Cuticular hydrocarbons for the identification and geographic assignment of empty puparia of forensically important flies.

Research in social insects has shown that hydrocarbons on their cuticle are species-specific. This has also been proven for Diptera and is a promising tool for identifying important fly taxa in Forensic Entomology. Sometimes the empty puparia, in which the metamorphosis to the adult fly has taken place, can be the most useful entomological evidence at the crime scene. However, so far, they are used with little profit in criminal investigations due to the difficulties of reliably discriminate among different species. We analysed the CHC chemical profiles of empty puparia from seven forensically important blow flies Calliphora vicina, Chrysomya albiceps, Lucilia caesar, Lucilia sericata, Lucilia silvarum, Protophormia terraenovae, Phormia regina and the flesh fly Sarcophaga caerulescens. The aim was to use their profiles for identification but also investigate geographical differences by comparing profiles of the same species (here: C. vicina and L. sericata) from different regions. The cuticular hydrocarbons were extracted with hexane and analysed using gas chromatography-mass spectrometry. Our results reveal distinguishing differences within the cuticular hydrocarbon profiles allowing for identification of all analysed species. There were also differences shown in the profiles of C. vicina from Germany, Spain, Norway and England, indicating that geographical locations can be determined from this chemical analysis. Differences in L. sericata, sampled from England and two locations in Germany, were less pronounced, but there was even some indication that it may be possible to distinguish populations within Germany that are about 70 km apart from one another.

Time Flies-Age Grading of Adult Flies for the Estimation of the Post-Mortem Interval.

The estimation of the minimum time since death is one of the main applications of forensic entomology. This can be done by calculating the age of the immature stage of necrophagous flies developing on the corpse, which is confined to approximately 2-4 weeks, depending on temperature and species of the first colonizing wave of flies. Adding the age of the adult flies developed on the dead body could extend this time frame up to several weeks when the body is in a building or closed premise. However, the techniques for accurately estimating the age of adult flies are still in their beginning stages or not sufficiently validated. Here we review the current state of the art of analysing the aging of flies by evaluating the ovarian development, the amount of pteridine in the eyes, the degree of wing damage, the modification of their cuticular hydrocarbon patterns, and the increasing number of growth layers in the cuticula. New approaches, including the use of age specific molecular profiles based on the levels of gene and protein expression and the application of near infrared spectroscopy, are introduced, and the forensic relevance of these methods is discussed.

Mites (Acari) as a Relevant Tool in Trace Evidence and Postmortem Analyses of Buried Corpses.

This report interprets the presence of mite species in three clandestine graves in Europe, evaluating their potential use as trace evidence or markers. Grave 1 (Sweden): Two mite species Rhizoglyphus robini Claparède, 1869 and Parasitus loricatus (Wankel, 1861) were recovered from the surface of a body buried in a shallow grave in an area surrounded by trees, in close vicinity to house gardens. Grave 2 (Germany): Phoretic deutonymphs of Gamasodes spiniger (Trägårdh, 1910) were attached to an adult fly (Diptera: Sphaeroceridae) found within a shallow grave containing two human bodies covered in soil and dung. Grave 3 (France): P. loricatus were recovered from the soil around a body buried in a deep grave (80 cm under). In graves 1 and 3 both corpses were undergoing advanced decay and skeletization, the locations match with the subterranean habit of P. loricatus, highlighting the value of this species as a marker of graves or burials in soil and during late decomposition. R. robini is a soil mite that feeds on decayed roots and bulbs; this mite species confirms the location of the corpse within top soil, agreeing with a more specific type of superficial burial, a shallow grave. In case 2, the presence of both coprophiles, the mite G. spiniger and the carrier fly confirm association of remains with dung or animal feces. The three mite species are reported for the first time in human graves. There are no previous records of R. robini from Sweden.

An empirical comparison of decomposition and fly colonisation of concealed carcasses in the Old and New World.

The level of exposure or concealment of a cadaver is known to have an important impact on insect colonisation and decomposition but has been the subject of few investigations. In the present study, 30 pig carcasses were stored in three different types of containers (suitcases, trashcans and drums) with different levels of access for necrophagous insects at two different geographic locations for 100 days. The decomposition proceeded in a similar way in both geographic locations in all three types of container. Both in trashcans and suitcases, the decomposition process was characterised by bones and greasy, brown decomposition fluids left in the containers and an overall moist decomposition. In contrast, decomposition in the drums was characterised by a long bloating phase followed by a slow transition from bloated to deflation. Tissue and the carcasses as a whole were still present till the end of the experiment. Insect occurrence patterns and species composition on suitcases and trashcans were similar for both countries. Mainly flies and some beetles were present in suitcases and trashcans until day 45, with blow flies (Diptera: Calliphoridae) dominating the fauna. There was no insect colonisation in the drums. Our study contributes to the knowledge about insect accessibility of concealed cadavers and the impact of concealment on the speed and type of decomposition. It clearly shows that the degree of exposure of a cadaver is a key factor in decomposition and insect colonisation, which had a larger effect here than the biogeographical location.

Correction to: Myiasis in humans-a global case report evaluation and literature analysis.

It was brought to the authors' attention that a citation within the published article was cited incorrectly.

Myiasis in humans-a global case report evaluation and literature analysis.

Myiasis refers to the infestation of living humans and vertebrates with fly larvae that feed on necrotic or vital tissue of the host. Since the invasion of new fly species in Europe is currently being observed, which live obligatorily parasitically or are close relatives of such species, the aim of this study is to obtain a global overview of the distribution of myiasis-causing fly species in times of climate change and to assess the possible consequences for Western Europe. A systematic literature search was conducted using Pubmed/Medline for the years 1997 to 2017 and a total of 464 international case reports from 79 countries were evaluated. The described cases were caused by 41 different species. In 99.4% of the cases, it was a colonization by just one species, a maximum of three species were detected in a human. Casuistics from Western Europe mostly describe myiasis as a "holiday souvenir" from tropical regions. Reports of autochthonous cases are rare in comparison to other regions. With regard to rising temperatures and the invasion of new fly species, a noticeable increase in the number of cases in Western Europe is to be assumed, which could be an increasing problem in the clinical area and in care, which is why thorough monitoring for professional identification and treatment seems important in the future.

Species diversity and tissue specific dispersal of necrophagous Diptera on human bodies.

In forensic entomology, many studies analyze fly activity and succession on dead bodies by using pig cadavers and a variety of small baited traps. Data on real human bodies are very rare. To address this shortcoming, we analyzed the fly fauna of 51 human bodies in Germany. Sex, age, place of discovery, and presumed time of death were noted. Larvae were sampled during autopsy according to body region or tissue. For every infested region, the total number of fly larvae were estimated and classified into categories of 1-10, 11-50, and 50+. All samples were identified to the species level. Besides a descriptive analysis of their occurrence patterns, a categorical PCA was performed, and multispecies generalized linear models and a latent variable model were run. Our results highlight the most forensically important blow flies on human bodies in Central Europe (Lucilia sericata, L. ampullacea, Phormia regina, Calliphora vicina); prove, for the first time, the general transferability of species lists based on succession studies on pig cadavers; recommend a certain set of species, such as the so-far neglected L. ampullacea, for future developmental studies; and reveal competitive occurrence of up to six species on the same body as a potential factor of influence. Assignment to a certain body region was often possible and our data clearly indicate certain tissues, e.g. brain, as of high interest for future developmental studies. Focusing on real scenario settings helps direct research to forensically relevant questions and appraises the plausibility of vital laboratory studies.

Same, same but different!-matching entomological traces to a human food source by stable isotope analysis.

Blow flies are important tools in forensic entomology, as they feed and develop on human bodies. This fact can be used to estimate the minimum post mortem interval (PMImin), by classifying the age of the immature stages found on corpses. The capacity to also be able to classify the age of adult flies, or even empty puparia, could increase the timeframe for a PMImin. However, the association of older developmental stages, such as adult flies or their empty cases, with a human cadaver may be challenged, as such specimens could stem from another food source. Analyzing the stable carbon (δ13C) and nitrogen (δ15N) isotopes in such specimens could be helpful here, as the isotope signatures reflect those of the food sources. We compared the δ15N and δ13C signatures of tissue from humans and 12 additional species by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). We did the same for adult flies and empty puparia of the blow fly Lucilia sericata, which were derived from juvenile stages developed on tissues from the mentioned species. The isotope signatures for the adult flies and their empty puparia linearly mirrored the isotope signatures for the respective tissues on which they developed as larvae. Results such as these are useful to indicate the flies' nutritional history, but they do not provide the same kind of evidence that a DNA analysis would. As individual dietary habits, global distribution and environmental effects can influence isotope ratios, we strongly recommend to analyzing entomological traces and muscle tissues of potential donors or for other food sources.

Multispecies blow fly myiasis combined with hypothermia in a man assumed to be dead.

We describe the case of a man who was found with severe hypothermia and advanced myiasis involving five species of blow flies, which eventually led to a transtibial amputation of the man's right leg. A case of such a heavy and species-rich infestation with fly larvae in an urban environment is extraordinary and has, to our knowledge, never been described so far. Best practice in cases such as this one demands accurate species identification not only to ensure appropriate treatment and pest management but also, from a forensic point of view, to explore the possibility of third-party responsibility. The cooperation between physicians and forensic entomologists is highlighted.

Estimating the age of the adult stages of the blow flies Lucilia sericata and Calliphora vicina (Diptera: Calliphoridae) by means of the cuticular hydrocarbon n-pentacosane.

Age estimation of insects like blow flies plays an important role in forensic entomology and can answer questions in regard to time of death. So far the focus is on the immature stages of these insects, but recently the adult fly became a target of interest. It has been established that the profile of specific cuticular hydrocarbons (CHCs) changes in a consistent pattern as adult insects age; thus, their analysis could be a promising tool for the age estimation of adult insects. We investigated the CHC n-pentacosane (nC25) on the legs of the adult blow flies Lucilia sericata and Calliphora vicina with gas chromatography-mass spectrometry. The flies were kept at room temperature (17°C±2°C) and 12:12 L:D from Day 1 to Day 20 post-emergence. For each of five flies per species, the amount of nC25 on all legs was determined daily. The amounts of nC25 on C. vicina increased linearly (R2=0.949). No significant difference between sexes could be detected. While L. sericata showed the same linear increase in general, we found significant (p<0.001) differences in the amount of nC25 between males and females. Although the amounts of nC25 increased linearly for both sexes (males: R2=0.948; females: R2=0.920), female L. sericata produced more nC25 than males. An equation for the prediction of fly age is constructed from these data. Although the influence of various environmental factors, e.g., fluctuating temperatures, still needs to be tested, nC25 seems to be a promising tool for the age estimation of adult flies.

Quantitative pteridine fluorescence analysis: A possible age-grading technique for the adult stages of the blow fly Calliphora vicina (Diptera: Calliphoridae).

Age estimation of adult flies could extend the possible window of time for calculating the minimal postmortem interval (PMImin) by means of entomological methods. Currently, this is done by estimating the time required by necrophagous Diptera to reach certain juvenile developmental landmarks, and the method only works until the end of metamorphosis and emergence of the adult fly. Particularly at indoor crime scenes, being able to estimate the age of trapped adult flies would be an important tool with which to extend the calculable PMI beyond the developmental period. Recently, several promising age-dependent morphological and physiological characteristics of adult insects have been investigated in medical and forensic entomology, but the results are still preliminary and restricted to a few species. We examined adults of the forensically relevant blow fly species Calliphora vicina and investigated the fluorescence levels of pteridine, a group of metabolites that accumulates in the eyes during aging. From Day 1 to Day 25 post-emergence, flies were kept at three different temperature regimes (20°C, 25°C, and fluctuating temperatures in the context of a field study) and 12:12 L:D. From Day 1 until Day 7, the fluorescence of pteridine was determined on a daily basis, and thereafter, every three days. The achieved fly age was multiplied with the relevant temperature and converted into accumulated degree-days (ADD). The fluorescence level of pteridine increased linear with increasing ADD (females: R2=0.777; males: R2=0.802). The difference between sexes was significant (p<0.001). Neither head weight nor temperature had an effect on pteridine fluorescence. Because the variation in pteridine fluorescence increased with increasing ADD, it seems favorable to combine several aging methods for more precise results. In context, we emphasize that different body parts of the same specimen can be used to analyze cuticular hydrocarbons (legs), pteridine fluorescence (head/eyes), and gonotrophic stage (female abdomen).

About the influence of burial on the development of forensically relevant blow fly larvae.

The determination of the minimum post-mortem interval (PMImin) based on the age estimation of necrophagous blowflies is an established forensic technique. Blow fly larvae are able to continue their development to the adult insect on buried corpses. However, it is not clear which factors influence their viability under these circumstances or to what extent. This was investigated in the present paper for Calliphora vicina, one of the most common blowflies in Germany. In respect to forensic casework, an explicit question was if reference values obtained by rearing blowfly larvae under laboratory conditions could be applied to individuals growing in a shallow grave. Under variable ambient temperature conditions, hatched flies exhibited a slightly lower length of the wing veins (0.2 mm) and of the tibia (0.1 mm) than the larvae which had been buried. The period of development, expressed as accumulated degree days (ADD), was 399.8 in buried larvae and 406.1 in larvae not covered with soil as control. Under constant temperature conditions no significant differences in the body length of the larvae and the average time of development (20.3 respectively 20.4 days) was observed. The results of the present study thus suggest that the data published on the development of C. vicina larvae can be used to calculate the PMI of a corpse found in a shallow grave of not more than 20 cm depth.