Decomposition and entomological colonization of charred bodies – a pilot study

Aim To use forensic entomological approach to estimate the post mortem interval (PMI) in burnt remains. Methods Two experiments were performed in a field in the outskirts of Milan, in winter and summer 2007. Four 60-kg pigs were used: two for each experiment. One pig carcass was burnt until it reached the level 2-3 of the Glassman-Crow scale and the not-burnt carcass was used as a control. In order to describe the decomposition process and to collect the data useful for minimum PMI estimation, macroscopic, histological, and entomological analyses were performed. Results In the winter part of the experiment, the first insect activity on the burnt carcass began in the third week (Calliphora vomitoria) and at the beginning of the fourth week an increase in the number of species was observed. In the summer part, adult flies and first instar maggots (Phormia regina) appeared a few minutes/hours after the carcass exposure. Both in winter and summer, flies belonging to the first colonization wave (Calliphoridae) appeared on burnt and control pigs at the same time, whereas other species (Diptera and Coleoptera) appeared earlier on burnt pigs. Conclusion In forensic practice, burnt bodies are among the most neglected fields of entomological research, since they are supposed to be an inadequate substratum for insect colonization. Entomological approach for PMI estimation proved to be useful, although further studies on larger samples are needed.

Estimation of the post mortem interval (PMI) plays an important role in forensic investigation. In the early post mortem period, PMI can be estimated by temperaturebased methods, but when decomposition begins, this estimation can be influenced by several variables (1,2). In addition, in cases of concealment, body dismemberment, explosion, and burning there is no standardized method based on experimental studies for deriving time since death from morphological characteristics of the corpse.
Entomological approach is a well known and widely accepted method to estimate the minimum PMI (3). However, in the literature there are only a few cases referring to charred bodies (4)(5)(6)(7)(8). Gruenthal et al (9) found dung fly Scathophaga stercoraria, larvae of Calliphora vicina and Calliphora vomitoria, and immature beetle forms, not further identified in 24 pig carcasses charred up to Glassman Crow scale-1 (GCS-1) for the head, neck, limbs, and CGS 2 for the torso. Catts and Goff (6) observed a few days' delay in the arrival of blowflies on a corpse burnt and charred inside an open-topped metal drum, and a week's delay in the case of a pig burnt inside a car that was set afire (6). Introna et al (4) also highlighted that burnt flesh delayed the arrival of blowflies. Due to the relevant lack of literature, the aim of our study was to report the results of an experimental approach to burnt bodies using pigs (Sus scrofa) as models.

MATEriAl AnD METhoDS
Two experiments were performed in a field in the outskirts of Milan, in Northern Italy (45° 20' N; 09 13' E), in the winter and summer of 2007. The weather at the site was hot and damp in the summer and cold in the winter with moderate surface winds. Meteorological data were collected from the closest meteorological station located 1.5 km from the studied area and compared with the measurements performed during the sampling.
Four 60-kg pigs were obtained from the Department of Veterinary Medicine (University of Milan); each animal died from causes independent from the experimental project. For each experiment, a pig carcass was burnt on a wooden pyre until it reached the level 2-3 of the Glassman-Crow scale (19), corresponding to the destruction of the extremities, initial charring of the skin, and a substantial preservation of the corpse. The second pig of similar weight, not burnt, was used as control. In both experiments, the carcasses were maintained at a 50 m distance from each other to avoid reciprocal contamination and a wire mesh (5 cm mesh size) was placed over each carcass to prevent vertebrate depredation. The animals were placed in the same place during the winter and summer experiments.
In order to perform the morphological evaluation of the exposed pigs, samples for histological analyses (square in shape, 1 cm wide) were taken from the charred skin areas, and fixed in 10% formalin and then paraffin-embedded according to standard histological technique. Four microtome-thick sections were cut from paraffin blocks and stained with standard hematoxylin eosin stain and Trichrome stain. All observations were made using a light microscope equipped with a digital camera and DP software for computer-assisted image acquirement and managing (Wild Heerbrugg, Switzerland).
Eight insect pitfall traps containing a saturated NaCl solution and soap were placed at 50 cm all around the carcass. Moreover, entomological samples were collected by hand on the carcasses, under them, and where and when possible in the carrion cavities. Insect identification was performed using specific entomological key and description (21)(22)(23)(24) and by comparison with specimens stored in the private collection of one of the authors (SV). Zoological nomenclature followed Minelli et al (25).

rESulTS
The first part of the study was conducted from February to June. The average temperature in this period was 16.0°C (min 1.0°C, max 32.4°C) and the rainfall was considerable during March (48 mm) and May (152 mm). The second part of the study was conducted from June to August. The aver-age temperature was 25.5°C (min 12.8°C, max 36.0°C). The rainfall during this time was negligible.
Macroscopic ( Figure 1 and Figure 2), histological, and entomological observations were carried out in order to describe the decomposition processes and insect colonization. The list of the saprophagous and saprophilous insects collected on the carcasses is shown in Table 1.
During the first two weeks of the winter part of the experiment, after the charring process, no clear external modifications occurred on the carrion, there was no decomposition fluid, and no insect egg depositions.     Dermestes frishii) were present in all the body regions. In the sixth week, all the bones were exposed, with disarticulation of leg bones.
The control pig in the summer period was exposed on the same day as the burnt one. The decomposition processes during the next 2 months followed the typical pattern with the bloated, active, and dry decay stage and the beginning of the skeletonization phase. The most important concentration of maggots (Ph. regina, L. sericata) was detected in the abdominal area. After 6 weeks, the control pig showed about 40% skeletonization. Coleopterans (Staphylinidae, Carabidae, Silphidae, Histeridae, Anthicidae, Dermestidae) were collected during the whole decomposition period.
Histological screening of the charred skin fragments showed that the outer crisp surface was completely destroyed macroscopically; but frequently underneath there was a thin layer of dehydrated skin, which showed moderate preservation of cellular patterns. The dermis, subcutaneous adipose, and muscle tissues were always visible dur-ing the whole experiment, both in the winter and summer part (Figure 3).

DiSCuSSion
The entomo-fauna that was collected during the two experiments in both not-burnt and burnt carcasses included a large number of species typical of all colonization waves (23). The first flies (Calliphora spp, Ph. regina, L. sericata) arrived on burnt and not-burnt carcasses at the same time, whereas other species (Diptera and Coleoptera) arrived earlier on burnt carcasses. The colonization of burnt carcasses showed a classic pattern for the first wave insects and anticipation of other waves, with insects usually attributed to different waves arriving at the same time. This was probably a result of the carbonization process, which brought about two main advantages for fly colonization; first, the breaking up of charred soft tissues, with multiple fissures of the skin exposing the viscera, which therefore become immediately available for fly colonization. This may explain why the flies attacked the charred carcass earlier than the control carcass. In addition, the disruption of the skin surface and fast decomposition of the viscera during the first days creates a substratum of tissues in different phases of decomposition and with different water concentrations. This complex environment may explain the arrival of different waves of insects within a short period of time, which in cases of standard decomposition usually arrive sequentially. Moreover, the burning process caused the transformation of several molecules resulting in a wide spectrum of odors (volatile molecules), attractive for different insects.
Entomological approach proves to be a reliable method for the time since death determination. PMI estimation is of utmost importance in cases involving charred corpses, where decomposition processes often have different dynamics, and tissue destruction prevents evaluation based on morphological appearance or chemical variation, although some studies on accumulated degree days produced useful results (9,26). Some authors recently calculated a correction factor of the original formula, which adequately takes into account the carbonization process, but at the moment the morphological approach for PMI estimation is experimental, and there are still doubts concerning the standardization of specific carbonization variables (temperature, use of accelerants, etc) (27).
In this study, decomposition of burnt carcasses stopped quite soon, which is in contrast with four stages of decomposition observed by Avila and Goff (7). Both in winter and summer, charred tissues did not show what could be referred to as actual decomposition; they became more brittle and were affected by progressive crumpling, which lasted during the entire experimental period, reducing the bodily area covered by the soft tissues and exposing the bone surface (7). Charred corpses were better conserved probably due to the loss of water, with a decrease in bacterial activity, which was confirmed by histological analysis showing that heat retains the tissues, probably through dehydration. In fact, the observed coartation of the dermis and the presence of large gas bubbles in the dermis and picnotic nuclei in the epithelium are reported to be changes caused by heat (28)(29)(30)(31)(32)(33)(34)(35). The results are in contrast with data reported by Gruenthal et al (9), which verified the decomposition rate in charred pig carcasses, and observed that, although the general decomposition trend was similar both in charred and uncharred carcasses, a more advanced pattern was visible in bodily regions highly affected by fire (9). The differences in the decomposition process found in these two studies may be explained by the lower carbonization degree and a limited charred area in the study by Gruenthal et al (9).
In conclusion, our results showed that in burnt remains, entomological approach can be used for estimation of the minimum PMI in the presence of insects belonging to the first colonization wave (mainly Calliphoridae). Our study does not support the claim that the burnt corpse is hardly colonized by flies (3,6) and indicates that the "classic" insect waves of colonization model reported by several authors cannot be applied to burnt remains. Further research is needed to evaluate the influence of temperature of carbonization, accelerants use, environment, and body size in PMI estimation in charred bodies and fill in the gap in this important field of forensic practice.
Ethical approval received from the University of Milan ethics committee.
Declaration of authorship SV planned the entomological sampling, performed the species identification, and wrote the manuscript in collaboration with CC and DG. EZ performed the collection of the entomological samples and the thanatological observation, part of the manuscript is related to her final year project. DG, AT, SA, and CC made a substantive contribution to writing and drafting of the manuscript.

Competing interests All authors have completed the Unified Competing
Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work. references