Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

An illustrated identification key to early instar larvae of forensically important Muscidae (Diptera) of the western Palaearctic region.

There is a significant gap in the availability of comprehensive identification keys for the early larval stages of forensically important fly species. While well-documented identification keys exist for the third instar larvae, particularly for the Calliphoridae, Muscidae and Sarcophagidae families, there is a notable scarcity of keys for the first, except Calliphoridae, and the second instar larvae, with no such resources available for muscid species. The second instar larvae suffer the most from the lack of morphological descriptions and available identification keys. The Muscidae is one of the most frequently reported dipteran families of forensic importance colonising animal cadavers and human corpses. Nevertheless, descriptions of the morphology of their early instars remain scarce and limited to only a few species, thus their larval identification is challenging or impossible. Considering the numerous challenges associated with studying small-sized entomological material, we tested whether it is feasible to identify muscid flies to the species or at least genus level based predominantly on the details of the cephaloskeleton. To overcome the obstacle of observing details of small sclerites, especially their shapes and interconnections, we effectively employed confocal laser scanning microscopy (CLSM) as a supplementary method for light microscopy (LM). This study provides an identification key for first and second instar larvae of forensically important muscid species from the western Palaearctic (Europe, North Africa, Middle East). The proposed key primarily utilises details of the cephaloskeleton with only addition of external morphology.

Discrimination of Diptera order insects based on their saturated cuticular hydrocarbon content using a new microextraction procedure and chromatographic analysis.

The nature and proportions of hydrocarbons in the cuticle of insects are characteristic of the species and age. Chemical analysis of cuticular hydrocarbons allows species discrimination, which is of great interest in the forensic field, where insects play a crucial role in estimating the minimum post-mortem interval. The objective of this work was the differentiation of Diptera order insects through their saturated cuticular hydrocarbon compositions (SCHCs). For this, specimens fixed in 70 : 30 ethanol : water, as recommended by the European Association for Forensic Entomology, were submitted to solid-liquid extraction followed by dispersive liquid-liquid microextraction, providing preconcentration factors up to 76 for the SCHCs. The final organic extract was analysed by gas chromatography coupled with flame ionization detection (GC-FID), and GC coupled with mass spectrometry was applied to confirm the identity of the SCHCs. The analysed samples contained linear alkanes with the number of carbon atoms in the C9-C15 and C18-C36 ranges with concentrations between 0.1 and 125 ng g-1. Chrysomya albiceps (in its larval stage) showed the highest number of analytes detected, with 21 compounds, while Lucilia sericata and Calliphora vicina the lowest, with only 3 alkanes. Non-supervised principal component analysis and supervised orthogonal partial least squares discriminant analysis were performed and an optimal model to differentiate specimens according to their species was obtained. In addition, statistically significant differences were observed in the concentrations of certain SCHCs within the same species depending on the stage of development or the growth pattern of the insect.

Exploring the ethical dimensions in forensic entomology: From codes of conduct to professional accountability.

This article investigates a critical part of professional careers: adhering to a code of ethics that defines proper behavior within the context of one's specialized practice. It aims to improve the setting of forensic entomology work by highlighting the ethical issues that frequently emerge. The primary goal is to uncover common ethical issues involved in this field's everyday activities and provide informed advice on appropriate resolution options. This investigation dives into the complex interaction of religious beliefs, cultural aspects, and issues of ethics, evaluating their relevance in forensic entomology. It examines a wide range of ethical issues, from the ethics involved in collecting samples at crime scenes and morguesto the ethics created into scientific investigations, report writing, and court testimony. These guiding principles are critical in setting scientific standards and building public trust in forensic entomology's ability to provide exceptional services. Notably, regardless of whether the forensic entomology service provider is accredited by any validating organization, these ethical requirements remain critical, emphasizing their global application.

The influence of altitude on the abundance and occurrence of species of the family Calliphoridae (Diptera) in corpses in a Mediterranean area.

A carcass is defined as a temporary resource that can support high levels of diversity compared to other resources. The level of diversity often depends on the environmental conditions in which the corpse is found. Calliphoridae (Diptera) are the most important necrophagous insects used in forensic investigations because this family is common, widespread, abundant, and usually the first to colonize a corpse. However, understanding the processes responsible for variation in calliphorid species abundance and richness along gradients in ecology remains a key challenge. In this study, we analyzed the influence of altitude on the abundance and diversity of Calliphoridae species in corpses at three different altitudes (400, 900, and 1500 m a.s.l.) in a mountainous area of southeastern Spain. The results revealed a gradient of decreasing abundance with increasing altitude. An altitudinal substitution of species was observed, with Chrysomya albiceps being found at the lowest altitude and Calliphora vicina at the highest altitude. Seasonal variation was also noted, with Ch. albiceps being the dominant species in the warmer months and C. vicina being the dominant species in winter. Our results confirm the importance of Calliphorids as seasonal, altitudinal, and environmental indicators, given the wide distribution and abundance of this family. This information is of great interest on the interpretation of data in forensic practice.

Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework.

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.

Morphometric analysis for determination of larval instars in Dermestes frischii Kugelann and Dermestes undulatus Brahm (Coleoptera: Dermestidae).

Dermestes frischii Kugelann, 1792 and Dermestes undulatus Brahm, 1790 are the most abundant species worldwide at outdoor or indoor crime scenes during the dry and skeletal stages of decomposition. The attribution of larval age in these beetles is problematic due to the variable number of instars, which is influenced by environmental factors. In this study, a morphometric approach was used to look for potential morphological features as evidence of larval stages. Breeding and monitoring were performed for both species in an incubator with a preset temperature of 28°C ± 0.5 without a photoperiod. Morphometric measurements were made on 10 larvae per instar for each species using length, width, and thickness parameters. Linear discriminant analysis was then used to generate decision boundaries that clearly separated larval stages. The cross-validation procedure demonstrated that the morphometric approach successfully discriminated adjacent larval stages in both species with high values of sensitivity and specificity. This less-invasive approach could improve the ability to estimate minPMI in forensic studies of Dermestidae beetles. Future studies may extend this approach to other species and establish good practices for collecting and storing specimens for morphometric analysis.

In vitro comparison of two food choice methodologies for necrophagous species of the genus Thanatophilus (Coleoptera: Staphylinidae: Silphinae).

To accurately model the food webs, we need to acquire precise data on food ecology of the interacting species. This allows better understanding of the trophic interactions and for the necrophagous species this information could be used in medico-legal investigations. For this reason, we recently proposed standardized laboratory methodology to assess the foraging strategies based on parallel testing of 2 food items (meat, dead larvae) (Jakubec et al. 2021). The original methodology had 2 shortcomings. It was not suited for testing living larvae, which could prove predatory behavior of the species. The methodology was also based on parallel experimental design, where the food items are tested together, which could underestimate the maximum consumption of the tested subject for some items. To test if these concerns are valid, we improved original methodology allowing testing living larvae as well as a new sequential experimental setup, where consumption of each item is tested individually in a random order, thus theoretically giving an unbiased maximum consumption estimate. These methodologies were tested head-to-head on 3 forensically relevant species from the genus Thanatophilus (Thanatophilus micans (Fabricius 1794)(Fabricius 1794), Thanatophilus rugosus (Linnaeus, 1758), and Thanatophilus sinuatus(Fabricius, 1775)). The experiments have confirmed that all 3 species are almost strictly necrophagous, although they were capable of predation, despite the presence of preferred food (meat). The comparison also showed that the sequential design has indeed improved capability to quantify the maximal consumption of the given food item. Thus, we suggest following this methodology in future studies.

Morphological changes of larvae and pupae of Lucilia sericata (Diptera: Calliphoridae) reared at two temperatures and on three food types.

Determining the minimum postmortem interval (minPMI) from an entomological perspective relies mainly on development data recorded for various species of flies collected from a crime scene or suspicious death. This study focused on the larval and pupal development of Lucilia sericata (Meigen), with an emphasis on the changes of the external morphology of the puparium and its pupal content throughout the duration of metamorphosis. Colonies of L. sericata were reared on 3 types of swine tissue (skeletal muscle, liver tissue, and heart tissue) at 2 different temperature regimes; 24 ±â€…1 °C and 30 ±â€…1 °C. The overall developmental time, larval width and length, and inner and outer pupal morphology changes were observed and recorded. The results show that: (i) temperature significantly influenced overall development time, as well as changes in larval width and length, but this effect was not dependent on tissue type; (ii) larval development duration was longest on heart tissue, and shortest on skeletal muscle for both temperatures; and (iii) pupation was longest for larvae reared on skeletal muscle at 24 ±â€…1 °C, and on liver tissue at 30 ±â€…1 °C, while those larvae reared on liver tissue at 24 ±â€…1 °C and heart tissue at 30 ±â€…1 °C had the shortest pupation period. A seven-character checklist plus 4 landmark stages were developed comprising the external morphology of the puparium and pupal content changes of L. sericata. In conclusion, the study provides larval and pupal development timetables, as well as checklists and photo guides for pupal character development that may be useful for future postmortem determinations.

Initial blow fly (Diptera: Calliphoridae) colonization of cats (Felis catus) in Indiana.

Many disciplines are utilized within the field of veterinary forensic sciences, including forensic entomology. Understanding the initial colonization period by flies of forensic importance can contribute to estimating the minimum postmortem interval. There is limited data regarding the time of colonization of animals with fur, and the interpretation of this data is difficult due to the variation in animal models used. The purpose of this study was to examine the initial insect colonization of cats (Felis catus), with light and dark fur. Twelve domestic short-haired cats were placed in cages 15.2 m apart in a grassy field in West Lafayette, IN, United States. Weather data (temperature, precipitation, sun/cloud exposure, humidity), insect activity, time to oviposition, and decomposition changes were documented. Eggs from initial oviposition events were collected and reared to identify the primary colonizing species. Although the time of first oviposition event was not different between the treatments, fur color did affect fly colonization, and cats with dark fur had more oviposition events than cats with light fur (t = 2.639, df = 4, P = 0.029). Three species of Lucilia (Diptera: Calliphoridae) colonized the cats on the initial day of placement. Further studies in cats should include the decompositional studies to understand the unique characteristics that occur during each stage of decomposition, which could aid in developing a scoring system for animals with fur. Additional studies could include analyzing how fur length would affect colonization.

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.

Effects of population variations and temperature on Chrysomya megacephala (Diptera: Calliphoridae) development: implications for estimating the postmortem interval.

Forensic entomology requires knowledge of the developmental rates of the species that colonize a body after death to estimate the postmortem interval (PMI). These developmental rates may vary depending not only on the species but also on the geographic location due to population differences. Therefore, the objectives of this work were to determine the developmental duration of the forensically important fly Chrysomya megacephala under constant controlled and field condition temperatures and to compare these results, through a meta-analysis, with data reported by other authors on populations from different localities. For this, C. megacephala colonies were established in the laboratory, and the duration of the life cycle was studied at two controlled temperatures (25 °C and 27 °C) and field conditions (27.5 ± 3.2 °C). Analysis of variance was performed to determine differences in developmental time and larval length between constant laboratory temperatures and field conditions. A generalized linear model was performed with predictor variables extracted from the literature (diet, relative humidity, latitude, longitude) to evaluate the effect of population variation on developmental times. The results showed significant differences in developmental times between 25 and 27 °C. As expected, the complete life cycle of C. megacephala was shorter at 27 °C. Finally, the meta-analysis suggested differences between the developmental times of different populations, based on temperature and geographic location. The results of this study provide fundamental developmental data to use C. megacephala in PMI estimations. Finally, we suggest that, when making expert reports, information from local populations should be used to determine a more accurate and reliable PMI.

First observation of Afromorgus chinensis (boheman, 1858) (Coleoptera: Trogidae) on a rabbit (Oryctolagus cuniculus L., 1758) carcass and its implications in forensic entomology.

Beetles (Coleoptera) are known to constitute forensic evidence in medico-legal investigations as their presence can be used to date human remains in almost all decomposition stages. Many forensic studies focus on the successional colonization pattern of flies (Diptera); however, beetles have not so far been studied extensively for this aspect. A beetle of the genus Afromorgus Scholtz, 1986, A. chinensis (Boheman, 1858) (Scarabaeoidea: Trogidae), was found beneath a late decaying rabbit carcass at Paya Indah Wetland, Dengkil, Malaysia, for the first time. Both genus and species are already known to occur in Malaysia from literature.

Application of omics techniques in forensic entomology research.

With the advent of the post-genome era, omics technologies have developed rapidly and are widely used, including in genomics, transcriptomics, proteomics, metabolomics, and microbiome research. These omics techniques are often based on comprehensive and systematic analysis of biological samples using high-throughput analysis methods and bioinformatics, to provide new insights into biological phenomena. Currently, omics techniques are gradually being applied to forensic entomology research and are useful in species identification, phylogenetics, screening for developmentally relevant differentially expressed genes, and the interpretation of behavioral characteristics of forensic-related species at the genetic level. These all provide valuable information for estimating the postmortem interval (PMI). This review mainly discusses the available omics techniques, summarizes the application of omics techniques in forensic entomology, and their future in the field.

Direct relationship between evapotranspiration rate (ETO) and vertebrate decomposition rate.

When vertebrate scavenging is excluded, the Evapotranspiration Rate (ETo) of a given geographic region directly regulates the decomposition rate of unclothed vertebrate carrion, with any deviation attributed to insect activity. We conducted four decomposition experiments using pig carrion (Sus scrofa domesticus) over the span of two years (2018-2020) at a location in Davis, California. We used ETo, a variable that accounts for five climatic parameters (wind, temperature, humidity, solar radiation, and altitude) as the rate-determining variable of the decomposition process. We found ETo to have a strong (R2 = 0.98) predictive relationship with the decomposition rate. To account for maggot activity decomposing the carrion, we measured maggot weight in 2019 and 2020 using a novel method, and in 2020 we used FLIR imagery to measure maggot mass temperatures as a surrogate measurement of total maggot activity. Maggot activity was a significant predictor (p < 0.0001) of the decomposition rate, while maggot weight was not (p > 0.1). We hope to show the forensic entomology community the potential of using ETo. Future projects can incorporate ETo as a baseline to decomposition studies to determine if ETo remains the most accurate descriptor of decomposition and ultimately increase certainty in the Postmortem Interval (PMI).

New developmental data of Chrysomya megacephala (Diptera: Calliphoridae) in tropical temperatures and its implications in forensic entomology.

The estimation of the postmortem interval (PMI) is an essential information in death investigations. It is necessary to know the developmental data of the most important necrophagous insect species in every geographical area. Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most common species associated with human body decomposition, especially in the tropics, so a precise knowledge of its life cycle is crucial. However, despite its ubiquity, developmental data in a range of tropical temperatures is scarce. For this reason, the aim of this study is to provide the developmental data of the blowfly, C. megacephala, in a range of tropical temperatures and to apply these data in forensic entomology. Four temperatures were examined (i.e., 27.0°C 29.5°C, 32.0°C and 34.5°C) and the time of developments from egg to adult were recorded. To build the growth curves, five larvae per day were measured with a digital caliper. Accumulated degree-days were calculated and the isomorphen diagram for this species was plotted. As we predicted, the results showed that the life cycle of this species was affected by the increasing temperature. The implications of these new data for determining the minimum PMI in forensic entomology were discussed.

Descriptive analyses of differentially expressed proteins during intrapuparial stage based on the label-free proteomics technique between Chrysomya megacephala and Synthesiomyia nudiseta.

The lack of rapid and accurate species identification methods on pupae restricts the practical application of forensic entomology. It is a new idea to construct portable and rapid identification kits based on the principle of antigen/antibody interaction. Screening differentially expressed proteins (DEPs) of fly pupae is a basis of solving the problem. Here, we used the label-free proteomics technique to discover the DEPs and further validate using the parallel reaction monitoring technique (PRM) in the common flies. In this study, we reared the Chrysomya megacephala and Synthesiomyia nudiseta at constanttemperature, and then we sampled at least four pupae at 24 h intervals until the end of the intrapuparial stage. We found 132 DEPs between Ch. megacephala, and S. nudiseta groups, with 68 and 64 proteins being up-regulated and down-regulated between the two groups. Among the 132 DEPs, we selected five proteins having potential for further development and utilization, such as C1-tetrahydrofolate synthase, Malate dehydrogenase, Transferrin, Protein disulfide-isomerase, and Fructose-bisphosphate aldolase, for further validation using PRM-targeted proteomics, with the trends of PRM results being consistent with the label-free data for corresponding proteins. The present study investigated DEPs via the label-free technique during the pupal development in the Ch. megacephala, and S. nudiseta and provided reference data for development of rapid and accurate identification kits.

[Injuries after the biological exposure]. / Povrezhdeniya ot biologicheskogo vozdeistviya.

The aim of the scientific work is to justify the need to highlight the topic of injuries after the biological exposure in forensic medicine. The definition of the «biological trauma¼ concept is presented - it means impairment of the body structure and function as a result of a special injury effects typical for wildlife representatives (animals, plants). Biological exposure includes antigenic, toxin, allergic, bioelectric, bioorganic exposures and their combinations. Biological injuries should be distinguished from mechanical injuries caused by small, medium, and large mammals and reptiles. Options of the antemortem and postmortem biological factor effects are considered. The qualitative limits of the postmortem period are defined. A new method of forensic reconstruction of postmortem conditions is proposed. Forensic entomological, forensic microbiological examinations, and their complex combination with forensic examination are isolated as independent methods.

The effect of postmortem penetrative trauma on oviposition behavior of Calliphora vicina.

Flies in the family Calliphoridae are purported to demonstrate a strong attraction to and preferential colonization of wounds when present on human corpses or carrion. This well-circulated concept in Forensic Entomology is based on surprisingly few empirical studies that have examined the oviposition behavior of necrophagous Diptera toward wounds of vertebrate animals. In the present study, the oviposition behavior of Calliphora vicina toward piglets inflicted with postmortem sharp force trauma was examined during a 10-h test period under controlled laboratory conditions and in an outdoor urban environment. Three species of flies (C. vicina, C. livida and Cynomya cadaverina) deposited eggs on wounded and non-wound piglets during the field studies, although more than 80% of all eggs laid were by C. vicina regardless of wound status. For all species, oviposition occurred predominately on the head, including in eyes, ears, nose and mouth, and less frequently in other locations on the body. Eggs were never found in or near wounds. Similar oviposition patterns were displayed by C. vicina under laboratory conditions, with the exception for limited egg deposition in wounds under overcrowded conditions. In this latter scenario, egg deposition represented only 0.14% of the total reproductive output of adult flies. The findings do not support the contention that calliphorids preferentially oviposit in wounds resulting from sharp force penetrating trauma.