observations it is necessary to clear the larvae. All larval instars are provided of cephalopharingeal skeleton and this structure is useful to distinguish species in all instars [9, 17]. However, this structure is mainly known for instar III, and there are quite few species in which this structure has been described for all larval instars. Today, the scientific movement preparing identification keys for Diptera species of forensic importance in instar III continue to consider the cephalopharingeal skeleton as a specific structure that is necessary to know. Light microscopy is also useful to study the external morphology of larvae in all instars but, sometimes, the resolution of this device is inappropriate for observing in detail some structures (Figs. 3e and 4c-d). SEM has shown these structures as having a very rich ultrastructure with many morphological details (Figs. 3f and 4e-f) [20]; some of 1553

7 A. them could be species selective. The pseudocephalon and anal division are the larval body segments in which the main specific structures appear (Fig. 3e-d), while the remaining segments show a smaller number of structures, very important in some families. Cephalic lobes, antennae, maxillary palpus, ventral organ, oral ridges, labial lobe are some of the structures that can be observed in the pseudocephalon of instar III using light microscopy (Fig. 3a-b, e). However, in order to observe these structures in instars I and II or to describe its detailed morphology it is necessary their study at SEM (Figs. 3e-d, f and 5a,c). This allows to determine that cephalic lobe shows many sensillae; antenna is composed of a dome and a basal ring with an external sensilla, maxillary palpus consists of several sensillae located in different places, ventral organ is a complex structure, but the most important consideration is that all these structures can be studied in all instars with a good resolution (Fig. 5a,c). Anal division has many structures of taxonomic value and some of them can be differentiated macroscopically (Figs. 1d and 4c-d). Posterior spiracles, posterior papillae, anal pad are some of the structures of taxonomic interest that can be studied at light microscopy, specially in instar III. However, for describing the respiratory slits of posterior spiracles, the peristigmatic tufts, the peritrema ornamentation, the sensillae in posterior spiracle, or simply the sculpturing of tegument, it is necessary to use SEM (Fig. 4a-b, e-f). The description of these structures by SEM allows their correct interpretation and to establish the proper correlations with the light microscopy observation, for the most possible accurate identification of species. The anterior spiracle morphology, spines arrangement in segments and tegument sculpturing are also larval features of interest to identify species and, although some of them can be observed by light microscopy, SEM provides exceptional images to be described. On the other hand, the description of structures by SEM is indispensable for an effective correlation of larval morphology of one species in its three instars [20, 21, 22, 23]. For instar I the light microscopy is insufficient to describe the external morphology. Moreover, instar I may present a structure arrangement very different to instars II and III, which are very similar morphologically (Fig. 5). Several studies have been published which focused in the description of instar I in many species [13, 24, 25] and the relationship with other larval instars because, sometimes, this larval stage is the only evidence in a forensic case and, usually, the specimens arrive dead to the researcher, being impossible their breeding to adults. Fig. 6 Ultrastructure of pupae of C. vicina. a) Pseudocephalon collapsed. b) Respiratory horn and traces of bubble membrane. c) General morphology of anal division. d) Detail of morphology of posterior spiracle. 3.3 Morphology of pupae Usually, the pupa of Diptera of forensic importance shows most of the morphological characters of larval instar III [9, 17], except for some collapsed parts, such as the pseudocephalon (Fig. 6a). The arrangement of spines in segments and anal division structures are more or less conserved in the pupa (Fig. 6c), specially the posterior spiracles (Fig. 6d), allowing the correlation to larval morphology for identifying purposes. These structures can be observed by light microscopy although it is better described by SEM. In fact the main description of pupae has been given by this 1554

8 A. technique. Moreover, pupae morphology shows exclusive structures, such as the bubble membrane and respiratory horn, which are very close related [9, 17]: bubble membrane appears in young pupae and disappears when the respiratory horn is developed, since both structures are placed in the same location (Fig. 6b). Although some papers describe the bubble membrane by light microscopy, according to Liu and Greenberg [17] SEM is the best technique to describe this membrane because the bubble protuberance may be very tiny. Therefore, for a correct study of pupae both microscopy techniques are necessary. 4. Conclusions and general remarks Currently, the description and identification of immature stages of Diptera useful for forensic sciences at the specific level requires the complementary use of light microscopy and SEM, because each one supplies different information completely useful for these purposes. For a long time, light microscopy has been the most used technique and continues to be important, since the procedures to observe the samples are faster than SEM. In fact, the keys developed by some scientists for the identifications of larvae are based mainly on the body observations by this technique. However these keys are limited to larval instar III. Although some species can be identified in larval instars I and II by light microscopy, this technique is very limited for a complete description of their morphology, especially in instar I, and for finding taxonomical characters in uncharacterized species to allow the correct specific identification. For these reason, SEM is increasingly being adopted by forensic entomologist as a new device to obtain precise information about larval body morphology, not only in eggs and instars I and II, but also for recharacterizing instar III and pupae. The joint use of both microscopical techniques is appropriate for a complete knowledge of larval morphology in all instars. This circumstance is allowing to establish new specific characters based on SEM observations that can be visualized by light microscopy for a quick identification of the specimens and, also, to correlate all the immature stages in each species. However, further efforts are necessary, especially by SEM, to characterize the immature stages in all species of Diptera of forensic interest. Acknowledgements This contribution has been supported by the projects 00848/CV/01 of Fundación Séneca of the Comunidad Autónoma de la Región de Murcia and CGL /BOS of Ministerio de Educación y Ciencia of the Spanish Government. References [1] Walker M. Entomology and Palynology. Evidence from the Natural World. Broomall, Pennsylvania: Mason Crest Publishers; [2] Kiely, T.F. Forensic Evidence. En: James SH, Nordby JJ. Forensic Science. 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