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.

Survival of Calliphora vicina (Diptera: Calliphoridae) embryos under cold temperature conditions: forensic implications.

Most blow flies (Diptera: Calliphoridae) species are sarcosaprophagous during the larval stage, primarily feeding on the soft tissues of carcasses during the early stages of decomposition, making them valuable forensic indicators for minimum post-mortem interval (minPMI) estimations. Like other insects, their developmental rates are strongly influenced by the environmental temperature. Although several studies have examined the influence of temperature on the development of different blow fly species, the impact of cold temperatures remains largely unstudied, despite its potential forensic implications. The present study investigates the effect of three cold temperatures (0, -2.5 and -5°C) on the survival of Calliphora vicina embryos of five different ages (0%, 20%, 40%, 60% and 80% of the total embryonic development) and two exposure times (6 and 24 h). Our results revealed significant differences in egg survival at the earliest embryonic stages (0% and 20% of the total embryonic development), resulting in high mortality rates. While at 20% of the total embryonic development high mortality was only observed under -5°C, at 0% of the total embryonic development high mortality rates were observed at all the temperatures tested. Although C. vicina embryos demonstrate tolerance to cold temperatures once they have completed the first 20% of the total embryonic development, potentially mitigating the impact of cold weather events, the possibility of minPMI underestimations due to the death of the first egg batches should not be disregarded. Additionally, considering that the embryonic development stages may last for several days under low temperatures, caution should be taken in the analysis of entomological evidence if a cadaver is discovered following cold weather episodes.

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.

Morphological and molecular characterization of puparia of Piophilidae species of forensic relevance.

Piophilidae are a small family of Diptera with a worldwide distribution and which are historically associated with human activities. In addition to their economic importance, piophilid larvae can also be of medical and legal relevance. Within a medicolegal context, piophilids are frequently associated with cadavers in advanced stages of decomposition, thus being potentially useful forensic indicators and they have been reported also from archaeo-funerary contexts. An accurate species identification is therefore an essential prerequisite to ensure the reliable analysis of insect material in medical, forensic and archaeological investigations. Identification of the adult piophilid flies is possible because of the availability of identification keys, in contrast immature insects, especially puparia, have been poorly investigated and described. In this paper, puparia of 11 species of forensic interest (Piophila casei, Piophila megastigmata, Parapiophila atrifrons, Parapiophila flavipes, Parapiophila vulgaris, Protopiophila litigata, Liopiophila varipes, Prochyliza nigrimana, Prochyliza xanthosoma and Stearibia nigriceps in subtribe Piophilina and Centrophlebomyia furcata in subtribe Thyreophorina) are described and a molecular analysis, based on the COI sequencing, is presented to show the potential of the molecular approach in their identification.

Long-term insect successional patterns on pig carcasses in central Spain.

Baseline data on the insect successional patterns on carcasses can be a valuable estimation tool in the investigations of suspicious deaths, particularly when the post-mortem interval is longer than months or years. However, although carrion insect succession is a recurrent topic in forensic science research, the duration of the published studies is typically shorter than 1 year, with only one published study from central Europe documenting successional patterns beyond the first year of decomposition. We provide here the first data on the long-term insect successional patterns in southern Europe, using pig carcasses exposed in the four seasons of the year and documenting the carrion entomofauna during the second and third year of decomposition. Our results confirmed previous observations from central Europe that several Coleoptera species are able to recolonize cadavers during the second and third year after death, with their larvae residing on the remains for long periods. The season of cadaver exposure appears to be a main factor determining the composition of the insect fauna that exploits the remains during subsequent years. Our results suggest that it might be possible to estimate the year and season of death analyzing the composition of the insect fauna occurring on a cadaver. The present data highlight the need for further studies on the long-term insect succession on cadavers in different seasons and geographical areas, in order to fully understand its patterns and ensure its proper application in PMI estimations.

Larval identification key to necrophagous Coleoptera of medico-legal importance in the western Palaearctic.

Several necrophagous Coleoptera species are frequently collected on cadavers, may occasionally act as intermediate or paratenic hosts of parasites, as vectors of pathogens or as allergens, and can also represent major pests of preserved animal products. However, despite their medical, veterinary and economic importance, there is a lack of reliable species identification tools for the larval stages (usually the only entomological evidence associated with medicolegal investigations), thus severely limiting their potential application as forensic indicators. Here, we provide an identification key to the larvae of the necrophagous Coleoptera species which have been recorded on carrion in the western Palaearctic region, based on easily observable morphological characters. In total, we provide diagnostic characters for the reliable identification of 23 necrophagous Coleoptera species within four different families (Cleridae, Dermestidae, Nitidulidae and Silphidae). In addition to the aforementioned families, we provide diagnostic characters for the identification of the larvae of families Histeridae, Staphylinidae and Trogidae, which can also be collected on cadavers. It is expected that the present key will facilitate the identification of larval material of necrophagous Coleoptera collected either in carrion succession studies or during medicolegal investigations, in order to further advance in the potential use of this insects as forensic tools.

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.

Estimating the age of Calliphora vicina eggs (Diptera: Calliphoridae): determination of embryonic morphological landmarks and preservation of egg samples.

Blow fly eggs may sometimes be the only entomological evidence recovered in a forensic case, especially in cooler weather when hatching might take several days: hence, a method for estimating their age is greatly needed. However, developmental data on blow fly eggs are mainly limited to records of the time to larval hatching. The current paper describes the morphological changes occurring during embryogenesis of the blow fly Calliphora vicina Robineau-Desvoidy and their timing in relation to temperature, in order to determine those characters which can be used for simple egg age estimation using light microscopy. At 7.3 and 25 °C, 15 easily visualised morphological landmarks were determined in C. vicina living embryos, allowing for their age estimation with a resolution of 10-20% of total egg developmental time. The observed age intervals were compared to the embryonic stages described for the fruit fly Drosophila melanogaster Meigen, which are used as reference data in multiple developmental studies. Moreover, current guidelines for preservation of egg samples, which recommend the placement of living eggs directly into 80% ethanol, were tested against the hot water killing (HWK) method prior to preservation in 80% ethanol, recommended for larval and pupal specimens. Direct placement of eggs into 80% ethanol caused marked decomposition of samples, and no morphological landmarks were discernible. On the other hand, HWK fixation prior to preservation in 80% ethanol enabled visualisation of 11 of the 15 age-specific morphological landmarks that were discernible in living embryos. Therefore, HWK fixation prior to preservation in 80% ethanol is recommended for egg samples, thus unifying the protocols for collecting entomological evidence.

Patterns of diversity and abundance of carrion insect assemblages in the Natural Park "Hoces del Río Riaza" (central Spain).

The patterns of diversity and abundance of the carrion insect species in the different habitats of the Natural Park "Hoces del Río Riaza" (central Spain) were studied with the use of carrion-baited traps. Representativeness of the inventories was assessed with the calculation of randomized species richness curves and nonparametric estimators. Coleoptera families, Silphidae and Dermestidae, and Diptera families, Calliphoridae and Muscidae, were dominant in every sampling habitat, but differences in the patterns of diversity and abundance were found. Lusitanian oakwood and riparian forest were the most diverse habitats with high abundance of saprophagous species, whereas more open (i.e., exposed to continuous sunlight during the day) habitats showed lower diversity values and a different species composition and distribution of species abundance, favoring thermophilous species and necrophagous species with high tolerance to different environmental conditions. Differences in the bioclimatical features of the sampled habitats are suggested to explain the composition and diversity of the carrion insect assemblages in different environments.

Influence of photoperiod on the developmental times of the forensically relevant blow fly species Calliphora vicina (Diptera: Calliphoridae).

Blow flies (Diptera: Calliphoridae) are frequently used in forensic investigations due to their rapid colonization of cadavers. As with other insects, environmental temperature strongly influences their developmental rates. While published research has typically explored not only the impact of the environmental temperature, but also of other factors like tissue type and drug presence on developmental rates, the influence of photoperiod on the developmental rates of forensically relevant blow fly species has remained largely underexplored. Understanding the relationship between photoperiod and developmental times is crucial, as neglecting this aspect could compromise the accuracy of minimum post-mortem interval (minPMI) estimations. The present study investigates the impact of three photoperiod conditions (0:24, 8:16, and 12:12 light:darkness) on the developmental rates of Calliphora vicina, focusing on the duration of the different immature stages and on the total developmental time. Our results revealed significant variation in the intra-puparial stage and total development time across different photoperiods. Notably, a 12:12 photoperiod led to a significantly prolonged intra-puparial stage and total development time compared to the 0:24 photoperiod, suggesting that Calliphora vicina develops faster in total darkness. These findings highlight the importance of considering photoperiod in both laboratory rearing protocols and forensic casework to improve the accuracy and reliability of minPMI estimations. In this regard, preliminary guidelines and recommendations are provided.

Sex-biased captures of sarcosaprophagous Diptera in carrion-baited traps.

The use of carrion-baited traps is a common and widely extended practice in the study of sarcosaprophagous Diptera. However, it implies different areas of bias, one of them being the different responses of males and females to carrion bait, which results in possible biased sex ratios in the captures. In the present study, the use of carrion-baited traps revealed significant female-biased captures in the families Calliphoridae, Muscidae, and Sarcophagidae, whereas the collected species of the families Piophilidae, Heleomyzidae, and Ulidiidae showed different patterns in the observed sex ratios. Possible explanations according to existing literature and the types of mating behaviors of the different families are discussed.

The immature stages of the necrophagous fly, Prochyliza nigrimana: comparison with Piophila casei and medicolegal considerations (Diptera: Piophilidae).

Flies of family Piophilidae have been recorded as major pests in the food industry, as agents of human myiasis and typically associated with carcasses in advanced stages of decay, being thus important in forensic entomology. Despite that the cosmopolitan species Piophila casei is the most cited in entomological studies, many other piophilid species develop on both carrion and animal products from the food industry. One of those species is Prochyliza nigrimana, widely distributed throughout the Holarctic and Neotropical regions. In this study, the morphological features of the immature stages of P. nigrimana are described for the first time and compared with those of P. casei. The third-instar larvae and puparium of P. nigrimana are significantly shorter than those of P. casei; the contrary pattern is observed in egg length. The number and arrangement of the lobes of anterior spiracles, which had been used as a distinctive character of P. casei in some keys, are the same in both species. Morphological features of the cephaloskeleton (such as the general shape and the distance between the tips and the base of the mouth hooks/base of the mouth hooks ratio), the arrangement of anal segment in third-instar larvae and the appearance of the ventral creeping welts in the puparium are the main characters allowing for identification of both species.

Looking Back to Move Forward: How Review Articles Could Boost Forensic Entomology.

The Locard's exchange principle (1930) holds that the perpetrator of a crime leaves traces behind that can later be sampled and used as forensic evidence [...].

First report of the forensically important fly, Stearibia nigriceps (Diptera: Piophilidae) in South Korea: Confirmation of specimens from human corpses based on cytochrome c oxidase subunit I barcodes.

Piophilidae are a relatively small family of Diptera that is frequently associated with cadavers at advanced stages of decomposition and are, therefore, considered potentially useful forensic indicators. However, their use in forensic investigations is typically hampered by a deficiency in reliable identification tools. This is particularly evident in countries such as South Korea, where forensic entomology is still in its infancy and the diversity of forensically relevant insect taxa remains largely undocumented. In the present study, we used cytochrome c oxidase subunit I (COI) barcodes to identify samples of piophilid larvae collected during medicolegal investigations performed in South Korea. A total of 174 COI sequences were obtained and have been made publicly available, thus augmenting the reference barcode library for forensically important Piophilidae species. Of the 174 sequenced samples, 172 were identified as Stearibia nigriceps (Meigen), whereas the two remaining samples may represent a previously unsequenced piophilid species. Stearibia nigriceps is recorded from South Korea for the first time, and our results suggest that it might be a particularly relevant forensic indicator in certain case types and scenarios in that country. The findings of this study highlight the utility of COI barcodes for achieving accurate identification of entomological samples, even by non-specialist forensic practitioners. They also contribute to the further development and consolidation of forensic entomology in South Korea and eastern Asia.

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.

Wing morphometrics for the identification of Nearctic and Palaearctic Piophilidae (Diptera) of forensic relevance.

The Piophilidae (Diptera) are a family comprising about 80 species, several of them of high economic and forensic relevance. An unequivocal species identification is crucial for designing effective control measures or to provide reliable estimations of the minimum post mortem interval. However, the identification may sometimes not be possible, either because the diagnostic morphological characters are not easily observable or very fragile, or because of a poor DNA quality and/or unavailability of reference molecular signatures. In the recent years, the use of wing morphometric techniques has emerged as a powerful tool for the identification of different families of forensically important Diptera. The present study applies, for the first time, this technique to the identification of 11 Piophilidae species of forensic relevance in the Nearctic and Palaearctic regions, as well as to the discrimination of sexes, distinct geographical populations and, in the case of the dimorphic species Prochyliza nigrimana (Meigen), seasonal morphs. Wing shape variation was analysed using 14 landmarks located at wing vein junctions and a cross-variation analysis was used to test the reliability of identifications. The present results demonstrate that wing shape can be used to identify most species with relatively high success, whereas cross-validation analyses indicated that discrimination between sexes, populations or morphs was less effective, even if significant differences were observed in every comparison. We conclude that wing morphometrics can be a powerful identification tool that might be used in combination with other methods in order to achieve accurate and reliable species identifications-independently of the sex, geographic origin or colour variation of the samples-even with those piophilid species that have been object of frequent misidentifications.

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'.