Skipping clues: forensic importance of the family Piophilidae (Diptera)

Among the insects which are typically considered of forensic interest, the family Piophilidae (Diptera) is frequently cited because of its common occurrence on carcasses in different stages of decay. Piophilids are mainly known from the cosmopolitan species Piophila casei, which can be also a major pest for the food industry and an agent of myiasis. However, many other species of Piophilidae occur frequently on carrion, including human corpses; hence, it is essential to ensure a careful identification of specimens. Reviews of relevant published information about the Piophilidae species of potential forensic use, including recent interesting records, are presented.     

Insects of forensic significance in Argentina

Records from forensic expertises and trappings with beef baits conducted in Buenos Aires, Argentina (34 degrees 36'S), show that the dominating species are widespread ones (Calliphora vicina and Phaenicia sericata), with different behaviour in each large latitudinal zone. It is suggested that the range of the yearly photoperiod variation has an influence in the behaviour of the blowflies, making up for differences in the succession patterns. The Calliphorid blowflies Cochliomyia macellaria and Chysomya albiceps were found on indoors corpses; the latter also on outdoors corpses when blood was shed, and in that case as primary. Three species of beetles of the genus Dermestes, which had been associated with mummified remains, appeared 10-30 days after death. The Silphid beetle Hyponecrodes sp. cf. erythrura was found on outdoor copses in rural environments. The Nitidulid beetle Carpophilus hemipterus was found in association with the cheese skipper Piophila sp. (Diptera: Piophilidae) in medullar cavities of bones after ca. 30 days; to this association is often added the Clerid Necrobia rufipes. Lepidoptera Tineidae appear on the head of mummified indoors corpses. North of parallel 32 degrees S, the Muscid grave-fly Ophyra sp. was found breeding on a corpse outdoors in summer. A division by latitude and climate is proposed for Argentina, and an extended system is proposed for the world.     

Occurrences of flesh flies (Diptera: Sarcophagidae) on human cadavers in Switzerland, and their importance as forensic indicators

From 1993 to 2008, criminal investigations were conducted in the western part of Switzerland with special attention to blowfly and flesh fly species in order to estimate the post-mortem interval when requested by the police authorities. Flesh flies were found in only 33 cases out of 160. Five species of the genus Sarcophaga were identified (S. africa, S. argyrostoma, S. caerulescens, S. similis and S. sp.). The main species found on corpses (larval stage) was S. argyrostoma. The thermal constant (K) calculated for this species in Switzerland is 380.6 ± 16.3 (mean ± S.D.) degree-days. With the exception of S. caerulescens, found three times in the larval stage on corpses, the three other species are of minor forensic importance. S. argyrostoma is found during summer and indoors. This species colonises dead bodies, usually the same day as blowfly species, and it could be used to estimate the post-mortem interval. Other species are discussed in the light of current knowledge on their biology and ecology. It is recommended that voucher material be deposited in a museum, allowing further studies by relevant specialists, thereby helping investigators and avoiding misidentifications.     

DNA Barcoding in Forensic Entomology – Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species,

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High‐quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1‐5P’ DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high‐quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.     

Artifacts Caused by Leaf-Cutting Ants of the Genus Atta (Hymenoptera: Formicidae): Postmortem Bite Injuries and the Tearing of Clothes

Ants are one of the first insects to find an exposed cadaver and can be present during all stages of decomposition. Although these organisms are not commonly used in postmortem interval estimates, they are to be taken into account on criminal investigations involving human corpses, since they can leave bite marks that can be mistaken for antemortem or perimortem injuries, which could be misleading when ascertaining the occurrence of abuse or physical altercation during a crime. A few studies report the action of ants on human cadavers and even though leaf-cutting ants of the genus Atta are frequently encountered in succession studies that use animal carcasses, there are no records of these fungus-growing species on human corpses. Atta is a genus restricted to the New World, ranging from northern Argentina to southern United States and acts as one of the most conspicuous neotropical herbivores. In this study, we report three cases of violent death that illustrate the impact of ants, especially those of the genus Atta, in a forensic setting. We compare the patterns displayed by postmortem bite injuries caused by leaf-cutter ants and other common species with less robust mandibles. We also present the capability of Atta ants to create artifacts by cutting victim’s clothes in a crime scene, contributing to the knowledge of ant-mediated confounding factors in crime scene investigation.     

Determination of postmortem interval by arthropod succession: a case study from the Hawaiian Islands

A postmortem interval of 34 to 36 days was established for remains recovered on the island of Oahu, Hawaii, based on interpretations of patterns of arthropod succession on the remains. This interval was primarily based on the presence of adult specimens of Philonthus longicornis (family Staphylinidae), mature larvae of Piophila casei (family Piophilidae), and empty puparial cases of Chrysomya rufifacies (family Calliphoridae). Species and developmental stages of two additional Coleoptera species and three additional Diptera species were also present, which was consistent with the estimated interval, although not definitive.     

DNA-based and taxonomic identification of forensically important Sarcophagidae (Diptera) in southeastern Spain

Studying dipterans at the scene of a death can provide essential information for interpreting the evidence and help to reconstruct the events happened to a corpse in the past. Molecular tools have been employed for identification at specific levels in the cases of cryptic species or poorly conserved specimens. Identification of specimens is essential in forensic entomology since each species has a specific growth rate, which determines the calculation of the minimum post mortem interval (minPMI). In addition, phylogeographic reconstruction within a species can help to differentiate the haplotypes from a geographic area, thereby helping to clarify the possible relocation of a corpse. The morphological identification of Sarcophagidae species is often difficult, especially for the females. This is an important Diptera family since some of its species are among the first to reach a corpse, especially in warm areas. In this study, we compared the sarcophagids found in human corpses in forensic cases in Alicante (southeast of Spain) with specimens collected from baited traps in the same area and surrounding provinces. In total, 189 specimens were collected, comprising 72 from forensic cases and 117 from baited traps. Molecular identification was conducted by sequencing the cox1 mitochondrial gene and analyzing the sequences using ABGD, GMYC, and BIN species delimitation methods. The median joining algorithm in the PopART program was used to construct phylogeographic networks. Eight species in the family Sarcophagidae were identified. The most widely collected species were Sarcophaga argyrostoma and Sarcophaga tibialis. The haplotype networks obtained for these species did not indicate a clear geographic distribution of haplotypes. The S. argyrostoma samples from Alcoy were clearly isolated. The results demonstrated that this method is useful for identifying Sarcophagidae samples in forensic investigations and it can be employed for minPMI estimation.     

The Red Swamp Crayfish Procambarus Clarkii (the Louisiana Crayfish) as a Particular Scavenger on a Human Corpse

An 85-year-old man was found deceased floating in an irrigation ditch 18 days after his disappearance. During crime scene investigation, specimens of the red swamp crayfish Procambarus clarkii were found in proximity of the body. The feeding activity of these nonendemic crayfishes contributed to the formation of specific injuries on the body and in the production of a large substance defects inside the corpse. The aim of this paper was to illustrate the scavenging activity of P. clarkii on a human body and highlight the potential postmortem artefacts caused by this species. This is the first report on a real case of postmortem injuries produced by P. clarkii crayfishes on a submerged human body. So far, crustaceans are not considered useful for the estimation of the minimum postmortem interval. However, the important modifications on the corpses deriving from the activity of these animals should be kept in consideration.     

Use of PCR-RFLP for differentiation of calliphorid larvae (Diptera,Calliphoridae) on human corpses

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.     

Shells and Bones: A Forensic Medicine Study of the Association of Terrestrial Snail Allopeas micra with Buried Human Remains in Brazil

Little is known regarding the scavenger fauna associated with buried human corpses, particularly in clandestine burials. We report the presence of 20 shells of the terrestrial snail Allopeas micra, within hollow bones of human remains buried for 5 years, during the process of collecting DNA material. The fact that a large number of shells of A. micra had been found in the corpse and in the crime scene supports the assumption that there was no attempt to remove the corpse from the area where the crime occurred. Despite this, our observations cannot be used to estimate the postmortem interval because there is no precise knowledge about the development of this species. This is the first record of a terrestrial snail associated with a human corpse and its role in this forensic medicine case.     

First Report of Blaesoxipha plinthopyga (Diptera: Sarcophagidae) from a Human Corpse in the U.S.A. and a New State Geographic Record Based on Specimen Genotype

Carrion flies in the taxonomic family Sarcophagidae are often recovered from a human corpse. However, because such specimens are difficult to identify, the forensic literature on this taxon is quite limited compared with that of the commonly employed Calliphoridae. Faced with a sarcophagid larva that could not be identified microscopically from a death investigation in the state of Idaho, we generated cytochrome oxidase one DNA sequence data from the specimen. Comparison to a reference data set of forensically significant sarcophagids from Canada and the U.S.A. confirmed that this was the first discovery of Blaesoxipha plinthopyga in a human corpse in the U.S.A. and the first record of this species in Idaho. Because B. plinthopyga occurs from the Northern U.S.A. to the Neotropics, it is potentially useful for estimating time since death at many locations.     

Succession pattern of carrion-feeding insects in Paramo, Colombia

The minimum postmortem interval can be estimated based on knowledge of the pattern of insect succession on a corpse. To use this approach requires that we take into account the rates of insect development associated with particular climatological conditions of the region. This study is the first to look at insect succession on decomposing carcasses in the high altitude plains (Paramo) in Colombia, at 3035 m above sea level. Five stages of decomposition were designated with indicator species identified for each stage: Callíphora nigribasis at the fresh stage; Compsomyiops verena at the bloated stage; Compsomyiops boliviana during active decay; Stearibia nigriceps and Hydrotaea sp. during advanced decay and Leptocera sp. for dry remains. A succession table is presented for carrion-associated species of the region, which can be used for estimating time since death in similar areas. Compsomyiops boliviana is reported for the first time in Colombia.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.     

Towards a "crime pollen calendar" - pollen analysis on corpses throughout one year

A palynological study was carried out on 28 corpses brought in one year (June 2003-May 2004) to the morgue of the Institute of Legal Medicine of Parma (Northern Italy). This preliminary research focuses on the date of death, which was known for all corpses examined. Pollen sampling and analyses were made with the first aim of comparing the pollen grains found on corpses with those diffused in the atmosphere in the region in the same season as the known date of death. Eyebrows, hair-line near the forehead, facial skin and nasal cavities were sampled. Most of the corpses had trapped pollen grains, with the exception of two December corpses. All pollen grains were found with cytoplasm and in a good state of preservation. In this way, a series of reference data was collected for the area where the deaths occurred, and we examined whether pollen grains on corpses could be an index of the season of death. To verify this hypothesis, the pollen analyses were compared with data reported in the airborne pollen calendars of Parma and the region around. Pollen calendars record pollen types and their concentrations in the air, month by month. The quantity of pollen recorded on corpses did not prove to be directly related to the quantity of pollen in the air. But qualitatively, many pollen types which are seasonal markers were found on corpses. Main corpse/air discrepancies were also observed due to the great influence that the local environmental conditions of the death scene have in determining the pollen trapped by a corpse. Qualitative plus quantitative pollen data from corpses appeared helpful in indicating the season of death. A preliminary sketch of a "crime pollen calendar" in a synthetic graphic form was made by grouping the corpse pollen records into three main seasons: A, winter/spring; B, spring/summer; C, summer/autumn. Trends match the general seasonal trend of pollen types in the air.     

Practice of forensic entomology--usability of insect fragments in the estimation of the time of death

Forensic entomology analyzes and evaluates the presence of necrophagous or necrophilic insects on human corpses to estimate the post-mortem interval. The three cases presented are good examples illustrating that not only the various stages of development of the necrophagous insects from egg via larva to pupa are useful for forensic assessment. Depending on the situation in the individual case, even inconspicuous remains of past insect activity such as empty pupal cases or larval skins may supply important entomological information. For this purpose it is indispensable to preserve such evidence, which may appear only as fragments or pieces at first sight, for proper entomological assessment.     

Soil Fungi: Their Potential use as a Forensic Tool

As a grave is an anomalous environment and differs from its surroundings, criminal investigators employ different techniques for locating, recovering, and analyzing clandestine graves. In this study were identified the fungi found in the soil under corpses in decomposition with an aim at relating the copresence of human remains and different fungal species. Were isolated the fungi in three ways: soil washing, serial dilutions, and moist chamber growth. Dichotomomyces cejpii, Talaromyces trachyspermus, Talaromyces flavus, and Talaromyces sp. were the representative species found—with those belonging to the ammonia group, whose fungi are the first in the succession of cadaver decomposition directly in the ground. The mycobiota found at the present study area clearly differs to mycobiota identified in control sample and from previously described species for other areas of Buenos Aires Province, Argentina. Further forensic examples of this type are needed to develop fully the detailed use of mycology as a forensic tool.     

DNA typing for identification of some species of Calliphoridae. An interest in forensic entomology.

To determine precisely post mortem interval, larvae and puparium species found on a corpse have to be identified. Among more than 200 cases examined at the entomology department of the Institut de Recherche Criminelle de la Gendarmerie Nationale, two-thirds concerned corpses less than one month old. Therefore, insects from first and second screwworms are the most frequently found [1]. Some species commonly found in France, such as different Lucilia and Calliphora vicina Robineau-Desvoidy, are easily identifiable at an adult stage, but are almost impossible to differentiate at immature stages when only fragments of puparium or necrosed first instar larvae are available. For this reason, an easy and objective method of identification was thus searched by genetic analysis of these insects. Sequencing of partial gene of sub unit I of cytochrome oxydase has been used to predict restriction sites. Restriction enzyme cleavage of PCR products with Dde I allowed us to differentiate these species.     

Medical forensic investigations of body fragments in the situations of the massive arrivals of corpses

We propose for the first time a set of methods for the determination of sex, face shape, body length and weight for the purpose of forensic medical investigations of fragmented corpses from a mass fatality catastrophe site. The study has demonstrated a high degree of reliability of the assessment of the personality group signs based on a wealth of somatometric data with the use of the multivariate statistic methods. This work has no analogy in forensic medical studies.     

Experimental validation of forensic evidence: a study of the decomposition of buried pigs in a heavy clay soil.

In a murder investigation, where the victim had been strangled and buried in a shallow grave, there were discrepancies between the post mortem interval (PMI) as estimated from entomological studies and estimations determined from other evidence. This inconsistency provided the impetus for examining the decay process using pig carcasses as analogues for the human cadaver. The pigs were buried in the immediate vicinity of the original burial site in December 1996, which was the month when the victim was purported to have been interred in the previous year. The buried pigs were then monitored for 5 months which, based on the evidence other than the entomological, was the period over which the corpse was thought to have lain in the ground. The pig corpses were disturbed by scavengers in mid April: this was the same time that the human corpse was discovered in the previous year by scavengers. Insects played no role in the decomposition process until the pig carcasses had been exposed by animals. Blowflies, notably Calliphora vomitoria, were attracted to the exposed tissues and laid eggs from which larvae developed. Calliphora vomitoria is a species often used to estimate PMI. This investigation has shown that soil conditions and low seasonal temperatures had preserved the pig carcasses for longer than might be expected. Using the blowfly larvae to estimate PMI would have produced erroneous results had not the burial environment and exhumation history been investigated.     

Significance of skin metallization in the diagnosis of electrocution

The differentiation of minute current marks from the faint traces of burns may offer some difficulties. Marcinkowski and Wojciechowski (1973) treated the skin of corpses with the same metal objects either heated to a high temperature or exposed to 250 V of alternating current, and determined (by an electrographic method) that metallization appeared only after applying the electric current.The continuation of these observations is linked with the actual experimental studies of Pankowski. By treating the skin of corpses with alternating and direct current of 10, 50, 100 and 250 V for 0.3 sec, 1 sec, 30 sec and 1 min passing through a radioactive electrode of ⁶⁰Co, he has shown that the radioactivity of the skin at the site of electrode contact increases with the elevation of the voltage and its duration. In the case of direct current the rise was 630 – 54000-fold at the site of the positive electrode.Using electrodes of copper, aluminium and iron (not radioactive) it has also been shown (by an electrographic method) that metallization intensifies under the same conditions of time and voltage.Metallization could be detected even when no current marks on the skin were evident. Electrography appears to be extremely useful in the detection of metallization. No metallization was detected at the site of the negative electrode (as refers to direct current).