How to prove neglect in the context of the post-mortem examination.

Understanding the causes, extent, and period of neglect is not only a medical but also a forensic task when it comes to legal investigations. In this study, we evaluated 46 autopsied cases where there was clear evidence of physical neglect during the last period of the deceased's life. The age of the deceased ranged from 21 to 96 years; most of them were female (71.7%). The majority of cases (89.9%) took place in a domestic environment, with partners or relatives providing care. The most frequent post-mortem findings were pressure sores, followed by inflammatory skin changes, and signs of malnutrition and dehydration. Neglect was the cause or co-cause of death in 23% of the cases. More than half of the deceased showed severe contamination of the skin surface by excrement, and in almost 40% of the cases, fly infestation was found. The majority of insects belonged to the group of house flies (Diptera: Muscidae), mainly the common house fly, Musca domestica. By analyzing the entomological evidence, it was possible to prove an insect infestation period of at least several days ante-mortem. Since the period of neglect may be relevant in terms of legal proceedings, the present work demonstrates the particular importance of insect traces in providing this evidence. While prosecution and conviction of caregivers remain challenging, it is all the more essential that entomology and legal medicine collaborate on the analysis of findings of neglect.

Morphological changes and protein degradation during the decomposition process of pig cadavers placed outdoors or in tents-a pilot study.

The delimitation of the postmortem interval (PMI) is of utmost importance in forensic science. It is especially difficult to determine the PMI in advanced decomposition stages and/or when dead bodies are found under uncommon circumstances, such as tents, or other (semi-) enclosed environments. In such cases, especially when insect access is restricted, morphological assessment of body decomposition is one of the remaining approaches for delimitation of the PMI. However, as this method allows only vague statements/indications about the PMI, it is required to develop new and more reliable methods. One of the most important candidates is the biochemical analysis of protein degradation. In this regard, it has been demonstrated that specific skeletal muscle protein degradation patterns characterize certain time points postmortem and thus can be used as markers for PMI estimation. In order to test this method in different micro-environments, a pilot study using ten pig carcasses was conducted in summer in Northern Germany. The cadavers were openly placed outside (freely accessible for insects), as well as enclosed in tents nearby, and left to decompose to investigate decomposition processes over a time course of 10 days. Muscle samples of the M. biceps femoris were collected on a regular basis and processed via SDS-PAGE and degradation patterns of selected proteins identified by Western blotting. In addition, morphological changes of the cadavers during decomposition were assessed using the total body score (TBS). Results showed that postmortem protein degradation patterns are largely consistent between treatment groups (open field versus tents) despite major morphological differences in the decomposition rate. This field study provides evidence that muscle protein degradation is mostly unaffected by different levels of exposure, making it a sufficient candidate for PMI delimitation under various circumstances.

Age-dependent gene expression of Calliphora vicina pupae (Diptera: Calliphoridae) at constant and fluctuating temperatures.

Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5-15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data.

How should living entomological samples be stored?

Sampling and storing insect evidence alive are important tasks in forensic entomology as it can impact survival and growth rates. To investigate the effect of cooling and storing of insect evidence before its arrival in the laboratory, samples of all three larval stages of the blow fly species Lucilia sericata and Calliphora vicina were analyzed. A first group was stored at room temperature and a second one in a refrigerator (~ 5 °C) for 16 h, all without air, supply of food, and sawdust. Afterwards, they were kept at 6-8 °C in a Styrofoam box for 8 h, simulating a transport situation. Mortality rate (MR) was calculated and 25% of the surviving larvae were killed and measured to check for interim growth. The remaining alive specimens were reared at 25 °C until adult's eclosion for estimating a possible storage impact on survival during later development. The results were then compared with a control which was not temporarily stored and chilled but left feeding in boxes with an air-permeable lid on food substrate at 25 °C.A 24-h temporary storage stopped the larval growth in comparison with the control especially in early larval stages in both species. A high MR of up to 100% for third instar (L3) larvae stored both at room temperature and in a cold environment without air supply was found. Oxygen supply can reduce significantly the MR at least for L3 larvae of L. sericata. Findings provide scientific evidence for the recommendation to store larval samples at cold temperatures with both oxygen and food supply. The high MR for samples of the last larval stage clearly shows the need for a fast delivery after sampling and a more sophisticated storage procedure like, e.g., providing air supply. Storing live samples at room temperature without air access should be avoided.

Of pigs and men-comparing the development of Calliphora vicina (Diptera: Calliphoridae) on human and porcine tissue.

Over the last decades, studies on juvenile development of forensically important Diptera were performed by using non-human tissues (e.g., beef liver) as a nutrition medium. Such developmental data are used as a reference in determining the age of juvenile necrophagous insects sampled from a human body and thereby to estimate the minimum postmortem interval (PMImin). Despite the acceptance of these studies in the forensic community, some might ask whether such data appropriately reflect the growth of blow flies on human tissue. We, therefore, studied larval growth rates and development times of Calliphora vicina (Diptera: Calliphoridae) on human muscle tissue as well as on pork loin, pork liver, and minced pork at 25 °C. Larval growth rates were significantly (p < 0.001) slower on pork loin and pork liver compared to human muscle tissue. Nonetheless, the time at which the examined developmental landmarks "post-feeding larvae," "pupae," and "adult fly" were reached was similar for all tissues, with significant delays in first fly eclosion only for specimens reared on pork loin (p = 0.027) and pork liver (p = 0.036). Our results highlight the fact that not all porcine tissues are similarly suitable for producing sound growth data for necrophagous Diptera. At present, we recommend the use of minced pork as a non-human nutrition medium, since our results show no developmental differences on this diet compared to human tissue.

Identification of medically and forensically relevant flies using a decision treelearning method.

Blow flies, flesh flies, and house flies can provide excellent evidence for forensic entomologists and are also essential to the fields of public health, medicine, and animal health. In all questions, the correct identification of fly species is an important initial step. The usual methods based on morphology or even molecular approaches can reach their limits here, especially when dealing with larger numbers of specimens. Since machine learning already plays a major role in many areas of daily life, such as education, business, industry, science, and medicine, applications for the classification of insects have been reported. Here, we applied the decision tree method with wing morphometric data to construct a model for discriminating flies of three families [Calliphoridae, Sarcophagidae, Muscidae] and seven species [Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Chrysomya (Ceylonomyia) nigripes Aubertin, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), Musca domestica Linneaus, and Parasarcophaga (Liosarcophaga) dux Thomson]. One hundred percent overall accuracy was obtained at a family level, followed by 83.33% at a species level. The results of this study suggest that non-experts might utilize this identification tool. However, more species and also samples per specimens should be studied to create a model that can be applied to the different fly species in Thailand.

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.

Evaluation of blowfly larvae (Diptera: Calliphoridae) as possible reservoirs and mechanical vectors of African swine fever virus.

In 2014, highly virulent African swine fever virus (ASFV) was introduced into the Baltic States and Poland, with new cases being reported almost every week from wild boar and also from domestic pigs. Contrary to initial predictions that the disease would either die out due to the high virulence of the virus strain or spread rapidly in westerly direction, the infection became endemic and spread slowly. The unexpected disease epidemiology led to the hypothesis that hitherto unconsidered factors might contribute to virus persistence and dispersal. To check whether arthropod species feeding and developing on infected carcasses might be involved, larvae of two commonly found blowfly species, Lucilia sericata and Calliphora vicina, were experimentally bred on ASFV-infected spleen tissue. After different time intervals, developing larvae and pupae were tested for infectious virus and viral DNA. By qPCR, contamination of the blowfly larvae and pupae with ASFV-DNA could be demonstrated even after several washing steps, proving the uptake of virus during feeding in the larval stage. However, infectious virus could never be isolated. By contrast, the larvae appeared to have inactivated ASFV in the offered tissue, which might be explained by the known anti-biotic effect of salivary secretions. It is concluded that immature blowfly stages do not play a relevant role as reservoirs or mechanical vectors of ASFV.

The importance of Saprinus semistriatus (Coleoptera: Histeridae) for estimating the minimum post-mortem interval.

In forensic science, the use of entomological evidence to estimate the minimum post-mortem interval can be crucial. However, not all cadaver-visiting insects are equally useful. Our focus is on the histerid beetle Saprinus semistriatus (Scriba 1790) (Histeridae; Coleoptera). Histeridae are common predators that feed mainly on dipteran larvae on carrion and dung. We review 23 publications mentioning this species and provide new experimental data on its temporal pattern beneath and on hanging pig cadavers. In a field experiment near Neuchâtel, Switzerland, we recorded the abundance of S. semistriatus on ten decomposing pig cadavers (Sus scrofa) over a 32-day period in summer 2013 (July, 01- August; 02). Five cadavers were placed on the ground and five cadavers were hung one metre above the ground. Insects were collected from pitfall traps and by manual sampling. The abundance of S. semistriatus was significantly higher during the active stage than during the fresh and the bloated stages of decomposition in both, ground and hanging pigs. However, S. semistriatus was more abundant on the ground than on the hanging cadavers. The literature and our new data show that S. semistriatus is present on cadavers during a relatively short period of time (approximately two weeks), mainly during the active decay stage, but it may also occasionally occur in the bloated stage. Identifying key indicators such as S. semistriatus can help optimise forensic research by focusing on the most informative taxa. A few key indicators for each decomposition stage may constitute an optimal toolbox for forensic entomologists.

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 insect's 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.

Forensic entomology in Germany.

Forensic entomology (FE) is increasingly gaining international recognition. In Germany, however, the development of FE has been stagnating, mainly because of the lack of cooperation between police, forensic medicine and entomology. In 1997 a co-operative research project 'Forensic Entomology' was started in Frankfurt/Main at the Center of Legal Medicine and the Research Institute Senckenberg. The aim of this project is to establish FE in Germany as a firmly integrated component of the securing of evidence from human cadavers in cases of suspected homicide. For this purpose we developed a forensic insect collecting kit, and policemen are educated for greater acceptance and better application of FE. The scientific programme focuses on the investigation of the insect succession on cadavers in urban and rural habitats. This also includes new indicator groups (e.g. parasitic wasps) for a more precise calculation of the late post mortem interval. Recently a DNA-based reliable and fast identification method especially for the immature stages of necrophagous insects became part of the project. Preliminary results are reported and two case studies presented.

The history of forensic entomology in German-speaking countries.

Megnin's book "La fauna des cadaveres" published in 1894 in France is generally accepted as a mile-stone in forensic entomology. It is hardly known that at the same time this topic was likewise explored in the German-speaking countries. Even PMI estimation based on developmental data of blowflies was performed. After a more descriptive period in the first half of the 20th century the complexity and variability of insects' biological behavior were detected and formally investigated. Improved technical facilities, enhanced comprehension of scientific studies and multidisciplinary cooperation, enabled rapid progress in forensic entomology during the last decades. With the European Association for Forensic Entomology founded in 2002 the frame work for a high standard of competency at an international level was constituted.

Therapeutic compliments: setting the stage for successful therapy.

Compliments are often viewed primarily as a linear event in which one person expresses approval or admiration of another. Far less attention has been given to the circular nature of compliments and the manner in which they enhance the positions of both the giver and the receiver of the compliment. Therapeutic compliments have proven to be highly effective means of motivating clients, while at the same time increasing therapeutic leverage. This article proposes that compliments should be purposefully given, and that the type of compliment should vary with the stage of therapy and the intended response of the client to the compliment.

THE MYTH OF THE UNFEELING STRATEGIC THERAPIST.

This paper explores what the authors consider to be a widespread myth: that strategic therapists ignore, avoid, or neglect client feelings in treatment. This myth is promulgated by trainers' admonitions and strategic theorists' injunctions against dealing with client affect. It is also cultivated by omission of this topic in the strategic literature. The myth is destructive in that it misrepresents what strategic practitioners actually do in a therapy session. Seven elements of the myth are delineated and the corresponding fallacies are illustrated.

[Practice of forensic entomology. Part 1: Insect collection at the site of discovery of the corpse]. / Praxis der forensischen Insektenkunde. Teil 1: Insektenaufsammlung am Fundort einer Leiche.

The life cycle of a fly illustrates that the late immature stages of necrophagous insects frequently leave the body, and are therefore not found on the corpse itself. Taking into account these stages (i.e. pupae) while investigating a death scene is very important. Two case histories demonstrate the effects of not taking that fact into consideration when determining the post-mortem interval. Since there is no standardized method in Germany for collecting insects at a death scene, an entomological collection kit with a short instruction is introduced.

The nocturnal oviposition behaviour of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications.

Numerous factors may cause delayed colonisation of a corpse by blowflies, leading to a discrepancy between the entomologically determined post-mortem interval (PMI) and the time of death. Blowflies, for example, are considered to be inactive at night, however, published observations are contradictory. In the present study, several field experiments and one type of indoor experiment were conducted in summer of 2004 and 2005 in order to investigate the nocturnal ovipositional behaviour of blowflies. In the field, two types of bait, dead hedgehogs and fresh beef liver, were placed at night in different urban and rural locations in Frankfurt and in Munich, Germany. For the indoor-experiments beef liver was placed in small plastic boxes containing caged Lucilia sericata females in the evening and left overnight. At night, no ovipositon was observed in the field (n=51, T=10-24 degrees C). Nocturnal oviposition in complete darkness occurred in the plastic boxes in two of six cases (T=25 degrees C). Considering the behavioural and physiological characteristics of flies we suggest that nocturnal oviposition of blowflies appears to be unlikely under natural conditions in Central Europe but may occur under certain circumstances, such as unusual high nightly temperatures and the presence of gravid flies with an appropriate arousal threshold.