Richard Zehner

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.

RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application

Abstract To identify common animal species by analysis of the cytochrome b gene a method has been developed to obtain PCR products of a large domain of the cytochrome b gene (981 bp out of 1140 bp) in humans, selected mammals and birds using the same specifically designed primers. Species-specific RFLP patterns are generated by co-restriction with the restriction endonucleases ALU I and NCO I. The RFLP patterns obtained are conclusive even in mixtures of two or more species. The results were confirmed by sequence analysis which in addition explained intraspecies variations in the RFLP patterns. The method has been applied to forensic casework studies where the origin of roasted meat, stomach contents and a bone sample has been successfully identified.

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.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen.

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

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.

Molecular studies on the ouabain binding site of the Na ,K -ATPase in milkweed butterflies

Summary. The Na+, K+-ATPase of the Monarch butterfly (Danaus plexippus) is insensitive to the inhibition by cardiac glycosides due to an amino acid replacement: histidine instead of asparagine at position 122 of the α-subunit representing the ouabain binding site. By PCR amplification of the DNA sequence of this site, a PCR product of 270 bp was obtained from DNA extracted from Danainae species (Danaus plexippus, D. chrysippus, D. gillipus, D. philene, D. genutia, Tirumala hamata, Euploea spp., Parantica weiskei, P. melusine), Sphingidae (Daphnis nerii) and mimics of milkweed butterflies (Hypolimnas missipus, Limenitis archippus and L. arthemis, Nymphalidae). Analysis of the nucleotide sequences revealed that the single point mutation in the ouabain binding domain (AAC-Asn for CAC-His) was present only in Danaus plexippus, but not in the other species investigated. Since these milkweed butterflies also store cardenolides, other structural modifications of the Na+, K+-ATPase may have occurred or other strategies of cardenolide tolerance have been developed.