Multiple colonization of a cadaver by insects in an indoor environment: first record of Fannia trimaculata (Diptera: Fanniidae) and Peckia (Peckia) chrysostoma (Sarcophagidae) as colonizers of a human corpse Simão Dias Vasconcelos, Thiago Ferreira Soares & Diego Leonel Costa International Journal of Legal Medicine ISSN 0937-9827 Int J Legal Med DOI 10.1007/s00414-013-0936-2 1 23 Your article is protected by copyright and all rights are held exclusively by SpringerVerlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. 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The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Int J Legal Med DOI 10.1007/s00414-013-0936-2 CASE REPORT Multiple colonization of a cadaver by insects in an indoor environment: first record of Fannia trimaculata (Diptera: Fanniidae) and Peckia (Peckia) chrysostoma (Sarcophagidae) as colonizers of a human corpse Simão Dias Vasconcelos & Thiago Ferreira Soares & Diego Leonel Costa Received: 30 July 2013 / Accepted: 23 October 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract We describe here a case of multiple colonization of a male cadaver found indoors in the municipality of Jaboatao dos Guararapes, Brazil. The body was colonized by six species of Diptera: Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria (Calliphoridae), Megaselia scalaris (Phoridae), Fannia trimaculata (Fanniidae), and Peckia (Peckia) chrysostoma (Sarcophagidae). The most abundant species were C. albiceps (65.0 % of all emerged adults) and C. megacephala (18.6 %). The case illustrates the ability of six insect species to simultaneously colonize a corpse in an indoor environment and represents the first collaboration between the forensic police and entomologists in Northeastern Brazil. We provide here the first record of two species, F. trimaculata and Peckia (P.) chrysostoma colonizing a human cadaver. We also report the first case of cadaver colonization by C. putoria and M. scalaris in Northeastern Brazil. Information on the development time of two species, C. albiceps and C. megacephala, were used to discuss the estimation of the post-mortem interval. Considering that the region harbors the highest rates of homicide in Brazil, implications of these findings for the consolidation of forensic entomology in the region are discussed. S. D. Vasconcelos (*) : T. F. Soares Insects of Forensic Importance Research Group, Department of Zoology, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil e-mail: simaovasconcelos@yahoo.com.br D. L. Costa Institute of Scientific Police, Government of Pernambuco State, Recife, Pernambuco, Brazil Keywords Legal medicine . Forensic entomology . Necrophagous insects . Post-mortem interval . Blow fly Introduction Insects of medicolegal importance include mainly Diptera species from the families Calliphoridae, Sarcophagidae, Muscidae, Fanniidae, and Phoridae, some of which have been used as evidence in criminal investigations [1]. Knowledge on the presence and the stage of development of insect larvae on a cadaver can help the forensic entomologist to estimate the minimum post-mortem interval (minPMI), to determine if the body was moved from the crime scene after death, and to detect the presence of illicit substances in the corpse [1]. In practice, however, the reliability of forensic entomology is often compromised by unrealistic experimental settings [2], which disregard the need for rigorous collection of immature insects on human bodies at the site of death. Legal, bureaucratic, and ethical restraints limit the use of cadavers for forensic studies so that access to homicide victims is usually limited to police personnel. Also, scientific guidelines such as those proposed by Amendt et al. [3] are not commonly used. The availability of human bodies is erratic, and procedures related to medicolegal practices, such as washing the cadavers, often eliminate entomological evidence [3]. This is the case of the Northeast Region of Brazil which has one of the highest homicide rates in South America [4]. In the state of Pernambuco, Northeastern Brazil, integrated programs to reduce the number of homicides have been recently Author's personal copy Int J Legal Med implemented, but new approaches to provide reliable tools for police investigators, such as forensic entomology, have been neglected. The use of entomological evidence in criminal investigations requires rigorous collection of information on the conditions of death, such as abiotic conditions and overall access to the cadaver. For example, Reibe and Madea [5] state that bodies located indoors can be colonized later and by a more limited number of Calliphoridae species when compared to outdoor situations. In some cases, the delay in discovering the body compromises the contribution of forensic anthropologists and entomologists [6]. As part of an initial cooperation between entomologists and the forensic police in Northeastern Brazil, we describe a case of multiple indoor colonization of a cadaver by species of Diptera. We present the first evidence for the use of human cadavers as substrate for the development of two species, which represents a new record of their geographical distribution and their forensic relevance. Methods The body of a 71-year-old Caucasian male was found in July 2012 in the municipality of Jaboatao dos Guararapes (08°06′ 46″S, 32°00′54″W, est. pop. 655,000), Pernambuco State, Brazil. The victim's flat (ca. 50 m2) was located on the first floor in a three-storey building, 400 m from an urban beach, in a neighborhood exposed to moderate accumulation of litter. The flat was relatively clean and tidy and windows were partially open, which allowed for limited access of insects. The deceased lived alone, and his disappearance had not been reported to local authorities. The police arrived following a phone call from the neighbors who noticed the strong odor coming from the apartment, which was locked from the inside. After the hypothesis of homicide had been discarded, the scientific police team, which included medical examiners, carried out an examination of the body at the scene. The deceased was found lying on the bathroom floor in a right lateral position dressed in shirt and shorts. The analysis revealed that death occurred in situ, and the report from the medical described the cadaver in an advanced stage of decay, with larvae coming out from the interior of the body, discreet tongue protrusion, mild edema of the scrotum, cyanosis of the nail beds, advanced stage of flaccidity, mydriatic pupils, absence of external injuries, and a strong putrid odor (Fig. 1). According to the physical appearance of the cadaver, death was estimated by the medical examiner to have occurred 5 days prior to the discovery of the body. After comprehensive analyses, the medical examiner decreed “undetermined cause of death.” The body was then transported inside a clean plastic bag to the Institute of Legal Medicine in the neighboring city Fig. 1 Maggot masses on a cadaver found in an indoor environment in Jaboatao dos Guararapes, Brazil: a head and upper torso, b head and left arm, c lower trunk and legs of Recife, the capital of Pernambuco. No toxicological analyses were performed. The larvae masses were distributed throughout the cadaver, with emphasis on the head and trunk. Abundant maggot masses were also located under and between the clothes. Third instar larvae were collected equally from several parts of the body using soft forceps in a 180-min sampling period. Approximately, 500 larvae were placed into plastic containers (ca. 10 insects/ pot) containing minced beef as food and sawdust as substrate for pupation, kept at glasshouse (mean temperature 26.4 °C, Author's personal copy Int J Legal Med relative humidity 80 %, 12 h:12 h light:dark cycle), and observed daily until emergence of adults. Each pot was kept into larger screen cages to prevent contamination by opportunistic species (e.g., Phoridae). Manipulation took place in controlled conditions in the laboratory. Food was monitored daily and replaced whenever necessary. Adults were identified using taxonomic keys [7–10]. Specimens have been deposited at the Entomological Collection at UFPE in Recife. Results As it was not possible to count all larvae in the corpse during the examination, a comparative visual estimate consisted of broad categories of abundance in increasing order: upper limbs (up to 1,000 larvae); lower limbs, especially under the shorts (1,000 to 3,000); head and neck (1,000 to 3,000); and trunk (>3,000 larvae) (Fig. 1). The head was the most decomposed part, at the advanced decay stage, compared to the rest of body, which was in late bloated/early advanced decay. Two hundred and thirty-seven adults emerged from the larvae reared in the laboratory. Six species belonging to four families of Diptera were recorded: Chrysomya albiceps (Wiedemann, 1830), Chrysomya megacephala (Fabricius, 1805), Chrysomya putoria (Wiedemann, 1830) (Calliphoridae), Megaselia scalaris (Lowe, 1866) (Phoridae), Fannia trimaculata (Stein, 1898) (Fanniidae), and Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Sarcophagidae) (Table 1). The most abundant species were C. albiceps (65.0 % of all emerged adults), C. megacephala (18.6 %), and M. scalaris (15.2 %). Emergence of adults began 4 days post-collection and lasted until the tenth day (Table 1). In the case of Chrysomya albiceps, adults emerged from 6–10 days post-collection of the larvae. The mean time until emergence of adults was 8.5 days. For C. megacephala, emergence of adults occurred from the sixth day (79.5 % of all individuals) until the eighth day (2.3 %), and the mean emergence time was 6.25 days. Discussion The family Calliphoridae comprises 1,526 fly species with a worldwide distribution and has been frequently associated with decomposing cadavers [11]. The dominant species reported here, C. albiceps and C. megacephala , have been observed on corpses in several countries, including Italy [12], Colombia [13], Malaysia [14], and Brazil [15] and have been effectively used in the determination of the minPMI [16]. Recently, both species were recorded on human cadavers in Northeastern Brazil [17]. In contrast, C. putoria is reported for the first time colonizing human corpses in the region despite its previous record in low abundance in traps baited with carrion [3]. The family Fanniidae comprises 285 species belonging to four genera [11]. So far, F. pusio, F. canicularis [18], and F. scalaris [19] have been recorded on cadavers, and F. canicularis was used as criminal evidence in a case of child neglect in Germany [20]. We present here the first record of F. trimaculata colonizing a human cadaver and also the first report of its presence in the Northeastern Region of Brazil. Phoridae is a cosmopolitan family that includes over 4,000 species [11], of which M. scalaris has been observed previously in human corpses and used as evidence in forensic cases [21]. The present study is the first record of M. scalaris on cadavers in Northeastern Brazil. Sarcophagidae is one of the most diverse families associated with cadavers: from the 3,073 described species, approximately 800 have been reported in the Neotropical region [11]. Their forensic relevance is limited by the paucity of taxonomic keys and of data on their life cycle. Recently, two species, Oxysarcodexia riograndensis (Lopes, 1946) and Ravinia belforti (Prado and Fonseca, 1932), were found in cadavers at the Legal Medicine Institute in Recife [17]. We provide here the first evidence of Peckia (P.) chrysostoma as colonizers of human bodies. The concentration of maggot masses on the head was partly due to the more advanced stage of decomposition of the head Table 1 Insects (Diptera) emerged from larvae collected from a human cadaver in Jaboatao dos Guararapes, Brazil, according to their abundance and previous use in forensic entomology cases Family Species Number of individuals Relative Frequency Time to emergence (days) Previous register in cadaver [Ref] Previous use in forensic entomology cases Calliphoridae Chrysomya albiceps Chrysomya megacephala Chrysomya putoria Megaselia scalaris Fannia trimaculata Peckia (Peckia) chrysostoma 6 154 44 1 36 1 1 237 65.0 18.6 0.4 15.2 0.4 0.4 100 8.49±1.67 6.23±1.38 9 4 4 7 Yes [17]; [19]; [26]; [27] Yes [13]; [14]; [17]; [26] Yes [15] Yes [14]; [28] No No Yes No Yes Yes No No Phoridae Fanniidae Sarcophagidae Total Author's personal copy Int J Legal Med when compared to the rest of the corpse, as well as to the fact that blow flies tend to oviposit in natural cavities such as mouth, ears, and eyes. The high abundance of calliphorid species reported here is a common pattern in cadaver colonization [13, 15]. Curiously, no native Calliphoridae species were reported here, a fact that reinforces the need of ecological studies to support the work of forensic pathologists and entomologists. The dominance of Chrysomya invasive species seems to reflect their ability to locate and colonize ephemeral resources. Also, the aggressive predatory behavior of C. albiceps larvae could hamper PMI estimation by the forensic entomologist as it could disrupt the post-mortem insect clock by clearing a corpse of early colonizers [16]. The period of insect activity (PIA) is of utmost importance for the estimation of the post-mortem interval since entomological evidence is most useful when there is an extended (>72 h) interval between death and the discovery of the body [22]. However, the PIA can be much shorter than the actual post-mortem interval in certain cases, such as when death occurs in enclosed environments [1]. In this study, most C. albiceps adults emerged 8.5 days after collection as third instar larvae. Data on C. albiceps development suggest a variable duration in larval stage depending mostly on the temperature. For example, the mean time from egg to third instar larvae was estimated to be approximately 3.3 days at 27 °C and 2.0 days at 32 °C [23]. This temperature range is typical of the death scene described in the present study, considering the climate data obtained from local official sources. Considering that death was estimated to have occurred 5 days prior to the discovery of the body using medicolegal methods, some inferences can be drawn from this scenario. An immediate interpretation should lead to the conclusion that the PMI was overestimated by approximately 12 to 24 h. Alternatively, a forensic entomologist may conclude that there was an interval of 1 to 2 days between death and cadaver colonization by insects, which was probably due to the limited access represented by the indoor environment. Reibe and Madea [5] demonstrated empirically that bodies located outdoors are colonized by blow flies more quickly than those located indoors, a pattern that has clear implications on the estimation of the PMI. More realistic estimates cannot be made until the effects of substrate and temperature on larval development have been determined. The time between the collection of larvae and adult emergence observed in this study confirms that the majority of larvae effectively were at third instar and that emergence tends to occur within a few days. It is also important to consider that the natural variations in temperature tend to be lower in indoor environments [1], which also have implications on the estimation of the PMI. C. megacephala reached adult stage 6.25 days after collection as third instar larvae. Data from laboratory studies using temperatures similar to those locally in Jaboatao dos Guararapes showed that the time from egg to early third instar was reported to vary between 2.5 and 4.0 days at 32 °C and from 2.5 to 5.0 days at 26 °C, although this variability can be magnified by the type of substrate in which larvae are reared in the laboratory [24]. In this case, a higher degree of consistency was observed between the estimated PMI based on entomological evidence and that proposed by the medical examiner when compared to C. albiceps . Naturally, models of insect development assume that the relationship between temperature and development time is not linear, but a reasonable degree of accuracy can be achieved when data generated from different temperatures are combined. In a recent review, Vasconcelos and Araujo [3] listed 16 species of Sarcophagidae, 11 of Calliphoridae, 3 of Fanniidae, and only 1 Phoridae (M. scalaris ) in the Northeastern Region of Brazil. The widespread use of these species as legal evidence, however, has not been accomplished in the country due mainly to the absence of reproducible data on development and ecology. Furthermore, rarely have several species been retrieved from a single cadaver in studies based on an indoor environment, which can also be a consequence of the high diversity of Diptera species in the Neotropical region. To our knowledge, this is the first documented case in the American continent that reveals such diversity of Diptera species colonizing a single human cadaver in an indoor environment. For the sake of comparison, the only other similar case reported occurred in Thailand, where six species were recovered from a mummified cadaver [25]. Our results expand the list of species recorded on corpses and add new locations to the known geographical distribution of several species. Despite the alarming rates of unsolved homicides, research on forensic entomology is incipient in Northeastern Brazil. The intricate police system, aggravated by the insufficient number of forensic professionals in the region, reduces the effectiveness of the cooperation among police investigators, pathologists, and local entomologists [3]. Caution is needed before indiscriminately using entomological evidence devoid of information not only about the deceased person but also on the local climate and particularly on the site of death (e.g., accessibility to insects, degree of human presence, and type of environment). Based on this experience, it is expected that further collaboration between the scientific police and university researchers will begin to consolidate forensic entomology practices in Northeastern Brazil. Acknowledgments We thank the staff from the Legal Medicine Institute (Recife) and from the forensic police from the Government of Pernambuco State for permission for the collection of biological material, Dr. Trev Williams (Inecol, Mexico) for the helpful comments on the manuscript, and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brazil) for the financial support. Author's personal copy Int J Legal Med References 1. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJR (2007) Best practice in forensic entomology—standards and guidelines. Int J Leg Med 121:90–104 2. Tomberlin JK, Byrd JH, Wallace JR, Benbow ME (2012) Assessment of decomposition studies indicates need for standardized and repeatable methods in forensic entomology. J Forensic Res 3: 147. doi:10.4172/2157-7145.1000147 3. Vasconcelos SD, Araujo MSC (2012) Necrophagous species of Diptera and Coleoptera in Northeastern Brazil: state of the art and challenges for the forensic entomologist. 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