Developmental differences in spatially distinct populations of the forensically relevant blow fly Lucilia sericata - About the comparability of developmental studies (and case work application).

The cosmopolitan blow fly Lucilia sericata is often used in forensic case work for estimating the minimum postmortem interval (PMImin). For this, the age of immature specimens developing on the dead body is calculated by measuring the time taken to reach the sampled developmental stage at a given temperature. To test whether regional developmental data of L. sericata is valid on a global scale, the time taken to reach different developmental stages was compared between a population from Mexico and one from Germany at two different constant temperatures. The German population of L. sericata was collected in Frankfurt/Main, while the Mexican population originated near Oaxaca de Juarez and was transported to Germany in the larval stage. Only the F1 generation was used to avoid adaption of the Mexican flies. Eggs were immediately placed at 20 °C and 30 °C. Five times 30 freshly eclosed larvae per replicate (n = 5) were then transferred to a cup of minced meat in separate containers. The larvae were checked every 8 h for migration, pupariation or emergence of adult flies. The time at which the first individual and 50 % of the specimens per container entered each of these stages, was recorded. Significant differences in the time of development between the two populations were observed at both temperatures. At 20 °C, the first specimens of the Mexican population reached all developmental stages a little (< 1 day to < 2 days) earlier than the German L. sericata. At 30 °C, the Mexican flies also reached the post-feeding stage slightly earlier (0.2 days). However, at 30 °C, the German flies started pupariation significantly earlier (after 5 days) than the Mexican flies (6.9 days) and the adults from Germany also emerged earlier (10.5 days compared to 13.1 days). The same pattern was observed when looking at 50 % of the total number of specimens per container. A comparison with previously published developmental studies was difficult as the experimental design varied widely between studies. However, the results were within the range of most studies. Our study has shown that age estimation can vary widely depending on the population on which the reference data used for the calculations are based. This highlights the importance of using local and population-specific developmental data for estimating the age of blow flies in case work.

Catch me if you can-emission patterns of human bodies in relation to postmortem changes.

The present study examines for the first time the emission patterns and olfactory signatures of 9 complete human corpses of different stages of decomposition. Air sampling was performed inside the body bags with solid sorbents and analysed by coupled gas chromatography-mass spectrometry after thermal desorption (TD-GC-MS). Furthermore, odour-related substances were detected by gas chromatography-olfactometry (GC-O). Sulfurous compounds (mainly dimethyl di- and trisulfide) were identified as most important to the odour perception. Around 350 individual organic substances were detected by TD-GC-MS, notably sulfurous and nitrogenous substances as well as branched alkanes, aldehydes, ketones, alcohols, carboxylic acids, carboxylic acid esters and ethers. A range of terpenes was detected for the first time in a characteristic emission pattern over all decomposition stages. Concentrations of the substances varied greatly, and no correlation between the emission patterns, the stage of decomposition and the cause of death could be found. While previous studies often analysed pig cadavers or only parts of human tissue, the present study shows the importance of analysing complete human corpses over a range of decomposition stages. Moreover, it is shown that using body bags as a kind of "emission test chamber" is a very promising approach, also because it is a realistic application considering the usual transport and store of a body before autopsy.

Deciphering the impact of microenvironmental factors on cuticular hydrocarbon degradation in Lucilia sericata empty Puparia: Bridging ecological and forensic entomological perspectives using machine learning models.

Blow flies (Calliphoridae) play essential ecological roles in nutrient recycling by consuming decaying organic matter. They serve as valuable bioindicators in ecosystem management and forensic entomology, with their unique feeding behavior leading to the accumulation of environmental pollutants in their cuticular hydrocarbons (CHCs), making them potential indicators of exposure history. This study focuses on CHC degradation dynamics in empty puparia of Lucilia sericata under different environmental conditions for up to 90 days. The three distinct conditions were considered: outdoor-buried, outdoor-above-ground, and indoor environments. Five predominant CHCs, n-Pentacosane (n-C25), n-Hexacosane (n-C26), n-Heptacosane (n-C27), n-Octacosane (n-C28), and n-Nonacosane (n-C29), were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The findings revealed variations in CHC concentrations over time, influenced by environmental factors, with significant differences at different time points. Correlation heatmap analysis indicated negative correlations between weathering time and certain CHCs, suggesting decreasing concentrations over time. Machine learning techniques Support Vector Machine (SVM), Multilayer Perceptron (MLP), and eXtreme Gradient Boosting (XGBoost) models explored the potential of CHCs as age indicators. SVM achieved an R-squared value of 0.991, demonstrating high accuracy in age estimation based on CHC concentrations. MLP also exhibited satisfactory performance in outdoor conditions, while SVM and MLP yielded unsatisfactory results indoors due to the lack of significant CHC variations. After comprehensive model selection and performance evaluations, it was found that the XGBoost model excelled in capturing the patterns in all three datasets. This study bridges the gap between baseline and ecological/forensic use of empty puparia, offering valuable insights into the potential of CHCs in environmental monitoring and investigations. Understanding CHCs' stability and degradation enhances blow flies' utility as bioindicators for pollutants and exposure history, benefiting environmental monitoring and forensic entomology.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

Preliminary study on prevalence of hemoprotozoan parasites harbored by Stomoxys (Diptera: Muscidae) and tabanid flies (Diptera: Tabanidae) in horse farms in Nakhon Si Thammarat province, Southern Thailand.

Background and Aim: Stomoxys and tabanid flies are of medical and veterinary importance because they play crucial roles in disease transmission as mechanical vectors of various hemopathogens. However, its role as a hemoprotozoan parasite vector in horse farms has not been studied. Therefore, we investigated the occurrence of hemoprotozoan parasites belonging to the genera Babesia, Theileria, and Trypanosoma in Stomoxys and tabanid flies using conventional polymerase chain reaction (PCR) and DNA sequencing. Materials and Methods: All samples (Stomoxys and tabanid flies) were collected using an Nzi trap for three consecutive days each month from November 2022 to March 2023. The flies were morphologically identified to the species level and separated according to sex. Individual (for tabanid flies) or pooled samples (consisting of three specimens of Stomoxys flies of the same species and sex collected from the same site) were used for DNA extraction. Conventional PCR was used to screen for hemoprotozoan parasite DNA, followed by Sanger sequencing to identify the species. Results: In total, 189 biting flies were collected, including four species of Stomoxys (Stomoxys bengalensis, Stomoxys calcitrans, Stomoxys indicus, and Stomoxys sitiens) and five species of tabanids (Atylotus cryptotaxis, Chrysops dispar, Tabanus megalops, Tabanus mesogaeus, and Tabanus rubidus). Stomoxys calcitrans was the most prevalent species, accounting for 58.7% (n = 111) of the collected flies. Ten (12.4%) of the 81 samples (individuals and pools) analyzed by PCR were positive for the 18S rRNA gene of the Theileria/Babesia species. Trypanosoma DNA was not detected in any sample. After performing Basic Local Alignment Search Tool searches and a phylogenetic analysis, only six samples (7.4%), including S. calcitrans (n = 2), S. sitiens (n = 2), T. megalops (n = 1), and A. cryptotaxis (n = 1), were found to be infected with Theileria sinensis. Furthermore, apicomplexan parasites, namely, Mattesia spp. and Colpodella spp., were found on S. indicus, the fungus Meira spp. was found on S. calcitrans, and the pathogenic green alga Helicosporidium spp. was found on A. cryptotaxis. Conclusion: This study is the first to report a variety of Stomoxys and tabanid flies collected from horse farms in Thailand, which were found to be infected with Theileria and Colpodella species that affect mammals, suggesting that Stomoxys and tabanid flies can be used to confirm the presence of hemoprotozoan parasites in the study area. Understanding the presence of hemoprotozoa in flies could help design vector control programs and manage various diseases in the study area.

Cuticular hydrocarbons as weathering biomarkers of empty puparia of the forensically important blowfly Calliphora vicina Robineau-Desvoidy, 1830 (Diptera: Calliphoridae) in soil v/s under room conditions.

Forensic entomology uses the age of insects, such as blow flies, to determine a minimum post-mortem interval (PMImin). Recent research has focused on using the analysis of specific cuticular hydrocarbons (CHCs) in adult insects and their empty puparia to estimate their age, as it has been shown that their profile changes are consistent with age. The current work is based on the weathering of five CHCs from empty puparia of Calliphora vicina that were stored in soil (field/outdoor) and non-soil (room/indoor conditions) based pupariation media for a total of six months. The experiment was conducted in a controlled environment chamber at a constant temperature of 25 ± 2 °C under constant darkness. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the cuticular hydrocarbons after they were extracted in n-Hexane. n-Pentacosane, n-Hexacosane, n-Heptacosane, n-Octacosane, and n-Nonacosane were the five CHCs investigated. Results showed that CHCs weathered more quickly in the soil than in the non-soil environment. It was also found that the abundance of Heptacosane increased in the samples during the fifth month when stored in a non-soil medium, while the abundances of all five CHCs were not detected after eight weeks onwards in soil pupation medium.

Decomposition and insect succession of pig cadavers in tents versus outdoors - A preliminary study.

The pattern of insect succession and the species composition on cadavers can be influenced by several parameters, one of which is concealment. It has been previously shown for cadavers inside containers (e.g. suitcases or vehicles) or various indoor situations, that concealment can lead to a delayed arrival, altered species composition and decreased number of taxa present at the cadaver. As no data is yet available for the specific environment of tents on these processes, we placed five pig cadavers inside closed two-person tents during the summer of 2021 in a mixed forest in Germany. Five control cadavers were freely accessible for insects. To minimize disturbances, tents were only opened every fifth day over a period of 25 days for estimating the temperature profiles, insect diversity and quantification of the cadavers' decomposition using the total body score (TBS). The temperature inside the tents was only slightly elevated compared to the ambient temperature during the study. Although the tents prevented direct access of adult flies and beetles, the cadavers were colonized because the flies oviposited on the zippers and fly screens of the inner tents. However, the resulting infestation of the cadavers by fly larvae was reduced and delayed, compared to the exposed cadavers. The dominant fly species on both the tent and exposed cadavers was the blow fly Lucilia caesar. Open cadavers showed the expected decomposition patterns with large larval masses. 25 days after placement, only bones and hair remained from the exposed pigs (TBS = 32), while most of the tissue of the cadavers inside the tents was still present (TBS = 22.5) and post feeding larvae were not able to leave the tents. Regarding the attraction of beetles to both treatments, open cadavers were predominately colonized by the silphid Oiceoptoma thoracicum, while the silphid Necrodes littoralis was the most dominant species in the pitfall traps surrounding the tents. Considering the prolonged pre-appearance interval of fly larvae colonizing the cadavers inside tents, the entomological evidence of forensic cases dealing with concealed bodies should be handled with caution, since the post mortem interval might be significantly underestimated.

Multimethod combination for age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) with implications for estimation of the postmortem interval.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMImin). Here, the developmental patterns of S. peregrina were investigated at 5 constant temperatures (15-35 °C). Morphological changes at different developmental stages of the pupa were observed at 4 constant temperatures (15-30 °C) by removing the puparium and staining the pupa with hematoxylin and eosin. Furthermore, differentially expressed genes (DEGs) were analyzed at 25 °C in the intrapuparial period to estimate the age of S. peregrina during the intrapuparial stage. S. peregrina completed development from larviposition to adult eclosion at 15 °C, 20 °C, 25 °C, and 30 °C; the developmental durations were 1090.3 ± 30.6 h, 566.6 ± 21.9 h, 404.6 ± 13.01 h, and 280.3 ± 4.5 h, respectively, while the development could not be completed at 35 °C. The intrapuparial period of S. peregrina was divided into 12 sub-stages on the basis of the overall external morphological changes; 6 sub-stages on the basis of individual morphological structures such as the compound eyes, antennae, thorax, legs, wings, and abdomen; and 10 sub-stages on the basis of internal morphological changes detected using histological analysis. The period of each sub-stage or structure that appeared was determined. Moreover, we found that 6 genes (NDUFS2, CPAMD8, NDUFV2, Hsp27, Hsp23, and TPP) with differential expression can be used for the precise age estimation of S. peregrina during the intrapuparial period. This study provided basic developmental data for the use of S. peregrina in PMImin estimation, and we successfully estimated PMImin in a real forensic case by using a multimethod combination.

Geographical Variation of Cuticular Hydrocarbon Profiles of Adult Flies and Empty Puparia Amongst Three Populations of Calliphora vicina (Diptera: Calliphoridae).

Blowflies (Diptera: Calliphoridae) are of great importance in forensic entomology and in determining the minimum post-mortem interval, as they may be the first group of insects to colonize decomposing remains. Reliable species identification is an essential prerequisite. Classically, morphological characters or DNA sequences are used for this purpose. However, depending on the species and the condition of the specimen, this can be difficult, e.g., in the case of empty fly puparia. Recent studies have shown that cuticular hydrocarbon (CHC) profiles are species-specific in necrophagous taxa and represent another promising tool for identification. However, the population-specific variability of these substances as a function of e.g., local climatic parameters has not yet been sufficiently investigated. The aim of this study was to determine the geographical variation of CHC profiles of the blowfly Calliphora vicina (Robineau-Desvoidy, 1830) depending on different countries of origin. Flies were reared in the United Kingdom, Germany, and Turkey in common garden experiments under ambient conditions. CHC profiles of the resulting adult flies and their empty puparia were analyzed using gas chromatography-mass spectrometry. Data were visualized by principal component analysis and clustered by population. The populations of the United Kingdom and Germany, both having similar climates and being geographically close to each other, showed greater similarities in CHC profiles. However, the CHC profile of the Turkish population, whose climate is significantly different from the other two populations, was very different. Our study confirms the high potential of CHC analysis in forensic entomology but highlights the need to investigate geographical variability in chemical profiles.

A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation.

The analysis of postmortem protein degradation has become of large interest for the estimation of the postmortem interval (PMI). Although several techniques have been published in recent years, protein degradation-based techniques still largely did not exceed basic research stages. Reasons include impractical and complex sampling procedures, as well as highly variable protocols in the literature, making it difficult to compare results. Following a three-step procedure, this study aimed to establish an easily replicable standardized procedure for sampling and processing, and further investigated the reliability and limitations for routine application. Initially, sampling and processing were optimized using a rat animal model. In a second step, the possible influences of sample handling and storage on postmortem protein degradation dynamics were assessed on a specifically developed human extracorporeal degradation model. Finally, the practical application was simulated by the collection of tissue in three European forensic institutes and an international transfer to our forensic laboratory, where the samples were processed and analyzed according to the established protocol.

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.

Wing morphometrics as a tool for the identification of forensic important Lucilia spp. (Diptera: Calliphoridae).

Blow flies of the genera Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) are considered forensically important species across several regions of the world. Due to the similarity of adults, especially females, the usual methods based on morphology or even molecular techniques can experience some limitations; therefore, alternative or supportive tools are required. Recently, the landmark-based geometric morphometric analysis has been applied to discriminate many insects on genus and species level. Herein, we focus on wing morphometric analysis as a tool in classifying five species of Lucilia; three species from Thailand - L. cuprina (Wiedemann, 1830), L. porphyrina (Walker, 1856) and L. sinensis Aubertin, 1933; and two species from Switzerland - L. caesar (Linnaeus, 1758) and L. illustris (Meigen, 1826). Canonical variate analysis of 233 right wings showed four overlapping clusters of L. cuprina, L. sinensis, L. caesar, and L. illustris with one distinct cluster of L. porphyrina. Eighty-eight to 100 percent of correct classification was achieved, with an UPGMA dendrogram analysis revealing clear-cut branch and sub-branch of five species determined. Results from this study suggested that wing morphometric analysis is a useful tool for the identification of adult Lucilia spp.

Dynamics of insects, microorganisms and muscle mRNA on pig carcasses and their significances in estimating PMI.

The accurate estimate of the postmortem interval (PMI) is of vital significance in the investigation of homicide cases. In this study, three pig carcasses were placed in the field to study the pattern of insect succession, the change of microorganisms and the degradation of muscle tissue RNA during the decomposition process. The results showed that insects could quickly colonize the carcasses and still exist on them until the end of the experiment (41 days). Their development and succession patterns are useful indicators for PMI estimation. The diversity of rectal microorganisms decreased with the decomposition time. In different decomposition periods, significant differences in the rectal and soil microbial composition and relative abundance were found, which could be used to estimate the PMI with an accuracy of 3-4 days. The RNA of muscle tissue was found to have a time-dependent relationship with the PMI. Ppia and Gapdh showed a linear upward trend within 10 h after the death, followed by a gradual downward trend from 10 to 240 h. The expression of ß-actin gene showed a gradual downward trend during 0-240 h. This is the first study in China to analyze the changes of insects, muscle RNA and microorganisms on pig carcasses in the same natural environment, which provide basic data for the PMI estimation.

The time of death in Dutch court; using the Daubert criteria to evaluate methods to estimate the PMI used in court.

When a capital crime is committed the post-mortem interval (PMI) is of particular importance in investigating a suspect's alibi in court. A forensic expert can use different methods to estimate the PMI. This research focuses on who is considered an expert in court and whether the methods used to estimate the PMI are reliable. In this study, the methods used to estimate the PMI and the experts consulted, available in Dutch jurisprudence, in the period 2010-2019 were investigated. Ninety-four judicial cases were included and multiple experts and methods of estimating the PMI were found. As part of this study, the methods that were used to estimate the PMI in court were subjected to the Daubert criteria. Of these methods, only the Henssge nomogram and entomological methods met the Daubert criteria. However, the methods are only useful when applied by the right forensic expert and in the right manner. Unfortunately, this was not always the case.

Influence of storage on larval length and age determination of the forensically important blow fly Lucilia sericata (Diptera: Calliphoridae).

One of the main tasks in forensic entomology is the determination of the minimum post-mortem interval (PMImin) based on the age of the juvenile insects feeding and developing on the dead body. An important task is to store the evidence appropriately so that the evaluation and expert report can be used in court. However, existing recommendations can be contradictory or lacking scientific validation, e.g. by proposing various preservation liquids without knowing whether and to what extent the period of storage in such a liquid has an effect on the length of the preserved larvae. Storage time can be an issue since, due to technical and procedural circumstances, killed larvae may be stored for hours, days, weeks or even longer prior length measurement. A changed body length would have consequences for the entomological report, as the age of the larvae is usually derived from their length. This study investigates the effect of four differently concentrated ethanol solutions (70%, 80%, 90% and 96%) during a storage period of up to 196 days on the body length of stored larvae of the forensically important blow fly species L. sericata (Diptera: Calliphoridae). Larvae of different ages (24 h, 48 h and 72 h after hatching) were killed by immersion in hot, non-boiling water (≥80 °C) for at least 30 s. Their lengths were measured immediately. Subsequently samples were stored in ethanol of appropriate concentration at room temperature (approx. 22 °C). Further length measurements were made at 16 different storage intervals between 1 and 196 days. Many specimens showed a length decrease for most storage conditions and all larval ages. However, there was a tendency for 48 h- and 72 h-old larvae to increase in length after the first days of storage of up to 1.1 mm which may lead to an erroneous overestimation of the PMImin using this kind of specimens. All changes in length within each cohort over total time were in the range of +7% to -9.1%. Significant differences in length changes within the first days of storage were found mainly in larvae stored in 70%- and 80%-ethanol, but larvae stored in 90%- and 96%-ethanol showed first significant differences on day 56 at the earliest. Our results lead to the recommendation that the measurements of fly larvae samples should be taken immediately after killing and before storage to avoid any effects. Ethanol ≥90% should be used for storage.

It is all about the insects: a retrospective on 20 years of forensic entomology highlights the importance of insects in legal investigations.

This study highlights the importance of insect evidence by evaluating 949 insect-associated cases, including 139 entomological reports, from 2001 to 2019 at the Institute of Legal Medicine Frankfurt/Germany. With a high number of cases in the summer months and a low number in the colder season, 78.5% of the bodies were found indoors, regardless of year or month. In more than 80% of the cases, where PMI information was available (n = 704), the presumed PMI ranged from 1 to 21 days, a period during which entomological evidence can provide a day-specific estimate of PMImin. In cases where insects have been identified to species level (n = 279), most bodies were infested by one or two species with a maximum of 10 different species. Overall, a total of 55 insect species were found. Information on biology, activity and distribution of the most abundant taxa is given and applied for 5 case histories estimating different PMImins of up to over 6 months. Despite proved importance and scientific development of forensic entomology, insects are still rarely considered as a tool in forensic case work. The main reasons are a lack of awareness and (too) late involvement of a forensic entomologist. Our work shows that forensic entomology is an independent discipline that requires specialist expertise.

Insect Decline-A Forensic Issue?

Recent reports have shown a dramatic loss in insect species and biomass. Since forensic entomology relies on the presence of insects, the question is whether this decline effects the discipline. The present review confirms that numerous studies document insect population declines or even extinction, despite the fact that the rates of decline and the methods used to demonstrate it are still much debated. However, with regard to a decline in necrophagous insects, there is little or only anecdotal data available. A hypothetical decrease in species diversity and population density in necrophagous insects could lead to a delayed colonization of dead bodies and a modified succession pattern due to the disappearance or new occurrence of species or their altered seasonality. Climate change as one of the drivers of insect decline will probably also have an impact on necrophagous insects and forensic entomology, leading to reduced flight and oviposition activity, modified growth rates and, therefore, an over- or underestimation of a minimum postmortem interval. Global warming with increased temperature and extreme weather requires a better understanding about necrophagous insect responses to environmental variations. Here, transgeneration effects in particular should be analysed in greater depth as this will help to understand rapid adaptation and plasticity in insects of forensic importance.

The Forensic Entomology Case Report-A Global Perspective.

Forensic practitioners analyzing entomological evidence are faced with numerous challenges when presenting their findings to law practitioners, particularly in terms of terminology used to describe insect age, what this means for colonization time of remains, and the limitations to estimates made. Due to varying legal requirements in different countries, there is no standard format for the entomological case report prepared, nor any guidelines as to the sections that are required, optional or unnecessary in a case report. The authors herein propose sections that should be considered when drafting an entomological case report. The criteria under which entomological evidence is analyzed are discussed, as well as the limitations for each criterion. The concept of a global, standardized entomological case report is impossible to achieve due to national legislative differences, but the authors here propose a basic template which can be adapted and changed according to the needs of the practitioner. Furthermore, while the discussion is fairly detailed, capturing all differences between nations could not be accomplished, and those initiating casework for the first time are encouraged to engage other practicing forensic entomologists or professional associations within their own nation or region, to ensure a complete report is generated that meets lab or national requirements, prior to generating a finalized report.

To Be There or Not to Be There, That Is the Question-On the Problem of Delayed Sampling of Entomological Evidence.

The aim of the current study was to analyze two major pitfalls in forensic entomological casework: delayed evidence sampling and the effect of low-temperature storage of the body. For this purpose, temperature profiles of heavily infested corpses during cooling and cases in which insect evidence was collected both at the scene and during autopsy were evaluated with regard to species composition and development stages found. The results show that the temperature in the body bags remained at higher average temperatures up to 10 °C relative to the mortuary cooler, therefore, sufficient for larval development, with significant differences in temperature between larval aggregations on one and the same body. In addition, we found large differences both in species number, species composition, and the developmental stages found at the scene and during the autopsy. These data and observations underscore the importance of sampling evidence at the scene and recording temperatures throughout the cooling period of a body.

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.