Jens Amendt

Best practice in forensic entomology—standards and guidelines

Forensic entomology, the use of insects and other arthropods in forensic investigations, is becoming increasingly more important in such investigations. To ensure its optimal use by a diverse group of professionals including pathologists, entomologists and police officers, a common frame of guidelines and standards is essential. Therefore, the European Association for Forensic Entomology has developed a protocol document for best practice in forensic entomology, which includes an overview of equipment used for collection of entomological evidence and a detailed description of the methods applied. Together with the definitions of key terms and a short introduction to the most important methods for the estimation of the minimum postmortem interval, the present paper aims to encourage a high level of competency in the field of forensic entomology.

Forensic entomology: applications and limitations.

Forensic entomology is the science of collecting and analysing insect evidence to aid in forensic investigations. Its main application is in the determination of the minimum time since death in cases of suspicious death, either by estimating the age of the oldest necrophagous insects that developed on the corpse, or by analysing the insect species composition on the corpse. In addition, toxicological and molecular examinations of these insects may help reveal the cause of death or even the identity of a victim, by associating a larva with its last meal, for example, in cases where insect evidence is left at a scene after human remains have been deliberately removed. Some fly species can develop not only on corpses but on living bodies too, causing myiasis. Analysis of larvae in such cases can demonstrate the period of neglect of humans or animals. Without the appropriate professional collection of insect evidence, an accurate and convincing presentation of such evidence in court will be hampered or even impossible. The present paper describes the principles and methods of forensic entomology and the optimal techniques for collecting insect evidence.

Genetic identification of forensically important flesh flies (Diptera: Sarcophagidae)

Unequivocal identification of fly specimens is an essential requirement in forensic entomology. However, not all species can be determined at every developmental stage, which is illustrated by the flesh flies (Diptera: Sarcophagidae), important members of the necrophagous insect fauna. Up to now no suitable key for the identification of the immature stages of this family of flies exists. DNA analysis of selected mitochondrial genes was applied to solve this problem. Sequence data of selected regions of the CO I and ND 5 genes of the most important European flesh fly taxa associated with cadavers are presented, which can act as reference standards for species determination.

The use of COI barcodes for molecular identification of forensically important fly species in Germany

Deoxyribonucleic acid (DNA)-based insect identification has become a routine and accurate tool in forensic entomology. In the present study, we demonstrate the utility of the mitochondrial DNA cytochrome oxidase I gene “barcoding region” as a universal marker for molecular identification of forensically important Diptera. We analyzed 111 specimens belonging to 13 species originating from Frankfurt am Main, Germany (Calliphoridae: Calliphora vicina, Calliphora vomitoria, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Lucilia sericata, Lucilia silvarum, Phormia regina, Protophormia terraenovae; Piophilidae: Parapiophila vulgaris; Muscidae: Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis). Intraspecific variation ranged from 0 to 1.17% and interspecific variation occurred between 1.17% and 15.21%. Although differences within species were generally less than among species, divergence percentages overlapped due to low interspecific nucleotide divergence of the recently separated sister species L. caesar and L. illustris. However, all species formed distinct monophyletic clades and thus the cytochrome oxidase 1 (COI) barcode has been shown suitable for clear differentiation and identification of forensically relevant Diptera in Germany.

Molecular identification of carrion-breeding scuttle flies (Diptera: Phoridae) using COI barcodes

Entomological evidence is often used in forensic cases for post-mortem interval (PMI) calculation. The most dominant species present on a corpse are typically blowflies. However, several cases have been reported where access to a corpse has been restricted for blowflies (e.g., on a buried or wrapped cadavers) but species of the family Phoridae were abundant. It has also been reported that some phorid species that exploit human corpses may also feature in cases of myiasis acquired ante-mortem. In all these cases, they may provide decisive evidence. As for blowflies, the precise identification of a phorid species collected from a corpse is necessary when estimating the PMI. Since morphological determination is often hampered due to similar characteristics especially in the larval and pupal stage, we used DNA-based methods to identify six phorid species (Megaselia scalaris, Megaselia giraudii, Megaselia abdita, Megaselia rufipes, Conicera tibialis, and Puliciphora borinquenensis) on the molecular level. We focused on a 658-bp-long region of the cytochrome oxidase I gene (COI), the most common molecular marker in forensic entomology. The amplified fragment is also used in DNA barcode approaches and was found to be suitable for identification of a wide range of insect taxa. The present study demonstrates that this region is also sufficient to distinguish between several species of scuttle flies.

Differential gene expression during metamorphosis: a promising approach for age estimation of forensically important Calliphora vicina pupae (Diptera: Calliphoridae)

Necrophagous blow fly larvae can provide accurate estimates of the minimum postmortem interval in death investigations. During larval development, predictable morphological changes occur and measurements of weight, length, and width are compared to species-specific growth curves for reliable age estimates. However, aging blow fly pupae is more challenging because morphological and anatomical changes are not visible with the naked eye. Thus, delicate preparation of the pupae or rearing to the adult stage seems unavoidable. Conversely, metamorphosis evokes a remodelling of the larval shape to adult structures, and gene expression analysis potentially serves as a molecular tool to mirror the ageing process of a pupa. The present study focuses on the differential expression of two newly described, arbitrarily named genes (15_2, 2014192) and two previously identified genes (actin, arylphorin receptor) during Calliphora vicina (Diptera: Calliphoridae) metamorphosis. Quantification through real-time PCR revealed significant up- and downregulation of these transcripts found to be temperature dependent and age specific, hence, a new possibility to age forensically important blow fly pupae.

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.

Advances in Entomological Methods for Death Time Estimation

The development of entomological methods for estimating the time of death has been rapid in the last decade, and new methods are on the horizon. These developments are reviewed with specific reference to experimental design, established and new techniques, and mathematical modelling for forensic retrodiction. The techniques include the use of electron microscopy, magnetic resonance imaging, computed tomography, cuticular hydrocarbon profiles, and real-time PCR to estimate the age of immature insects found on corpses, based on their stage of development. Near-infrared spectrograph and pteridine fluorescence techniques can be applied to this task on adult insects. The use of ecological succession in the carrion insect community is also introduced briefly. Finally, the creation and uses of standard techniques in forensic entomology is discussed. We recommend that two steps in this standardisation process are that physiological and ecological studies should be reported in physiological time wherever this is appropriate, and that the type of post-mortem interval being estimated should be stipulated more explicitly than is currently common.

De novo transcriptome analysis and highly sensitive digital gene expression profiling of Calliphora vicina (Diptera: Calliphoridae) pupae using MACE (Massive Analysis of cDNA Ends)

Determining a post-mortem interval using the weight or length of blow fly larvae to calculate the insects age is well established. However, to date, there are only a handful studies dealing with age estimation of blow fly pupae, in which weight or length cannot be used as a relevant parameter. The analysis of genetic markers, which indicate a certain developmental stage, can extend the period for a successful post-mortem interval determination. In order to break new ground in the field of age determination of forensic relevant blow flies, we performed a de novo transcriptome analysis of Calliphora vicina pupae at 15 different developmental stages. Obtained data serve as base to establish molecular age determination techniques. We used a new, deeper, and more cost-effective digital gene expression profiling method called MACE (Massive Analysis of cDNA Ends). We generated 15 libraries out of 15 developmental stages, with 3-8 million reads per library. In total, 53,539 distinct transcripts were detected, and 7548 were annotated to known insect genes. The analysis provides high-resolution gene expression profiles of all covered transcripts, which were used to identify differentially expressed genetic markers as candidates for a molecular age estimation of C. vicina pupae. Moreover, the analysis allows insights into gene activity of pupal development and the relationship between different genes interesting for insect development in general.

Prevalences of tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in Ixodes ricinus populations of the Rhine-Main region, Germany.

Tick-borne encephalitis (TBE) and Lyme borreliosis are the most common tick-borne zooanthroponoses in Germany. The federal risk map for TBE in this country is based on recorded cases of human infection, whereas information on the vector-based prevalence of either pathogen is fragmentary. In this study, a total of 12,497 host-seeking nymphal and adult Ixodes ricinus ticks (Acari: Ixodidae) were collected from March to October 2009 and April to June 2010, in 5 TBE non-risk and 4 TBE risk areas of the Rhine-Main region (Hesse) via flagging. A total of 3615 ticks was examined for infection with Borrelia burgdorferi sensu lato and 9115 ticks were analyzed for TBE virus (TBEV). Pathogens were detected by real-time polymerase chain reaction. Among 3615 questing ticks, 344 (9.5%) were found infected with B. burgdorferi sensu lato. Five Borrelia genospecies were identified by sequencing the OspA gene: B. afzelii (81.3%), B. garinii (14.0%), B. valaisiana (2.7%), B. spielmanii (1.3%), and B. bavariensis (0.7%). TBE infection of ticks differed between areas classified as TBE risk and TBE non-risk areas. While the prevalence of TBEV was between 0 and 0.2% (3 of 3947 ticks) in the TBE risk areas, no TBEV-infected tick was detected from TBE non-risk areas. The results show that B. burgdorferi sensu lato occurred in all 9 examined locations, indicating that Lyme borreliosis is prevalent in the Rhine-Main region, whereas TBEV was detected only in previously classified risk areas.