Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication.

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure.This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results.Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.     

Metrological framework for selecting morphological characters to identify species and estimate developmental maturity of forensically significant insect specimens

Accurate age estimates of immature necrophagous insects associated with a human or animal body can provide evidence of how long the body has been dead. These estimates are based on species-specific details of the insects’ aging processes, and therefore require accurate species identification and developmental stage estimation. Many professionals who produce or use identified organisms as forensic evidence have little training in taxonomy or metrology, and appreciate the availability of formalized principles and standards for biological identification. Taxonomic identifications are usually most readily and economically made using categorical and qualitative morphological characters, but it may be necessary to use less convenient and potentially more ambiguous characters that are continuous and quantitative if two candidate species are closely related, or if identifying developmental stages within a species. Characters should be selected by criteria such as taxonomic specificity and metrological repeatability and relative error. We propose such a hierarchical framework, critique various measurements of immature insects, and suggest some standard approaches to determine the reliability of organismal identifications and measurements in estimating postmortem intervals. Relevant criteria for good characters include high repeatability (including low scope for ambiguity or parallax effects), pronounced discreteness, and small relative error in measurements. These same principles apply to individuation of unique objects in general.

Key points

• Metrological rigour can increase in forensic entomology by selecting measurements based on their metrological qualities.
• Selection of high-quality features for morphological identification of organisms should consider these criteria: (1) pronounced discreteness of features (minimising group overlap or maximizing interval); (2) high repeatability of assessment (such as symmetrical width rather than asymmetrical length); (3) small relative error in measurement (selecting the physically largest continuous rigid feature for measurement).
• These metrological principles also apply to individuation of unique objects in general.

     

Practice of forensic entomology. Part 1: Insect collection at the site of discovery of the corpse

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.     

青岛地区常见嗜尸性蝇种及其季节性分布研究

目的观察青岛地区嗜尸性蝇种季节性演替规律,为死亡时间推断提供参考。方法在室外设立田间观察场所,2012年4月1日开始至2013年4月1日,每月第1天6时放置猪尸,20min后开始观察,至18时,期间每小时观察1次;第3天以后,分别于7时、12时、18时各观察1次。观察时记录尸体上出现的昆虫种类、虫态、数量并采集相应标本,进行分类鉴定。结果可以观察到有嗜尸性蝇类侵袭尸体的月份为4月至11月,共发现3科23种蝇类,其中丝光绿蝇、大头金蝇和亮绿蝇数量位列前三,分别占全部观察数量的40.71%、31.95%和7.12%。丝光绿蝇、大头金蝇中午时段出现的数量显著多于早上和傍晚。一年中各种蝇类在中午出现的数量均多于早上及晚上。4月上旬,蝇类早、晚与中午出现的数量相差显著;而在7、8月份差别较小。大部分嗜尸性蝇种集中在新鲜期和肿胀期。结论本文观察数据可为利用嗜尸性蝇类演替规律进行法医学死亡时间推断,以及建立地区嗜尸性蝇种类数据提供参考。     

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.     

The effect of antifreeze (ethylene glycol) on the survival and the life cycle of two species of necrophagous blowflies (Diptera: Calliphoridae)

Entomotoxicology involves the analysis of the presence and the effects of toxicological substances in necrophagous insects. Results obtained by entomotoxicological studies may assist in the investigation of both the causes and the time of death of humans and animals. Ethylene glycol (EG) is easy to purchase, sweet and extremely toxic. It may be consumed accidentally or purposefully, in an attempt to cause death for suicidal or homicidal intent. Several cases report fatalities of humans and animals. The present study is the first to examine the effects of EG on the survival, developmental rate and morphology of two blowfly species, (Diptera: Calliphoridae) typically found on corpses and carcasses: Lucilia sericata (Meigen) and L. cuprina (Wiedemann). Both species were reared on substrates (beef liver) spiked with three different concentrations of EG that could cause death in either a human or cat: 1/2LD₅₀ (T1), LD₅₀ (T2), 2LD₅₀ (T3), in addition to a control treatment (C) with no EG.Results of this research show that: a) both species are unable to survive if reared on a food substrate spiked with the highest concentration of EG (T3), while lower and medium concentrations (T1, T2) affect, but not prevent, the survival and the completion of the life cycle of such species; b) adults of L. sericata eclose only in C and T1, while adults of L. cuprina in both C, T1, T2; however, c) the developmental time of both species reared in T1 and T2 is statistically slower than the control; d) the body length of the immatures of both of the species reared in T1 and T2 is statistically smaller than the control.     

北京地区尸食性蝇类调查

目的了解北京地区目前尸食性蝇类数目及常见尸食性蝇类的季节分布。方法采用动物尸体或组织诱捕和饲养的方法采集尸食性蝇类标本,对其进行分类保存后进行观察和统计。结果北京地区尸食性蝇类为4科9亚科21属46种,其中有12种是北京新记录种。尸食性蝇类具有区域性分布和季节性分布的特性。结论北京地区尸食性蝇类及常见的尸食性蝇类季节分布数据,可为相关研究提供参考依据。     

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.     

An analytical evaluation of eight on-site oral fluid drug screening devices using laboratory confirmation results from oral fluid

A decaying cadaver emits volatile organic compounds that are used by necrophilous and necrophagous insects in order to find their brood substrate. Although volatile organic compounds (VOCs) that are released by carcasses have been identified, little is known about the specific compounds that are used by these insects while searching for a brood substrate. Therefore, we have investigated the chemical ecology involved in the attraction of the necrophagous hide beetle Dermestes maculatus, which feeds as an adult and larva upon decomposing carcasses. Our aims have been to identify the responsible compounds in the odours of the carcass that are important for the attraction of the beetles. Furthermore, we have studied sex- and age-related differences in beetle attraction and tested whether the hide beetle can distinguish between various stages of decomposition by means of the emitted odours. Headspace collection of volatiles released from piglet carcasses (bloated stage, post-bloating stage, advanced decay and dry remains), coupled gas chromatography-mass spectrometry (GC-MS), gas chromatography with electroantennographic detection (GC-EAD) and bioassays were conducted to identify the volatiles responsible for the attraction of the beetles. Freshly emerged male beetles were attracted by the odour of piglets in the post-bloating stage (9 days after death; T(mean) = 27 °C) and the EAD-active compound benzyl butyrate. Statistical analysis revealed a higher relative proportion of benzyl butyrate in the odour bouquet of the post-bloating stage in comparison with the other stages. We therefore conclude that this compound plays an important role in the attraction of hide beetles to carcass odour. This underlines the potential use of D. maculatus for the estimation of the post mortem interval. The decomposition stage at which the female beetles are attracted to the odour of a cadaver remains unknown, as does the nature of this attraction. Pheromones (sexual or aggregation pheromones) might play an essential role correlated with their attraction to carrion and consequently with their attraction to the substrate for mating and ovipositioning.     

Necrophagous Caterpillars Provide Human mtDNA Evidence*

Abstract: Decomposition of large mammalian carcasses is greatly accelerated through the action of insects. Specialized feeders capable of digesting keratin and collagen found in skin, hair, and tendons and ligaments are attracted to corpses in late stages of dry decomposition and include Tinea pellionella, the casemaking clothes moth, and Tineola bisselliella, the webbing clothes moth (Lepidoptera; Tineidae). Until now, details of the caterpillar behavior as necrophagous insects were vague. Here, we detail the behavior of each species and document the incorporation of human hair into the portable larval shelters constructed by the caterpillars of T. pellionella. Hair of the decedent used as building material for caterpillar shelters provided enough starting template to amplify and sequence the HVI and HVII sections of the control region (mtDNA) of the decedent.     

The succession and development of insects on pig carcasses and their significances in estimating PMI in south China

The succession of insect communities on carrion varies at local and global spatial scales. As such, ecological succession data obtained from corpses at one geographic location cannot necessarily be applied to other locations. Our study describes this succession in the far southern part of China to provide such data for forensic cases in this region. A total of 18 pig carcasses were placed in the field in four seasons, and the timing of the following events were recorded: appearance of larvae, onset of larval wandering, when most larvae had wandered, onset of pupariation, when most larvae had pupariated, onset of eclosion and end of eclosion. Our results indicated that all of the evaluated events could be used as accurate indicators of postmortem interval (PMI). The carcasses decayed fairly quickly in spring, summer and autumn, taking 225+/-75 h, 183+/-44 h, and 247+/-70 h, respectively, to decay from the fresh stage to skeletonisation. In winter, carcasses needed longer (1180+/-291) to decay as much. Carcasses attracted 47 species of insect, with flies predominating. The larvae were mainly Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart) and Hydrotaea (Ophyra) spinigera (Stein). Most necrophagous insects were found all year around, and there were no marked differences in species among the four seasons, except that Dermestes maculatus (De Geer) was absent in winter. Blowflies produced only one generation on a carcass before it became skeletonised, which simplified the estimation of PMIs.     

Entomological identification of the post-mortem colonization of wolf cadavers in different decomposition stages

Human-driven biodiversity loss is progressively becoming a problem with dramatic consequences for the conservation of vital ecosystems. The increasing number of illegal killings of the grey wolf (Canis lupus, Linnaeus, 1758), a threatened species, displays the need for investigation and prosecution of such offences. Forensic entomology makes use of the knowledge about necrophagous insects to estimate a minimum time-since-death interval of the deceased person or animal, which can give important information on a possible perpetrator. The cadaver fauna along five decomposition stages of wolves in Germany was investigated in the period 2014–2021. The insects from 70 wolf cadavers, originating from all over Germany, were provided by the Leibniz Institute for Zoo and Wildlife Research Berlin. The accumulated degree day (ADD) model was applied for the post-mortem interval estimation on wolf cadavers for the first time. A total of 20 coleopteran species and 14 different dipteran species were discovered and identified. Almost 99 % of all insect specimens were from the order of Diptera, and beetles (Coleoptera) accounted for only 1 % of the cadaver fauna. The blowflies (Calliphoridae) are of particular importance for forensic issues, accounting for about 66 % of all families. Carrion beetles (Silphidae) were found as the second most abundant family (about 21 %). In addition, combining all cases, a steadily increasing insect species richness S was detected from early decay to advanced decay (fresh S = 8; bloated S = 12; active decay S = 21; advanced decay S = 34). In the following remains stage, the species number decreased again (S = 24). However, no significant difference in the number of species was found between the stages of decay when the cases were considered individually. The temporal pattern of insect appearance was found to be congruent with those of previous studies. Furthermore, a time of death was determined for each case and compared to the pathologist's estimates. This study provides insights into the arthropod fauna of wolf remains for the first time, applies the ADD-Model for post-mortem interval estimation, and discusses the suitability of forensic entomology for wildlife death investigations.     

Puparial case hydrocarbons of Chrysomya megacephala as an indicator of the postmortem interval

The puparial case is one of the most common 'stages' of necrophagous flies encountered in crime investigations of highly decomposed corpses. If methods for determining the weathering time of these puparial cases are developed, it is possible that the postmortem interval (PMI) could be estimated accordingly. Gas chromatography coupled with mass spectrometry (GC-MS) was used to determine the changes with the weathering time in cuticular hydrocarbons of the puparial cases of Chrysomya megacephala in the laboratory. The results have shown that cuticular hydrocarbons of the puparial cases were a mixture of n-alkanes, methyl-branched alkanes, and dimethyl-branched alkanes. The carbon chain length ranged from C21 to C35, and the hydrocarbon composition showed significant regular changes with the weathering time. For the even numbered n-alkanes with low molecular weight, namely n-C22, n-C24 and n-C26, the abundance increased significantly with the weathering time. For n-C26, in particular, a linear increase in abundance with the weathering time was observed. In addition, for most of the other low molecular weight hydrocarbons (n-C26 or below), the abundance decreased significantly with the weathering time. It is concluded that, cuticular hydrocarbon is a potential indicator of the weathering time in C. megacephala, and possibly in other necrophagous flies, and might further be used to determine the PMI.     

The analysis of dyes in ball point pen inks on single paper fibres using laser desorption ionisation time of flight mass spectrometry (LDI-TOFMS)

A preliminary field study in the summer in a rural village in El-Qalyubiya Governorate (Egypt) compared the necrophagous insects colonizing the cadavers of two male rabbits (Oryctolagus cunicullus domesticus L.) killed by asphyxia (control), with two poisoned by the organophosphate (OP) pesticide pirimiphos-methyl (test). Decay of control carcasses was rapid since they reached the skeletal stage in only 19 days. Test carcasses did not decay completely, even 40 days post-killing. Insect species colonizing both carcasses types were not different, indicating that despite its odor, the OP were not masking the decomposition odors which were drawing the species to the bodies. The blowfly Chrysomya albiceps (Weidemann) constituted 76.6% of all samples collected. They were the first colonizers and played a major role in the decomposition process of control carcasses and in the partial decay of the test ones. They were significantly fewer numbers of immature stages developing on the test carcasses which probably contributed to their distinct lag and poor decomposition. Samples indicate that 17.3% of the insects were members of the Formicidae. This family was present in all carcasses, mostly in the early stages of decomposition. Formicidae may be considered omnivorous, and one of the fauna which use the cadaver as a refuge, to obtain humidity and food. This study provides additional knowledge in the context of Egyptian forensic entomology and the influence of OP which is of relevance to forensic science.     

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.     

利用嗜尸性蝇类生活史推测死亡时间

目的积累一些嗜尸性蝇类生活史的资料及群落组成的季节性变动规律。方法采用动物尸体作诱饵,分别对自然飞到诱饵上繁殖生长的8种嗜尸性蝇类的生活史及群落组成的季节性变动规律进行观察研究。结果夏季高温时段蝇类群落组成物种较为丰富,冬季低温时段蝇类种群较为单一。常见8种嗜尸性蝇类虫态经历时间在气温高时较短,气温低时较长。结论嗜尸性蝇类生活史及群落组成的季节性变动规律可为推测死亡时间提供依据。     

Diversity of Forensic Rove Beetles (Coleoptera,Staphylinidae) Associated with Decaying Pig Carcass in a Forest Biotope

Most forensic studies are focused on Diptera pattern colonization while neglecting Coleoptera succession. So far, little information is available on the postmortem colonization by beetles and the decomposition process they initiate under temperate biogeoclimatic countries. These beetles have, however, been referred to as being part of the entomofaunal colonization of a dead body. Forensic entomologists need increased databases detailing the distribution, ecology, and phenology of necrophagous insects, including staphylinids (Coleoptera, Staphylinidae). While pig carcasses are commonly used in forensic entomology studies to surrogate human decomposition and to investigate the entomofaunal succession, very few works have been conducted in Europe on large carcasses. Our work reports the monitoring of the presence of adult rove beetles (Coleoptera, Staphylinidae) on decaying pig carcasses in a forest biotope during four seasons (spring, summer, fall, and winter). A total of 23 genera comprising 60 species of rove beetles were collected from pig carcasses.     

Estimation of postmortem interval in real cases based on experimentally obtained entomological evidence

Using the entomological evidence obtained in several forensic cases analyzed in our laboratory for comparison, we evaluated the results of an experimental study carried out in a semiurban setting to determine the structure of the sarcosaprophagous fauna from a Mediterranean region of SE Spain. In all, 18 orders of arthropods were collected. The summarized experimental results refer to the most important taxa for estimating the postmortem interval. Thus, the seasonal character of certain Diptera species, such as Phaenicia sericata, Calliphora vicina, Chrysomya albiceps and Musca domestica, is cited. Among the Coleoptera, the role of Dermestidae as a necrophagous species, and of Staphylinidae and Histeridae as necrophilous, or Cleridae, Tenebrionidae and Nitidulidae as omnivorous, as well as their appearance on the corpse, is described. Among the Hymenoptera, Formicidae were as the most abundant group, acting as omnivores and not apparently related to any particular decomposition stage. The real cases are discussed using data from the literature and the experimentally obtained results. In every case, the most relevant factors for estimating PMI are briefly discussed, mentioning, when possible, the relation with the experimental results. We confirm the significance of the experimental results because they seem to be applicable to actual forensic cases, the details of which enlarge our very little knowledge on the subject in the Iberian Peninsula. The importance of regional faunistic studies of the sarcosaprophagous arthropod community, the results of which may be applied to forensic practice, is mentioned.     

Pedophilia on the Internet

The Internet provides adults and children with a medium for professional and personal communication throughout the world. As widespread communication in cyberspace grows exponentially, the potential for Internet-related crimes, such as cyberstalkng, has accelerated. Children are particularly vulnerable to sexual predators, such as pedophiles, on the Internet. This article reports cases of pedophiles who used the information superhighway to exploit child victims. Two models of pedophilia on the Internet are discussed: a trust-based seductive model and a direct sexual model. This article explores the role that mental health professionals may play in the assessment of Internet pedophilia. For example, mental health professionals may provide psychiatric treatment to victims of Internet pedophiles. Psychiatrists may be involved in the evaluation of perpetrators of Internet crimes in a clinical or forensic setting. Governmental agencies, the criminal justice system, school, or the media may request consultations by mental health professionals as part of a multi-disciplinary approach to prevention. Methods of government intervention are discussed.