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Bambaradeniya, T.B.; Magni, P.A.; Dadour, I.R. Concepts, Procedures and Techniques by Forensic Entomologists. Encyclopedia. Available online: (accessed on 24 April 2024).
Bambaradeniya TB, Magni PA, Dadour IR. Concepts, Procedures and Techniques by Forensic Entomologists. Encyclopedia. Available at: Accessed April 24, 2024.
Bambaradeniya, Tharindu B., Paola A. Magni, Ian R. Dadour. "Concepts, Procedures and Techniques by Forensic Entomologists" Encyclopedia, (accessed April 24, 2024).
Bambaradeniya, T.B., Magni, P.A., & Dadour, I.R. (2023, June 14). Concepts, Procedures and Techniques by Forensic Entomologists. In Encyclopedia.
Bambaradeniya, Tharindu B., et al. "Concepts, Procedures and Techniques by Forensic Entomologists." Encyclopedia. Web. 14 June, 2023.
Concepts, Procedures and Techniques by Forensic Entomologists

Forensic entomology is a branch of forensic science that incorporates insects as a part of solving crime. Insect-based evidence recovered at a crime scene can be used to estimate the minimum postmortem interval, determine if a carcass/corpse has been relocated, and contribute to the cause and manner of death. Sampling and preservation of insect material is paramount if this evidence is presented in court.  To this end, a qualified forensic entomologist should attend the crime scene, however this is not always the case and such evidence is collected and preserved by a proxy.  After reading this paper and following a number of essential protocols, a proxy should be able to submit insect evidence that a forensic entomologist may be able to use and present a best estimate of the time since death.

casework crime scene skill base entomology procedures

1. Introduction

Forensic science is a crucial constituent in crime-solving that agglutinates the investigation with the subsequent judicial activities [1][2]. Today, forensic science has diverged into numerous subfields; some of the more widely known sciences are forensic entomology, forensic toxicology, forensic anthropology, and forensic microbiology [2][3]. These disciplines provide principles and procedures to recover and analyse crime-associated evidence to reconstruct the actual circumstances of a crime scene [1]. This facilitates in the determination of a crime, which eventually results in a prosecution [4].
The physical evidence recovered from a crime scene is primarily used to verify offenders while exonerating any innocent persons, as well as how, when, and where the crime was committed [5]. The recovered physical evidence provides either observation-based clues or may need to be processed by advanced techniques to arrive at further conclusions. As an example, a microscopic analysis of hair could provide information such as the age, gender, and race of an individual present at a crime scene. This may be followed up with a DNA analysis and spectroscopic analysis, providing details relating to an individual’s identification and the presence of explosive and drug residues, respectively [6].

2. Forensic Entomology for Crime Scene Investigations

What is forensic entomology? It is known for using insects and other arthropods to aid in solving criminal and civil crimes [7][8]. Generally, forensic entomology involves three main subcategories: urban, stored product, and medicolegal entomology [6]. Urban entomology investigates the insect-related structural defects of buildings and medical myiasis [9][10]. Stored product entomology is used to assess biosecurity risks associated with food contamination and environmental pollution [11]. The last category, medicolegal entomology, uses insect-based evidence to investigate numerous criminal activities associated with homicides, suspicious fatalities, abuse of vulnerable people, hospital neglect, animal cruelty, contraband trafficking, and wildlife poaching [7][8][12].
The primary function of insects in crime solving is as biological indicators to estimate the time elapsed since death or the minimum postmortem interval (minPMI) [13]. Furthermore, the colonisation time of dipteran larvae in myiasis wounds can provide an indication of the time of neglect for humans, pets, and livestock [14]. The two fundamental principles of utilising insects are determining the minPMI based on their predictable temperature-based development and the sequential colonizing patterns on carcasses [7]. Flies (Order Diptera) are the main protagonists of all the carrion arthropods that are predominantly considered for use in determining the postmortem interval due to their involvement in carcass decomposition as both early and enduring colonisers [15][16]. The role of flies in carcass decomposition interchanges between scavengers of decaying tissues and prey to other predacious invertebrates (e.g., beetles and ants) and vertebrates (e.g., hyenas and vultures) [16][17] (Figure 1a–d).
Figure 1. The different life stages of a fly. The measurements added are from Byrd and Tomberlin, 2020 [7]. (a) Fly egg raft (size range of single egg 1.2–2 mm). (b) Three larval instars (in ascending order; third, second, first instar (size range from 2–23 mm). (c) Fly pupa (size range from 6–23 mm). (d) Adult fly (size range from 6–14 mm).
Typically, the minPMI for early stages of carcass decomposition (i.e., 3–72 h following death) is estimated by pathological methods [18]. However, after this period, fly eggs, larval, and pupal specimens become one of the most reliable indices for the age determination of a carcass [19][20]. This is achieved by three methods: (1) calculating the accumulated degree day/hour units, (2) referring to life history tables, and (3) using species-specific isomegalen/isomorphen curves [21]. In addition, the minPMI in advanced stages of decomposition can be estimated by assessing the succession patterns of flies and other carrion-associated invertebrates, although this is generally less accurate [22]. However, estimating the minPMI based on fly development in advanced stages of decomposition has some drawbacks, for example, the fly larvae and full puparia sampled may not represent the earliest colonisers, which may have already completed their life cycle [23][24]. Generally, the predicted sequence of attracting flies to carrion begins with the Calliphoridae, followed by Sarcophagidae, Muscidae, Sphaeroceridae, Piophilidae, Fannidae, and Phoridae [7][21]. Carcasses are also colonised by beetles (Staphylinidae, Scarabaeidae, Carabidae, Silphidae, Cleridae, and Dermestidae); moths (Pyralidae and Tineidae); and mites (Pterygosomatidae and Omentolaelapidae), which can also be forensically significant [25][26]. Furthermore, the sequence of these invertebrates invading a carcass can vary based on external and internal influences, such as changes in the carcass characteristics (size, nutrient composition, existing decomposition stage, and xenobiotics), abiotic (temperature and humidity), and biotic (terrestrial and aquatic habitats) factors [24].
Apart from estimating the minPMI, entomological assessments in some cases can be made to determine the transfer of carcasses from their original crime locations [27]. This is underpinned by the concept that the entomofauna of a carcass resembles the overall insect diversity of the environment where it originally resided, and any secondary movement to an alternative place could change this crime location-specific entomofauna [28][29]. This is generally context driven, depending on the crime scene location and environmental conditions. In addition, disturbance to a carcass may cause any fly larval aggregations to disperse, which are especially visible when densely packed larval masses are present [7]. Additionally, there is a potential use of the characterised internal and external microbial profiles of larval masses infesting carcasses to verify the originality of a crime scene. This could be achieved by assessing the homogeneity of microbial profiles between the larval masses and the crime scene [30].
It has been widely demonstrated that fly tissues can be potentially used for victim identification and determining the cause of death under entomogenetics and entomotoxicology [31][32]. This is crucial when the tissues and fluids of victims are not available to recover from the body for analysis due to the occurrence of tissue degradation by larval feeding, scavenging, and burning [33][34]. An analysis of the contents of parts of the fly alimentary tract using DNA may also reveal the identity of a victim [35]. To identify fly species, DNA can also be extracted from flies at various stages of life, followed by sequencing species-specific nucleotides of their genes, and then referring these to a genetic database [31][32]. Moreover, the age estimation of larvae and pupae based on gene expression is another molecular approach [36].

2.1. Forensic Entomologist: An Apprentice to an Expert

The analyses of entomofauna at a crime scene is complex, involving sampling and identifying insects, determining species–habitat relationships, evaluating the development stages and successional patterns, and assessing insect ethology concerning variations in climatic parameters [37]. Becoming a practitioner in forensic entomology requires training in the fundamentals of entomology and ecology. To conduct these tasks, an FE must understand concepts such as insect physiology, taxonomic and phylogenetic relationships, the behavioural adaptation of insects for different ecological setups, and their impacts on human affairs [38][39]. This training should then be narrowed down to specific topics that directly address the diagnostic characteristics and interrelationships of forensically important insect groups. This subset of knowledge related to forensic entomology can be acquired by following an undergraduate or postgraduate level specialised course or a degree program [40].

2.2. Forensic Entomology in Crime Investigation: Complexity and Constraints

Insects play a pivotal role in carcass decomposition [41]. Flies and other arthropods visit a carcass if there are no physical barriers to block their access. When the food source (carcass) is exhausted, the insects leave [42]. The insect colonisation of a carcass primarily relies on the seasonal- and regional-based climate, including temperature, humidity, and photoperiod. It also relies on the intra and interspecies dynamics, including feeding preferences and competition and various habitat scenarios such as terrestrial and aquatic, urban and rural, indoor and outdoor, and burial and exposed [24]. In addition, secondary factors such as the method of death, including burning, poisoning, drowning, dismemberment, concealment, and hanging, as well as carcass features such as age, size, clothing, and trauma also influence the patterns of insect colonisation while impacting on the decomposition process.
Besides these carcass insect complexities, an FE must also deal with many regulatory challenges such as interdisciplinary cooperation with other investigators at crime scenes, including other scientists and forensic field agents, strict deadlines to submit expert witness statements, and courtroom conflicts [43][44].

2.3. Progression and Standardisation in Forensic Entomology

Several early classic works by Mégnin (1894) [45] and Payne (1965) [46] highlighted the importance of insects in contributing to the carrion decomposition process. These studies laid the conceptual framework to utilise insects in solving crime. Mégnin (1894) [45] proposed the first formal definition and testable mechanism of insect succession in carrion, and Payne (1965) [46] emphasised the significant involvement of insects in reducing carrion biomass in the early stages of decomposition. Following these formative works, many studies have been conducted in different regions of the world to introduce identification keys for carrion insects, document carrion-associated insect succession under different temporal and spatial conditions, record fly development periods under field and laboratory conditions, and unite forensic entomology with other related fields such as genetics, toxicology, and microbiology [13][47]. These findings have been incorporated into many crime investigations, published as casework in books and scientific journals, presented in conference proceedings, and sometimes proffered by popular media.
The standardisation of entomological methods developed for solving crime has strengthened the science of forensic entomology and has allowed this science to gain more visibility and acceptability among law enforcement agencies. Several crucial studies have been published recently that critically evaluate many successional and development studies, bridging the gap between research and casework. This has led to the improvement of standards and protocols for the FE to use in real crime situations [21][48][49][50].
Numerous attempts have been made to propose global standards to assist an FE in performing their work logically and systematically under field and laboratory conditions [7][49][51][52]. These standards outline the basic procedures and the equipment required to conduct entomology-associated tasks by an FE at a crime scene and in a laboratory to generate accurate estimates of the minPMI [7][52]. However, these standards are not rigid and can be modified based on the degree of training and experience of the practitioner, availability of resources, and the circumstances of the crime scene. The three standards presented here (Gold, Silver, and Bronze) do not substantially change the basic procedures but provide some alternative ways to limit any misinterpretations [7][53].

3. Crime Scene and Health Pathology Facility

The following procedures fall into two major categories based on the location where specific entomological interpretations are undertaken: crime scene/pathology facility and the entomology laboratory [7][54].
Generally, FEs or proxies collect insect evidence at a crime scene, or later at a health pathology facility, where autopsies are usually conducted. The remains are assigned to these facilities by police, pathologists, and medical examiners/coroners. A request then follows if insect material is evident to determine the minPMI and/or provide a general statement about the entomological context of the crime scene [8][53].
At any of these sites, an FE or proxy must perform several standard tasks, namely, the collection of the crime scene parameters (being present or via video and photos), including climatic parameters, insects, and their remnants (e.g., mouth parts, appendages, wings, empty puparia). Following collection, some insects may be preserved and stored, and some placed (40% of eggs larvae and full puparia) on a medium to be reared, followed by transporting these live and dead specimens to an entomology laboratory for further analysis [7][51][54].
An FE or proxy should adhere to the general guidelines set by the law enforcement agency controlling the crime scene [53]. An FE or proxy, similar to all other investigators at a crime scene, must wear the appropriate clothing and personal protection equipment (e.g., closed-toed shoes, long pants, scrubs, (disposable overalls) and masks) to avoid any possible contamination and biohazard risks [7][53]. They must visit the scenes with the necessary equipment and documentation to fulfil three major purposes. Firstly, collecting insects (live and/or dead): labelled plastic containers and vials, a handheld lens, hot water (insulated bottle), forceps, fined tipped artist paint brushes, an insect net, bedding materials (e.g., dry sand, sawdust, vermiculite), and food sources (e.g., beef, pork pieces) for rearing purposes, and ethanol (ethyl alcohol) for killing and preserving insects. Secondly, gather the microclimatic data: a portable weather gauge, an infrared thermometer, and a temperature data lodging device. Thirdly, document necessary information via prepared data entry sheets. An FE should have an a priori knowledge of the standard operating procedures of law enforcement at a particular location and this should determine if other devices such as a camera and a video recorder should be included equipment [7][53].

3.1. Collection of Onsite Data

The collection and preservation of entomological evidence at a crime scene should be carried out as soon as possible after a body is discovered [55]. The delayed recording and sampling of entomofauna could potentially generate misleading information. This is due to the development, succession, and orientation patterns of carrion insects, which continuously alter with climatic changes and other interruptions caused by inadvertent destruction by investigator activities and scavenging of the remains by other vertebrates and invertebrates [56].
Due to a possible high risk of damage and contamination, the insect evidence should be safeguarded by the law enforcement agency protocols at a crime scene. When law enforcement establishes a crime scene perimeter, it is essential to consider the area where insect activity may be evident in and around a carcass [57]. This is dependent on the decomposition stage and the age of the immature insects on the remains [53]. Specifically, eggs and feeding larval stages of flies are restricted to the carcass; however, post-feeding larval and pupal stages can be dispersed underneath the carcass, within the surrounding cadaveric soil, some distance from the remains and buried in the soil. In dwellings, these stages maybe located near physical objectives present in the vicinity of the carcass (e.g., under carpets, cupboards, and other household items) [7][54]
It is essential to request from the forensic investigator in charge any related information concerning the management of the scene. This should include photographs and video footage from any initial visits of investigators to the scene, and any evidence of physical alterations made to the immediate environment after the remains were discovered (e.g., opening windows, switching on/off heaters or air conditioners, and placing light sources to conduct investigations at night). A potential bias associated with a crime scene is informing the FE or proxy about the date of the last sighting of the deceased. Such information should ideally only be obtained after estimations of the minPMI have been ascertained by the FE [44].
Following the arrival at a crime scene and consulting with law enforcement, the FE or proxy should be able to view the remains and observe the patterns of insects on and around the carcass. Following this assessment, the FE or proxy can realise what tools are required to work the scene. Sometimes, large larval aggregations may be present in and around the natural orifices of carcasses, such as in and around the mouth, nostrils, ears, and the genital region [58]. These body regions provide protection and suitable soft tissues for larval feeding in the early stages of development [58][59]. These early colonizing sites can also occur on other parts of the body based on factors such as surface humidity, insulation, body orientation, clothing, and the wrapping of bodies and wounds [59].

3.2. Collection of Microclimatic Data at a Crime Scene

Insects are poikilothermic, their body temperatures fluctuate along with the ambient temperatures to be in homeostasis [60]. Therefore, entomological assessments at crime scenes should be conducted concerning ambient temperatures, as it affects the development and succession patterns of carrion insects [61][62]. Specifically, temperature impacts insect development by regulating their movement, metamorphosis, reproduction, and diapause [63]. In contrast, succession patterns change with temperature fluctuations as it affects the microbial activities associated with the carcass decomposition [64].
The best available source to obtain environmental temperature readings at a crime location is the crime scene itself matched to the temperatures from the nearest meteorological station [51]. Investigators can request daily or hourly temperature data for as many days as required prior to discovering the remains [7][51][65]. However, when cadavers are inside a dwelling, these meteorological station data are only helpful if the indoor space is open to the outdoor by windows, doors, and vents. In such situations, the outside temperature data must be paired with the indoor temperatures when constructing the thermal simulation model for the crime scene environment [66].
Apart from the environmental temperatures, body, water, soil, and larval mass temperatures should also be recorded based on the context of the crime. A contactless infrared thermometer can measure the body, and larval mass temperatures, and the soil and water body temperatures can be recorded by a temperature probe [7][53]. The decision of the temperature data most relevant for minPMI estimations must be made based on their degree of heat transfer for larval development. 

3.3. Recovery of Insects for Storage or Rearing Purposes

The specimens collected at a crime scene should be a clear cross-section of the different development stages (eggs, larvae, full and empty puparia, and adults) of various insect successional groups (flies, beetles, mites, and moths) wherever located on the carcass and its surrounding environment [51]. The number of individuals recovered at each stage of development must be adequate for conducting a range of entomological assessments in the laboratory (i.e., species identification, PMI estimation, and xenobiotic determination). However, these sampling numbers depend on their availability and could range from a single egg or larva to several egg rafts and larval masses [67].
Insects collected at a crime scene should be divided into two batches, one batch preserved and the other reared to adult. This is generally performed at the scene, but if appropriate equipment is unavailable, then they must be cooled (not frozen) to slow down development and dispatched rapidly to a laboratory [7]. Preserved specimens are used for identifying the species, determining their age at the time of sampling based on their instar stage, the larval length and width measurement, and extracting gut contents for genetic and toxicological analysis [68].

3.3.1. Preservation of Insects

As mentioned above, insects recovered from a carcass should be preserved at their collection point [7]. When kept alive in containers to preserve for a later time and not cooled, their age status will alter, may exacerbate some cannibalistic behaviour, and reduce the number of collected larvae for further analyses [69]. In such circumstances, the development rate is compromised, and such samples should not be used to estimate the minPMI [69]. However, if a complete record of the thermal history of a sample is provided, an FE should be able to backtrack the age of the insects at the collection time and extrapolate useful information concerning the minPMI for the investigators.
Fly eggs and adult stages can be difficult for estimating the PMI, as limited knowledge is available pertaining to only a few species [7][70]. However, sometimes, the only samples collected are these life history stages, and a practiced FE may be able to identify the morphological changes that occur during egg embryogenesis [71], as well as using some of the methods described by Tyndale-Biscoe (1984) [72] with regard to the age of adults, especially those captured inside a dwelling. Once eggs are located, they can be collected after differentiating them from soil and litter particles using a handheld lens and a fined-tipped artist paint brush [73]. In contrast, the adult flies at the scene can be trapped using an insect net. Following capture, they can simply be removed by dipping or spraying the fly with 70% alcohol and then transferring the dead specimen to a vial. If alcohol is unavailable, then the adult fly can be captured in the net and carefully placed into an insect killing jar prior to preservation [7].
Beetles collected at a crime scene should be handpicked using forceps and hot water, dipped to kill them before being placed in vials containing 70% ethanol. Beetles may be observed by thoroughly examining the litter surrounding the carcass as well as sieving the soil under the carcass to a depth of approximately 20 cm [74]. It is worthwhile to mention here that Silver and Bronze personnel should collect all insects associated with the corpse and allow the FE to make the necessary decisions on their relevance.
When dealing with a crime scene located in an aquatic environment, the larval stages of freshwater insects such as mayflies (Order: Ephemeroptera), stone flies (Order: Plecoptera), and caddis flies (Order: Trichoptera) should be collected from submerged and floating bodies into plastic vials (70% ethanol) using forceps. Once again, it is recommended that Silver and Bronze personnel collect all aquatic fauna that they observe associated with the corpse.

3.3.2. Preparation of Insects at the Crime Scene for Rearing Purposes

The live insects for rearing purposes collected at the crime scene should be placed on a dish (Petri dish, foil) with a suitable food source (e.g., meat). This dish is then placed on 2–3 cm of bedding material (sawdust, vermiculite, builders’ sand) inside a container with a mesh lid [7]. When preparing full puparia for transporting to the laboratory, they can be directly transferred into containers half filled with bedding material with a standard lid. No feeding source or ventilation is required if all insect material is transported inside a cooler or kept refrigerated.

3.4. Laboratory

The following sections pertain to the work that an FE must conduct on either the material collected by themselves, or the material collected by their proxy. To identify, measure larval, pupal, and adult external body characters and rear immatures to adult stage. An entomology laboratory should be equipped with the following basic apparatus: stereo microscopes, vernier callipers, growth chambers or temperature-regulated rooms, preservative chemicals (ethanol), gloves, forceps, scalpels, and Petri dishes.

3.5. Identification

The identification of specimens to species level should be completed first. Without this knowledge, other analyses will be compromised [75]. Generally, insect identifications are made using morphological and/or molecular methods. Other techniques such as hyperspectral or CT imaging, tomography, and chemical techniques may also be used; however, each has associated pros and cons according to expense and availability [76][77]. Nonetheless, whichever technique is used, it should provide the best identification outcome. In fact, the most informative and best identification is to send the collected specimens relevant to the minPMI to a dipteran taxonomist. The following sections discuss the two main techniques used to identify insects: the morphological identification and molecular identification.
The morphological identification of carrion insects to species level can be achieved using taxonomic keys and standard or electron microscopic images [78][79]. Dichotomous taxonomic keys provide a stepwise assessment using morphological characters that guide the user to identify the species [75]. The level of accuracy and confidence in using taxonomic keys for species determination develops with a skill base in entomology a
Ultrastructure studies of eggs, larvae, full and empty puparia, and adult stages of different fly species by scanning electron microscopes (SEM) aid in precise species identification. Typical egg-associated ultrastructures are micropylar plate, median area, respiratory plastron, hatching lines, and chorion sculpturing [80][81]. The suite of larval characters includes the posterior spiracles, respiratory slits, peritreme ornamentation, anterior spiracles, cephaloskeleton, and spine arrangement [81][82]. When identifying full puparia, characteristics such as bubble membranes and respiratory horns are used, along with spine arrangements and posterior spiracles that are correlated with their preceding larval structures [77][80][81][83]

3.6. Examining Larvae and Full Puparia for Age Determination

Development studies provide time frames required for immature flies that can be used as indications to estimate the minPMI [21]. The instar of fly larvae can be determined by a microscopic observation of the number of respiratory slits available at their posterior spiracles [84]. Typically, a first instar blow fly larva has a single slit, whereas second and third instar larvae have two and three slits, respectively. In contrast, pre- and post-feeding stages of third instar larvae are discriminated by their body colour change (translucent to opaque), a food distended crop, body compaction, and wandering behaviour away from the carcass [21].
In addition, the length and width of larvae can also be considered as an indication of age [85]. The length and width of larvae can be measured by a stage micrometre and a vernier calliper. The correlation of actual length and width data of a sampled individual with reference values given in previous publications aid in determining the age of the specimen, hence the PMI. However, temperature variations, the type of food substrates, and the presence of xenobiotics in it reflect on any of these age determinations.
Furthermore, in previous studies, length and instar stage variation of larvae under different constant temperature regimes were demonstrated using isomegalen and isomorphen curves, respectively. These curves are available for species such as, Calliphora vicina (Robineae-Desvoidy), L. sericata, Ch. albiceps, Protophormia terraenovae (Robineae-Desvoidy), Ch. megacephala, and Liopygia argyrostoma (Robineae-Desvoidy) [86][87][88].
Pupal morphogenesis can be considered for minPMI estimations as the full puparia coexist with carcasses over a prolonged period [89][90]. The extent of the external organ development of pupae can be examined via a stereo microscope after removing the puparium using a surgical knife and forceps. A photograph showing the status of the external organ development of a sampled pupa can be coupled with a reference photograph series given for the same species in a previous publication for the age determination. However, it is essential to consider the temperature variations of the environment that the full puparia were exposed to prior to sampling, as low temperatures can delay pupal morphogenesis [91].

3.7. Rearing

The rearing of eggs, larval, and pupal specimens until the adult stage can often facilitate a more accurate species identification [92]. Adult-level identification has a greater certainty in correct species determination, and they are well-described in taxonomic keys [93]. When rearing dipteran species in an insectary, they should be confined in insect cages and provided ad libitum with food, water, and oviposition substrates [15][21].
Immature stages are best reared in growth chambers whereby temperature, humidity, and photoperiod can be controlled. Such development studies are used to determine the time duration required to ascertain the development stage of the sampled species and their larval length and width changes. It is recommended to use swine muscle as larval food for these growth chamber studies as the domestic pig (Sus scrofa L.) is primarily considered as the proxy of humans when conducting development studies [94].

3.8. Xenobiotic Detection

The utilisation of insects for xenobiotic detection relies on the prolonged tissue retention of drugs, pesticides, and metals, leading to high sensitivity for analytical detection techniques [95][96].
The fly larval, pupal, and adult stages collected at a crime scene can be used as a source to extract and detect chemicals. Previous studies showed that larvae collected from internal organs such as liver, or from the head and muscles of a carcass retain high concentrations of chemicals [97].
The larvae sourced from these regions should be kept alive and starved for several hours prior to analysis to ensure the absorption of any chemicals into their metabolic system. It is recommended to store killed larvae under dry conditions at −20 °C. Such steps will ensure a higher retention and concentration of chemicals because storing in ethanol may diminish trace volumes of drugs in larvae [97][98].

4. Documentation

4.1. Referring to the Literature

The accuracy of establishing a successful entomological interpretation of the circumstances at a crime scene depends on the empirical analysis developed by an FE throughout their career [40]. One of the best ways of acquiring and upgrading the knowledge and skills associated with insects for solving crime is referring to previously published research and casework [99]. These publications essentially guide a practitioner to incorporate the available knowledge into procedures and provide a comprehensive guideline for researchers and academics to develop training models and conduct future research [99].

4.2. Fly Development Studies

Development studies outline the variations in the fly life history [21]. The data from these development studies can be directly incorporated to estimate the minPMI, as these publications essentially contain life history tables and isomegalen/isomorphen curves. The timeline data given in life history tables and the lower development threshold temperatures can be used to calculate the ADD/ADH [23][100], and hence, the minPMI. However, when selecting a previous development study to corroborate a minPMI estimation of a particular fly species, the FE should determine if the temperature regimes, photoperiod and humidity, and larval feeding tissue type, along with the location where the source colonies originated, are appropriate to use [101].

4.3. Insect Successional Studies

Insect successional studies provide a descriptive account of insect colonisation patterns concerning the decomposition changes in carcasses. These studies are either observational or experimental, and both are directed to gather information such as insect checklists, environment temperature, and rainfall data of the study period, and the characteristics and duration of the decomposition stage [102].

4.4. Casework

Occasionally, a case is published which is the combination of facts and opinions of an FE who investigated a particular crime scene and may be relevant to the current case under investigation. Reference to casework can provide guidance to produce a robust witness statement but can also provide insight into areas where further research may benefit better interpretations.


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