An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

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.

Larval anatomy of the digestive and excretory systems of the pharyngeal bot fly, Pharyngomyia picta (Diptera: Oestridae).

Oestrid flies (Diptera: Oestridae) are obligate parasites of mammals during their larval stage and show anatomical adaptations for the infestation of host tissues. Unlike the species that parasitize domestic mammals, those oestrid species that infest wild mammal hosts remain poorly known. With the use of x-ray micro-computed tomography, we describe for the first time the anatomy of the digestive and excretory systems of the second and third larval instars of Pharyngomyia picta (Meigen), a parasite of cervids that, like other species within the subfamily Oestrinae, causes nasopharyngeal myiasis. Both larval instars of P. picta show a pair of remarkably large salivary glands arranged in a characteristic 'glandular band', a convoluted and thickly uniform midgut and a greatly enlarged distal region of the anterior pair of Malpighian tubules. These anatomical features also have been described in other species within the subfamily Oestrinae, whereas they differ from the observations in other oestrid subfamilies. We discuss the potential functional significance of the anatomy of the digestive and excretory systems of Oestrinae larvae as specific adaptations to parasitize the nasopharyngeal cavities of mammal hosts.

Larval morphology of the avian parasitic genus Passeromyia: playing hide and seek with a parastomal bar.

The enigmatic larvae of the Old World genus Passeromyia Rodhain & Villeneuve, 1915 (Diptera: Muscidae) inhabit the nests of birds as saprophages or as haematophagous agents of myiasis among nestlings. Using light microscopy, confocal laser scanning microscopy and scanning electron microscopy, we provide the first morphological descriptions of the first, second and third instar of P. longicornis (Macquart, 1851) (Diptera: Muscidae), the first and third instar of P. indecora (Walker, 1858) (Diptera: Muscidae), and we revise the larval morphology of P. heterochaeta (Villenueve, 1915) (Diptera: Muscidae) and P. steini Pont, 1970 (Diptera: Muscidae). We provide a key to the third instar of examined species (excluding P. steini and P. veitchi Bezzi, 1928 (Diptera: Muscidae)). Examination of the cephaloskeleton revealed paired rod-like sclerites, named 'rami', between the lateral arms of the intermediate sclerite in the second and third instar larva. We reveal parastomal bars fused apically with the intermediate sclerite, the absence of which has so far been considered as apomorphic for second and third instar muscid larvae. Examination of additional material suggests that modified parastomal bars are not exclusive features of Passeromyia but occur widespread in the Muscidae, and rami may occur widespread in the Cyclorrhapha.

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

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.

Cuticular hydrocarbons for identifying Sarcophagidae (Diptera).

The composition and quantity of insect cuticular hydrocarbons (CHCs) can be species-specific as well as sexually dimorphic within species. CHC analysis has been previously used for identification and ageing purposes for several insect orders including true flies (Diptera). Here, we analysed the CHC chemical profiles of adult males and females of eleven species of flesh flies belonging to the genus Sarcophaga Meigen (Sarcophagidae), namely Sarcophaga africa (Wiedemann), S. agnata Rondani, S. argyrostoma Robineau-Desvoidy, S. carnaria (Linnaeus), S. crassipalpis Macquart, S. melanura Meigen, S. pumila Meigen, S. teretirostris Pandellé, S. subvicina Rohdendorf, S. vagans Meigen and S. variegata (Scopoli). Cuticular hydrocarbons extracted from pinned specimens from the collections of the Natural History Museum, London using a customised extraction technique were analysed using Gas Chromatography-Mass Spectrometry. Time of preservation prior to extraction ranged between a few weeks to over one hundred years. CHC profiles (1) allowed reliable identification of a large majority of specimens, (2) differed between males and females of the same species, (3) reliably associated males and females of the same species, provided sufficient replicates (up to 10) of each sex were analysed, and (4) identified specimens preserved for up to over one hundred years prior to extraction.

The Relationship between Research and Casework in Forensic Entomology.

Research is a vital component of all forensic sciences and is often stimulated by casework, which identifies gaps in our knowledge. In such a niche area of forensic science as entomology there should be a close and mutually beneficial relationship between research and casework: to some extent there is a continuum between the two and many forensic entomologists are involved in both to a greater or lesser degree. However, research and casework involve quite differing challenges, from the replicated, highly controlled, sometimes esoteric aspects of research to the very individual, sometimes chaotic and disruptive, but highly applied aspects of casework. Ideally casework will include the full involvement of a forensic entomologist, who will collect the insect and climate evidence at the scene and produce a robust expert witness statement based on a full analysis of this data. Unfortunately, it can also include situations where samples, if collected at all, are poorly preserved, not representative of the full cadaver fauna available and presented to the entomologist months or years after the event, without local temperature data. While research is recognised through publications and their citation indices, casework and its associated expert witness statements often receive no credit in an academic workplace, although they do have a positive societal impact and many other benefits of teaching and public engagement value. This manuscript examines the relationship between research and casework from a UK perspective, to raise awareness of the need to create an environment that values the contribution of both, for future generations to flourish in both areas.

Multidisciplinary investigation of two Egyptian child mummies curated at the University of Tartu Art Museum, Estonia (Late/Graeco-Roman Periods).

Two ancient Egyptian child mummies at the University of Tartu Art Museum (Estonia) were, according to museum records, brought to Estonia by the young Baltic-German scholar Otto Friedrich von Richter, who had travelled in Egypt during the early 19th century. Although some studies of the mummies were conducted, a thorough investigation has never been made. Thus, an interdisciplinary team of experts studied the remains using the most recent analytical methods in order to provide an exhaustive analysis of the remains. The bodies were submitted for osteological and archaeothanatological study, radiological investigation, AMS radiocarbon dating, chemical and textile analyses, 3D modelling, entomological as well as aDNA investigation. Here we synthesize the results of one of the most extensive multidisciplinary analyses of ancient Egyptian child mummies, adding significantly to our knowledge of such examples of ancient funerary practices.

The use of wing fray and sex ratios to determine the origin of flies at an indoor crime scene.

When adult flies are collected at indoor crime scenes, the question of their origin arises, i.e., whether they originated from the outdoors, coming in to oviposit on the body, or if they developed through the larval and pupal stages from eggs laid on the body. This is particularly important if no empty puparia were collected, potentially because they were overlooked, with a consequent impact on the accuracy of the minimum post-mortem interval estimation. This study used two methods to determine if flies sampled in various experimental conditions and at an actual crime scene were wild flies attracted to the body or had developed on the body, i.e., through the assessment of: (1) wing fray and; (2) sex ratio. A template was created to measure the amount of wing fray damage quantitatively in a simple, rapid and efficient manner. The experiments demonstrated that by combining the two methods it is possible to establish the origin of a population of adult flies at a crime scene.

Pigs vs people: the use of pigs as analogues for humans in forensic entomology and taphonomy research.

Most studies of decomposition in forensic entomology and taphonomy have used non-human cadavers. Following the recommendation of using domestic pig cadavers as analogues for humans in forensic entomology in the 1980s, pigs became the most frequently used model cadavers in forensic sciences. They have shaped our understanding of how large vertebrate cadavers decompose in, for example, various environments, seasons and after various ante- or postmortem cadaver modifications. They have also been used to demonstrate the feasibility of several new or well-established forensic techniques. The advent of outdoor human taphonomy facilities enabled experimental comparisons of decomposition between pig and human cadavers. Recent comparisons challenged the pig-as-analogue claim in entomology and taphonomy research. In this review, we discuss in a broad methodological context the advantages and disadvantages of pig and human cadavers for forensic research and rebut the critique of pigs as analogues for humans. We conclude that experiments using human cadaver analogues (i.e. pig carcasses) are easier to replicate and more practical for controlling confounding factors than studies based solely on humans and, therefore, are likely to remain our primary epistemic source of forensic knowledge for the immediate future. We supplement these considerations with new guidelines for model cadaver choice in forensic science research.

Estimating crime scene temperatures from nearby meteorological station data.

The importance of temperature data in minimum postmortem interval (minPMI) estimations in criminal investigations is well known. To maximise the accuracy of minPMI estimations, it is imperative to investigate the different components involved in temperature modelling, such as the duration of temperature data logger placement at the crime scene and choice of nearest weather station to compare the crime scene data to. Currently, there is no standardised practice on how long to leave the temperature data logger at the crime scene and the effects of varying logger duration are little known. The choice of the nearest weather station is usually made based on availability and accessibility of data from weather stations in the crime scene vicinity. However, there are no guidelines on what to look for to maximise the comparability of weather station and crime scene temperatures. Linear regression analysis of scene data with data from weather stations with varying time intervals, distances, altitudes and microclimates showed the greatest goodness of fit (R2), i.e. the highest compatibility between datasets, after 4-10 days. However, there was no significant improvement in estimation of crime scene temperatures beyond a 5-day regression period. The smaller the distance between scene and weather station and the higher the similarity in environment, such as altitude and geographical area, resulted in greater compatibility between datasets. Overall, the study demonstrated the complexity of choosing the most comparable weather station to the crime scene, especially because of a high variation in seasonal temperature and numerous influencing factors such as geographical location, urban 'heat island effect' and microclimates. Despite subtle differences, for both urban and rural areas an optimal data fit was generally reached after about five consecutive days within a radius of up to 30 km of the 'crime scene'. With increasing distance and differing altitudes, a lower overall data fit was observed, and a diminishing increase in R2 values was reached after 4-10 consecutive days. These results demonstrate the need for caution regarding distances and climate differences when using weather station data for retrospective regression analyses for estimating temperatures at crime scenes. However, the estimates of scene temperatures from regression analysis were better than simply using the temperatures from the nearest weather station. This study provides recommendations for data logging duration of operation, and a baseline for further research into producing standard guidelines for increasing the accuracy of minPMI estimations and, ultimately, greater robustness of forensic entomology evidence in court.

Visualization of insect metamorphosis.

Metamorphosis and, in particular, holometaboly, the development of organisms through a series of discrete stages (egg, larva, pupa, adult) that hardly resemble one another but are finely adapted to specific roles in the life cycle of the organism, has fascinated and mystified humans throughout history. However, it can be difficult to visualize the dramatic changes that occur during holometaboly without destructive sampling, traditionally through histology. However, advances in imaging technologies developed mainly for medical sciences have been applied to studies of insect metamorphosis over the past couple of decades. These include micro-computed tomography, magnetic resonance imaging and optical coherence tomography. A major advantage of these techniques is that they are rapid and non-destructive, enabling virtual dissection of an organism in any plane by anyone who has access to the image files and the necessary software. They can also be applied in some cases to visualize metamorphosis in vivo, including the periods of most rapid and dramatic morphological change. This review focusses on visualizing the intra-puparial holometabolous metamorphosis of cyclorraphous flies (Diptera), including the primary model organism for all genetic investigations, Drosophila melanogaster, and the blow flies of medical, veterinary and forensic importance, but also discusses similar studies on other insect orders. This article is part of the theme issue 'The evolution of complete metamorphosis'.

A molecular, morphological, and physiological comparison of English and German populations of Calliphora vicina (Diptera: Calliphoridae).

The bluebottle blow fly Calliphora vicina is a common species distributed throughout Europe that can play an important role as forensic evidence in crime investigations. Developmental rates of C. vicina from distinct populations from Germany and England were compared under different temperature regimes to explore the use of growth data from different geographical regions for local case work. Wing morphometrics and molecular analysis between these populations were also studied as indicators for biological differences. One colony each of German and English C. vicina were cultured at the Institute of Legal Medicine in Frankfurt, Germany. Three different temperature regimes were applied, two constant (16°C & 25°C) and one variable (17-26°C, room temperature = RT). At seven time points (600, 850, 1200, 1450, 1800, 2050, and 2400 accumulated degree hours), larval lengths were measured; additionally, the durations of the post feeding stage and intrapuparial metamorphosis were recorded. For the morphometric and molecular study, 184 females and 133 males from each C. vicina population (Germany n = 3, England n = 4) were sampled. Right wings were measured based on 19 landmarks and analyzed using canonical variates analysis and discriminant function analysis. DNA was isolated from three legs per specimen (n = 61) using 5% chelex. A 784 bp long fragment of the mitochondrial cytochrome b gene was sequenced; sequences were aligned and phylogenetically analyzed. Similar larval growth rates of C. vicina were found from different geographic populations at different temperatures during the major part of development. Nevertheless, because minor differences were found a wider range of temperatures and sampling more time points should be analyzed to obtain more information relevant for forensic case work. Wing shape variation showed a difference between the German and English populations (P<0.0001). However, separation between the seven German and English populations at the smaller geographic scale remained ambiguous. Molecular phylogenetic analysis by maximum likelihood method could not unambiguously separate the different geographic populations at a national (Germany vs England) or local level.

Impact of sample degradation and inhibition on field-based DNA identification of human remains.

The advent of DNA technologies for field-based application promises to provide rapid intelligence to aid investigations. Their validation and adoption by enforcement groups have demonstrated utility in sample screening and prioritisation, but field application in some areas of forensic science, such as human remains identification, is little evidenced. Assessing the ability of such approaches to provide meaningful data is critical as decomposition is likely to complicate analysis and limit the effective use of such field-based DNA interventions. This research assessed the ability to collect viable DNA data in the field using the ParaDNA Field Instrument and Intelligence Test chemistry. Different sample collection methods were assessed; direct from skin surface; direct from exposed tissue; indirect from muscle swab transferred to FTA card; and from larvae on the donors. Samples were collected and processed on-site at the Anthropology Research Facility, University of Tennessee. The data show that the muscle tissue provided the most effective sample template and, using this approach, it was possible to generate STR profiles from human remains in under two hours from the time of sample collection. STR profile data were collected up to four days from donor placement (114 Accumulated Degree Days). After this time there was a rapid decrease in the quality of the profiles collected due to the onset of decomposition. The data also show that effective sample recovery was not possible from the surface of the skin, exposed tissue or from carrion larvae. Inhibition studies in the laboratory suggest that by-products of the decomposition process are the primary mode of failure. Together these data suggests a possible application for screening and prioritisation in criminal casework but highlights issues that may affect the success of the approach.

3D virtual histology at the host/parasite interface: visualisation of the master manipulator, Dicrocoelium dendriticum, in the brain of its ant host.

Some parasites are able to manipulate the behaviour of their hosts to their own advantage. One of the most well-established textbook examples of host manipulation is that of the trematode Dicrocoelium dendriticum on ants, its second intermediate host. Infected ants harbour encysted metacercariae in the gaster and a non-encysted metacercaria in the suboesophageal ganglion (SOG); however, the mechanisms that D. dendriticum uses to manipulate the ant behaviour remain unknown, partly because of a lack of a proper and direct visualisation of the physical interface between the parasite and the ant brain tissue. Here we provide new insights into the potential mechanisms that this iconic manipulator uses to alter its host's behaviour by characterising the interface between D. dendriticum and the ant tissues with the use of non-invasive micro-CT scanning. For the first time, we show that there is a physical contact between the parasite and the ant brain tissue at the anteriormost part of the SOG, including in a case of multiple brain infection where only the parasite lodged in the most anterior part of the SOG was in contact with the ant brain tissue. We demonstrate the potential of micro-CT to further understand other parasite/host systems in parasitological research.

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography.

Minimum post-mortem interval (minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.

The 'dance' of life: visualizing metamorphosis during pupation in the blow fly Calliphora vicina by X-ray video imaging and micro-computed tomography.

The dramatic metamorphosis from larva to adult of insect orders such as Diptera cannot usually be witnessed because it occurs within an opaque structure. For the cyclorrhaphous dipterans, such as blow flies, this structure is the puparium, formed from the larval cuticle. Here, we reveal metamorphosis within the puparium of a blow fly at higher temporal resolution than previously possible with two-dimensional time-lapse videos created using the X-ray within a micro-computed tomography scanner, imaging development at 1 min and 2 min intervals. Our studies confirm that the most profound morphological changes occur during just 0.5% of the intrapuparial period (approx. equivalent to 1.25 h at 24°C) and demonstrate the significant potential of this technique to complement other methods for the study of developmental changes, such as hormone control and gene expression. We hope this will stimulate a renewed interest among students and researchers in the study of morphology and its astonishing transformation engendered by metamorphosis.

Looking into the puparium: Micro-CT visualization of the internal morphological changes during metamorphosis of the blow fly, Calliphora vicina, with the first quantitative analysis of organ development in cyclorrhaphous dipterans.

Metamorphosis of cyclorrhaphous flies takes place inside a barrel-like puparium, formed by the shrinking, hardening and darkening of the third-instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro-computed tomography (micro-CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra-puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X-ray images and micro-CT virtual sections. The origin of this gas bubble has been largely unknown, but micro-CT virtual sections show that it is connected to one of the main tracheal trunks. Micro-CT virtual sections also provided enough resolution for determining the completion of the larval-pupal and pupal-adult apolyses, thus enabling an accurate timing of the different intra-puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra-puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre-helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.

Optimising crime scene temperature collection for forensic entomology casework.

The value of minimum post-mortem interval (minPMI) estimations in suspicious death investigations from insect evidence using temperature modelling is indisputable. In order to investigate the reliability of the collected temperature data used for modelling minPMI, it is necessary to study the effects of data logger location on the accuracy and precision of measurements. Digital data logging devices are the most commonly used temperature measuring devices in forensic entomology, however, the relationship between ambient temperatures (measured by loggers) and body temperatures has been little studied. The placement of loggers in this study in three locations (two outdoors, one indoors) had measurable effects when compared with actual body temperature measurements (simulated with pig heads), some more significant than others depending on season, exposure to the environment and logger location. Overall, the study demonstrated the complexity of the question of optimal logger placement at a crime scene and the potential impact of inaccurate temperature data on minPMI estimations, showing the importance of further research in this area and development of a standard protocol. Initial recommendations are provided for data logger placement (within a Stevenson Screen where practical), situations to avoid (e.g. placement of logger in front of windows when measuring indoor temperatures), and a baseline for further research into producing standard guidelines for logger placement, to increase the accuracy of minPMI estimations and, thereby, the reliability of forensic entomology evidence in court.