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"Forensic Entomology"
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How to store a beetle larva? Comparing temporal effects of common fluid preservation methods on color, shape, and DNA quality
Proper fixing and long-term preservation of entomological evidence are essential in collections and research and crucial in applied fields such as forensic entomology. Incorrectly stored samples may lose important morphological features over time, rendering molecular analyses exceedingly difficult. The most effective method for preserving soft samples such as larvae is fluid preservation. It uses a combination of a wide range of fixatives and storage fluids. However, very little comparative work has been done to determine the effects of long-term storage on sample quality in terms of color, shape, and DNA stability. Moreover, the current golden standard in forensic entomology has been tailored for age estimation of larvae of Diptera, which differ from larvae of Coleoptera in morphology and subsequently in applied methods. We compared the effects of combinations of 6 commonly used fixatives and 6 commonly used storage fluids on midsized larvae of the forensically important beetle, Necrodes littoralis (Linnaeus, 1758), in terms of color, shape, and suitability for DNA analyses over a 2-yr period. We were looking for combinations that can preserve specimens in a satisfactory state, can be used on a regular basis, do not require advanced protection or skills of the personnel, and are not toxic or too harmful to the environment. We found not only several methods that scored significantly better in the tested parameters compared with the golden standard but also several common methods that should be avoided. The effects of agents on each tested category are discussed in detail.
Journal Article
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.
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•Blow flies used to determine PMImin; research focuses on CHCs in insects and puparia.•The study investigated weathering of CHCs in Calliphora vicina puparia stored in soil and non-soil environments.•Gas chromatography-mass spectrometry was used to analyze the cuticular hydrocarbons after extraction in hexane.•CHCs weathered faster in soil; Heptacosane increased in non-soil medium after fifth month.•The relative abundance of all five CHCs were not detected after eight weeks onwards in soil pupation medium.
Journal Article
Guidelines for laboratory rearing of insect evidence: the importance of air humidity for breeding of Necrodes littoralis (L.) (Coleoptera: Staphylinidae)
In forensic entomology, effective rearing of insect evidence is crucial as it increases the accuracy of post-mortem interval (PMI) estimation and facilitates species identifications of some evidence. However, virtually no single species has a forensically useful rearing protocol. This study investigates the importance of relative humidity on larval development and fitness of
Necrodes littoralis
(L.) (Staphylinidae), and proposes an all-inclusive rearing protocol for this forensically important beetle species. We hypothesized that high relative humidity enhances growth, shortens development and promotes thermogenesis. By manipulating relative humidity (50%, 70%, 90%) and the presence of adult beetles during the prelarval phase, we demonstrated that high relative humidity level significantly improved beetle fitness by increasing survival, beetle mass at eclosion and shortening development. Specifically, at 90% relative humidity, beetles showed a twofold increase in mass and a threefold increase in survival compared to 50%. Moreover, thermogenesis was higher at high relative humidity, further facilitating larval growth. These results highlight the key role of humidity for
N. littoralis
development. Finally, we proposed a comprehensive rearing protocol for
N. littoralis
to improve forensic investigations involving these beetles and to guide future developments of similar protocols for other insects of forensic importance.
Journal Article
Field validation of post-mortem interval estimation based on insect development. Part 2: Pre-appearance interval, expert evidence selection and accuracy baseline data
This is the second part of the results from the field experiment aimed at validating PMI estimation based on insect evidence. First, we investigated the accuracy gains of PMI after supplementing age estimates with PAI. Second, we compared the impact on PMI of various ways, in which an expert selects insect evidence for the estimation. Third, we provided baseline data regarding the accuracy of PMI as estimated using different methods, insect species and life stages. Insects were sampled from nine pig carcasses exposed in a forest habitat, one carcass every one or two months, with 8–11 samples taken from each carcass using standard techniques. It was found that insect age alone clearly underestimates the true PMI across insect species and life stages. When age estimates were supplemented with PAI, the estimated PMI became generally significantly closer to the true PMI. Averaging PMI across multiple different pieces of evidence yielded more accurate estimates than using single evidence. The best single evidence method tested in this study, i.e. the use of the oldest evidence from the latest colonizing species, yielded the average PMI errors of about 22 % or 25 % (depending on the type of PAI used). For the best multiple evidence method, i.e. averaging PMI for late life stages of early colonizing species and early life stages of late colonizing species, these errors were about 13 % and 16 %. PMI ranges derived using 30 % error rate covered a true PMI in 95 % of cases but only for the best multiple evidence methods. These results demonstrate that the compound entomological method for estimating PMI (including age and PAI estimates), when applied to mock human death cases, is satisfactorily accurate. Moreover, the present findings highlight the importance of PAI and the great benefits of using multiple insect evidence to estimate PMI.
•Estimating pre-appearance interval for insect evidence lowers PMI errors.•Averaging PMI estimates across multiple evidence increases accuracy.•The oldest evidence from the latest colonizer are the most beneficial when single pieces of evidence are used.•Combining estimates from late life stages of early colonizers and early stages of late colonizers improves PMI accuracy.•PMI range derived using 30 % error rate covers a true PMI in 95 % of cases but only when multiple evidence are used.
Journal Article
Deep learning method based on image recognition for intra-puparial age and postmortem interval estimation in the forensically important Sarcophaga peregrina (Diptera: Sarcophagidae)
Accurate estimation of intra-puparial age in necrophagous flies is essential for determining the postmortem interval (PMI) in forensic entomology. Traditional methods based on morphological observation of intra-pupal structures are widely used but rely on complex diagnostic criteria and are subject to observer bias, posing a technical bottleneck in PMI estimation using insect evidence. Deep learning, particularly image-based methods, offers a promising solution for objective and automated identification in forensic entomology. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a common necrophagous fly species. In this study, we propose an image-based deep learning framework for automatic classification of intra-pupal developmental age in S. peregrina to enhance the accuracy of PMI estimation. Pupae were reared at 25 °C, and samples from different developmental stages (Day 1 to Day 11) were collected. After removing the puparium, high-resolution images of intra-pupal morphology were captured to construct a dataset. A ResNet50 network was first employed to extract regions of interest, followed by a Vision Transformer (ViT) model for end-to-end classification of developmental stages. The proposed method achieved a classification precision of 94.00 %, recall of 93.41 %, and F1-score of 93.43 %. These findings demonstrate that deep learning can serve as an effective and objective alternative to manual morphological assessment, reducing reliance on expert experience in intra-puparial age estimation. The proposed approach establishes a viable AI-assisted pathway for standardized, rapid, and accurate PMI inference based on insect evidence, offering practical value for forensic investigations.
•Deep learning model classifies pupal age of Sarcophaga peregrina accurately.•Internal pupal images from Day 1–Day 11 were used to train and validate the model.•Combined ResNet50 and Vision Transformer (ViT) for ROI extraction and classification.•Achieved 94.00 % precision, 93.41 % recall, and 93.43 % F1-score in stage recognition.•Provides an AI-assisted method for objective PMI estimation in forensic entomology.
Journal Article
Development and age estimation of the intrapuparial period of Phormia regina (Diptera: Calliphoridae) for postmortem interval estimation
Phormia regina (Meigen, 1826; Diptera: Calliphoridae) is a Holarctic species that rapidly colonizes carcasses and has been used as an indicator for determining the minimum postmortem interval. However, studies using morphological methods to estimate the intrapuparial age of P. regina are lacking. In this study, morphological changes within the puparium were observed under a stereomicroscope at 7 constant temperatures ranging from 16 °C to 34 °C.The intrapuparial period was categorized into 12 substages. Morphological indicators, including compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were recorded in detail. The observed morphological changes were divided into 6–10 substages, and the duration of each substage was also recorded in detail.The results of this study provide primary data for using the intrapuparial morphology of P. regina when pupae are collected at a crime scene and estimating the minimum postmortem interval.
Journal Article
Interval estimation of thermal summation parameters in forensically important insects
Estimating time of death based on entomological evidence commonly relies on the “law of total effective temperature”, which requires developmental parameters of specific insect taxa. These are often calculated using the method of Ikemoto and Takai. However, this approach has key limitations. Most importantly, the lack of interval estimates may give the false impression of population homogeneity, which contradicts the substantial variation typically observed in insect populations. In this study, we propose an alternative method. It estimates interval values for developmental parameters while simultaneously identifying component populations within a dataset. The method involves fitting a finite mixture of Weibull distributions to development time data using the Expectation-Maximization (EM) algorithm. This allows for the inclusion of individual-level variability in the estimation process. We tested the method using previously published developmental data on two beetle species,
Creophilus maxillosus
and
Necrodes littoralis
(Staphylinidae). Our approach yielded 95% intervals with coverage close to the nominal level, in contrast to Ikemoto and Takai’s method, which captured only 59% and 75% of actual cases, respectively. These findings suggest that our method improves the accuracy of insect-based postmortem interval estimates in forensic entomology and, more broadly, provides a general framework for interval estimation of developmental parameters applicable in thermal ecology and applied entomology.
Journal Article
A forensic toolkit for the “cheese skipper”: Larval identification and thermal requirements of the immature stages of Piophila casei (L.) (Diptera: Piophilidae)
The ‘cheese skipper’, Piophila casei (L.) (Diptera: Piophilidae), is a cosmopolitan and synanthropic species that frequently colonises cadavers in advanced stages of decomposition. Despite its potential as a forensic indicator, the use of P. casei in casework is hampered by (i) some contradictory descriptions of its larval morphology, which limit the reliability of the identification of specimens collected at the forensic scene, and (ii) the lack of reference data and temperature-dependent models, which hinders the accuracy of minimum postmortem interval (minPMI) estimates based on the developmental rates of this species. Here we determine and illustrate a series of diagnostic morphological characters that allow for the unequivocal identification of P. casei larval specimens. The shape and degree of sclerotization of the larval cephalopharyngeal skeleton and the number and arrangement of the rows of spines of the ventral creeping welts are particularly useful allowing not only for species identification, but also for larval instar determination. In addition, we analyse the influence of temperature on the development of the different immature stages of P. casei, providing an isomorphen diagram and determining the thermal requirements (lower developmental threshold and thermal summation constant) for five developmental events, enabling accurate minPMI estimates in forensic casework. In addition, within the intra-puparial period, we determine a series of age-specific morphological markers that are easily observable through the translucent puparium of P. casei, thus increasing the accuracy of minPMI estimates and avoiding the potential risk of sample damaging during puparium dissection when using puparial samples.
•The morphology of the larval instars of Piophila casei was studied using LM and CLSM.•Diagnostic characters for a reliable identification of P. casei larvae are provided.•The influence of temperature on P. casei development is analysed.•Baseline developmental data for the different immature stages are provided.
Journal Article
The Entomo-Toxicological Effect Of Dichlorvos on Cuticular Hydrocarbon Profiles of Some Sarco-Saprophagous Insects for Forensic Applications
Forensic entomology has relied on species-specificity, quantitative and qualitative variations of cuticular hydrocarbons to successfully carry out chemotaxonomic identification of insects based on species, age and gender. This work studied the effects of dichlorvos on the cuticular hydrocarbon profiles of some adult sarco-saprophagous insects of forensic importance that fed on dichlorvos-poisoned carrions for utility during death investigations. Cuticular hydrocarbons (CHCs) were extracted from adult insects of the species
Chrysomya albiceps, Sarcophaga exuberans, Musca domestica
,
Hermetia illucens
,
Dermestes maculatus
and
Necrobia rufipes
from both dichlorvos-poisoned and control pig (
Sus scrofa
Linnaeus) carrions and subjected to chemotaxonomic profiling using Gas Chromatography-Mass Spectrometry (GC–MS). A total of 41 CHCs were successfully identified from insects of both the dichlorvos-poisoned and control carrions ranging from C8 to C33 carbons consisting of majorly the n-alkanes, methyl branched alkanes and an alkene. There was a higher abundance of CHCs present in the insects of dichlorvos-poisoned carrions than the control group. The highest mean peak concentration and abundance of the CHCs was recorded by 2,6,10,14 -Tetramethyl Pentadecane (10.38 ± 0.53 μg/mg for dichlorvos-poisoned carrions and 8.99 ± 1.13 μg/mg for the control carrions). The visualization of the species-specific differences in CHCs compositions showed less overlapping CHCs clusters and quantitative metrics of principal component analysis plots of the insects from both carrion groups with high eigenvalues > 3 which were indications of good species level discrimination. The study showed that insects’ CHCs profiles of dichlorvos-poisoned and control carrions exhibited uniqueness cum variations in terms of abundance and chemical identity.
Journal Article