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.

A geometric morphometric analysis of wing variations in shape and size of the blue bottle fly, Calliphora vicina (Diptera: Calliphoridae).

A geometric morphometric analysis was performed on the right wing of adult Calliphora vicina (Robineau-Desvoidy) collected across 4 altitudinal levels in Sicily. The objective of this study was to assess differences in shape and centroid size (CS) between females and males and across elevations. The wings analyzed in this study were removed from adults of C. vicina collected with baited traps at 20, 700, 1,153, and 1,552; for this study, 19 landmarks were identified in each wing. The coordinates of the landmarks were aligned and superimposed to prevent variations due to position, orientation, and scale; they were then scaled to the same CS and recentered. CS and Procrustes differences were, respectively, used to assess variations in size and shape. Significant differences were observed in wing shape between males and females but not between all altitudinal levels. Female wings were found to be significantly larger than males (P < 0.01). Wings of flies collected at the highest altitudinal level resulted in significantly larger wings than those collected at lower altitudes (P < 0.001), with CS values ranging from 12.1 to 14.1. Variation in wing shape can impact thermal regulation, and therefore, oxygen content, temperature, atmospheric pressure, and solar radiation can have an effect on an insect's body and activity levels. At high elevations and lower temperatures, larger wings could mean less energy expenditure when flying to increase body temperature.

Evaluating the impact of hot water killing larvae on gene expression using the transformer gene in Cochliomyia macellaria (Diptera: Calliphoridae).

In forensic entomology, determining the age of a larva from a body to estimate time since death is commonly performed through the measurement of a physical trait. Gene expression has been studied as an alternative age estimation approach, but the storage conditions required for these studies are different than those used in forensic entomological casework. Studies analyzing gene expression prioritize the preservation of RNA, which requires fresh tissue and ultra-cold storage. Casework, in contrast, utilizes hot water killing specimens that may not be analyzed for a long period after collection. In the current study, the impact of hot water killing on gene expression was assessed for larval samples of the forensically important blow fly, Cochliomyia macellaria. Successful amplification of the sex-determining gene, transformer, was tested across larvae ranging in size from 3.22 to 16.85 mm in length after storage times of 1-2 weeks, 4-5 weeks, and 8-9 weeks at 4°C in RNAlater. Larvae hot water killed were processed in tandem with larvae stored live to allow for a direct assessment of the impact of boiling on gene expression. As expected, the transformer gene was successfully amplified in all larvae stored live. For the hot water-killed larvae, the success rate was only slightly lower, with 3 out of 75 larvae not generating a sex-specific band pattern. The results show gene expression can be used for hot water-killed samples, though future work across different genes, species, and extending to quantitative gene expression methods is needed.

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

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

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.

Bionomics, reproductive traits and assessment of forensic relevance of Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae).

Peckia (Peckia) chrysostoma (Wiedemann, 1830) (Diptera: Sarcophagidae) is a colonizer of cadavers in the Neotropical Region. Nevertheless, data on development for the P. (P.) chrysostoma (e.g., instar duration) and behavioral strategies used by the species for locating and colonizing a corpse are scant. We aimed to explore bionomic and reproductive aspects of the flesh fly P. (P.) chrysostoma, and in this article we: (a) provide quantitative data on the life cycle of P. (P.) chrysostoma; (b) present bionomic measurements (length and weight) of larvae and pupae; (c) describe intrauterine egg and larvae development; and (d) analyze the ovo/larviposition behavior by gravid females. Females showed ovaries with discernible eggs and larvae between 8 and 10 days (x̅ = 23.3 eggs/female). This study reports the first observation of egg deposition, an atypical behavior for the species. The average development time for immature stages was 22.24 h and 21.36 h for 1st and 2nd respectively, and 3rd showed an average development time of 80.47 h. Pupa had the longest duration (x̅ = 295.69 h). A direct increase was observed in weight (P < 0.05) and length (P < 0.05) throughout time. The average survival time of males and females is approximately 30 days. This study expands the knowledge on P. (P.) chrysostoma, such as facultative ovoviviparity under laboratory conditions and the life cycle, which may benefit future studies for accuracy in entomology-based estimation of minimum post-mortem interval (min PMI).

A preliminary study of carrion insects in Greece and their attraction to three animal baits: a forensic entomology perspective.

The current study provides data on the composition of the dipteran and coleopteran fauna attracted to three different animal baits (chicken stomach, beef liver, fish) in the urban ecosystem of Athens, Greece. An additional objective was to examine the effect of bait choice by the collected taxa. Three trap locations were established within the Agricultural University of Athens. At each location two different types of baited traps were used to capture and record the local dipteran and coleopteran biodiversity. During the sampling period, a total of 2416 dipteran and coleopteran specimens representing 43 species were collected and identified. The three most abundant dipteran taxa were Sarcophaga sp. (Diptera: Sarcophagidae), Calliphora vicina Robineau-Desvoidy, Lucilia sericata (Meigen) (Diptera: Calliphoridae). The results showed that the structure of the local insect community associated with the decomposition of animal baits depends on the bait used for sampling. This study reinforced the feasibility of using animal tissues as attractants to a wide diversity of medical, parasitological and forensically important taxa. Overall, beef liver attracted the most insects with Diptera preferring beef liver and Coleoptera preferring chicken stomach. Furthermore, this study revealed that baits of one animal tissue did not attract all species but in combination a much better understanding of this biodiversity was achieved. Finally, from a forensic entomology perspective this study highlights the lack of understanding of the value of entomological data related to case work in Greece.

Colonization pattern and thermal needs of immature phases of Sarcophaga argyrostoma (Diptera: Sarcophagidae): Significance for estimating postmortem interval.

Members of Sarcophagidae are necrophagous and are commonly found on decaying carcasses; and their developmental forms are important indicators for the approximation of lowest postmortem interval (PMImin). This work describes the biological characteristics of Sarcophaga argyrostoma from Tehran, Iran. Various temperature regimes were applied to estimate the thermal summation constant (k) and thermal requirements for development of S. argyrostoma. Five growth proceedings, containing 1st ecdysis, 2nd ecdysis, wandering, pupariation and eclosion, were investigated under eight fixed temperature regimes (6-30 °C). The effects of fly age, freshness, and availability of oviposition substrate on oviparity and viviparity was studied. At 6 °C, no development occurs, and at 8 °C only the first ecdysis occurs. At temperatures between 10 and 30 °C, all immature stages proceeded to the adult stage, and thus immature development was analyzed at these six remaining temperatures. The development phases needed minimum (Dz ± SE) 5.4(0.4), 8.5(0.26), 5.3(0.44), 3.8(0.1), and 6.6 (0.6)°C to attain one of the succeeding developmental occasion, correspondingly. The approximated K for those five occasions were 15.04 ± 1.12, 12.62 ± 0.65, 140.36 ± 4.35, 14.59 ± 0.6, and 222.8 ± 4.18°-day (DD) accordingly. When the food substrate is available and fresh, the female prefers to lay eggs, no matter how old she is. However, the chance of ovoviviparity increased when no oviposition substrate was available. The truth that S. argyrostoma able to either larviposit or to lay eggs encompasses serious consequences for the precise estimation of the PMImin, as the existence of larvae resulting from eggs laid on the carcass could add hours (based on ambient temperature) to the PMImin.

Improving accuracy of age estimates for insect evidence-calibration of physiological age at emergence (k) using insect size but without "k versus size" model.

Postmortem interval may be estimated based on the age of insect evidence collected on a death scene. Reference data that are used in such estimation frequently comprise thermal summation constant (i.e. k), which is equal to the insect age upon completion of immature development expressed in accumulated degree-days or degree-hours (ADD or ADH). Essentially, k is a central point of an insect group and it may poorly represent insect evidence that is near the limits of variation for the group. Accordingly, it was postulated to calibrate k for particular insect evidence and insect size and sex were found to be useful for this purpose in some of the species. However, the calibration is only possible by using the model that correlates k with insect size. Since very few such models were published, this lack of data limits the calibration of k in forensic casework. In this article, we develop a formula that is useful for the calibration of k without the use of "k versus size" model (and related datasets). The formula uses k from the general thermal summation model for a species (with its standard error), size range for the species (retrieved from entomology literature), and size measurements for particular insect evidence. The calibration of k with the formula was validated using the Creophilus maxillosus (Coleoptera: Staphylinidae) and Necrodes littoralis (Coleoptera: Silphidae) datasets. It was particularly useful while analyzing unusually small and large insects, in case of which the formula reduced the inaccuracy of k from the general model on average by ~25 ADD in C. maxillosus and ~40 ADD in N. littoralis. We discuss the limitations and prospects of the calibration protocol that employs the formula.

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

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

Developmental differences in spatially distinct populations of the forensically relevant blow fly Lucilia sericata - About the comparability of developmental studies (and case work application).

The cosmopolitan blow fly Lucilia sericata is often used in forensic case work for estimating the minimum postmortem interval (PMImin). For this, the age of immature specimens developing on the dead body is calculated by measuring the time taken to reach the sampled developmental stage at a given temperature. To test whether regional developmental data of L. sericata is valid on a global scale, the time taken to reach different developmental stages was compared between a population from Mexico and one from Germany at two different constant temperatures. The German population of L. sericata was collected in Frankfurt/Main, while the Mexican population originated near Oaxaca de Juarez and was transported to Germany in the larval stage. Only the F1 generation was used to avoid adaption of the Mexican flies. Eggs were immediately placed at 20 °C and 30 °C. Five times 30 freshly eclosed larvae per replicate (n = 5) were then transferred to a cup of minced meat in separate containers. The larvae were checked every 8 h for migration, pupariation or emergence of adult flies. The time at which the first individual and 50 % of the specimens per container entered each of these stages, was recorded. Significant differences in the time of development between the two populations were observed at both temperatures. At 20 °C, the first specimens of the Mexican population reached all developmental stages a little (< 1 day to < 2 days) earlier than the German L. sericata. At 30 °C, the Mexican flies also reached the post-feeding stage slightly earlier (0.2 days). However, at 30 °C, the German flies started pupariation significantly earlier (after 5 days) than the Mexican flies (6.9 days) and the adults from Germany also emerged earlier (10.5 days compared to 13.1 days). The same pattern was observed when looking at 50 % of the total number of specimens per container. A comparison with previously published developmental studies was difficult as the experimental design varied widely between studies. However, the results were within the range of most studies. Our study has shown that age estimation can vary widely depending on the population on which the reference data used for the calculations are based. This highlights the importance of using local and population-specific developmental data for estimating the age of blow flies in case work.

Use of thermal drone in detection and assessment of larval mass temperature in decomposed rabbit carcasses.

Manual ground searches and cadaver dogs are traditional methods for locating remains, but they can be time- and resource-intensive, resulting in the decomposition of bodies and delay in victim identification. Therefore, thermal imaging has been proposed as a potentially useful tool for detecting remains based on their temperature. This study investigated the potential of a novel search technique of thermal drones to detect surface remains through the detection of maggot mass temperatures. Two trials were carried out at Selangor, Malaysia, each utilizing 12 healthy male Oryctolagus cuniculus European white rabbits and DJI Matrice 300 RTK drone China, equipped with a thermal camera; Zenmuse H20T to record the thermal imaging footage of the carcasses at various heights (15, 30, 60-100 m) for 14 days for each trial. Our results demonstrated that the larval masses and corresponding heat emissions were at their largest during the active decay stage; therefore, all the carcasses were observable in thermal images on day 5 and remained until day 7. Statistical analyses showed that (1) no statistically significant differences in thermal images between clothed and unclothed subjects (p > 0.05); (2) 15 m above ground level was proven to be the optimal height, as it showed the greatest contrast between the carcass heat signature and the background (p < 0.005). Our data suggested the potential window of detection of thermal signatures was detectable up to 7 days post-deposition. This could be an important guideline for the search and recovery teams for operational implementation in this tropical region.

The utility of blow fly (Diptera: Calliphoridae) evidence from burned human remains.

Burning of human remains is a common method to conceal or destroy evidence associated with homicides and illegal activities. However, data regarding blow fly colonization of burned remains are scarce, with all previously published empirical studies focusing only on non-human animals. It is critically important to obtain basic data on blow fly colonization patterns of burned human remains as such evidence may represent the only feasible method for PMI estimation in cases of burning. In this study, we thermally altered six human donors to a Crow-Glassman Scale Level 3 (CGS-3) and placed them at the Anthropology Research Facility at the University of Tennessee in Summer 2021, Spring 2022, and Summer 2022. Six unburned human donors were used as controls. Observations for insect activity began within 24 h of placement and continued twice weekly through decomposition. Age estimations were performed with immature blow flies to estimate the time of colonization (TOC), and accuracy was assessed against the time of placement for each donor. All burned donors examined in this study were colonized by blow flies. No significant difference in species composition was determined between treatments, though TOC estimations from burned donors were slightly (but significantly) less accurate than TOC estimations from unburned donors (80% vs. 83% accuracy; χ2 = 0.041, df = 1, P = 0.840). These results indicate that blow flies can successfully colonize human remains burned to CGS-3 and that accurate TOC estimations can still be generated from larval specimens. Though several limitations to this study exist (e.g., inconsistent donor BMI, lack of donor temperature data), our results underscore the utility of entomological evidence in cases of burned human remains.

Geometry of posterior larval spiracles to identify medically and forensically important calliphorids in Thailand.

Fly identification is the primary step of analysis in forensic entomology. Although morphology and molecular techniques are considered satisfactory methods, some constraints may arise from a financial or even human point of view. Over the past decade, the geometric morphometric approach has been increasingly advocated for the classification and identification of arthropods. This study explored the method for species identification of 800 third-instar larvae of eight blow fly species of medical and forensic importance: Chrysomya chani Kurahashi, Chrysomya megacephala (Fabricius), Chrysomya (Ceylonomyia) nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya (Achoetandrus) rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia cuprina (Wiedemann), and Lucilia porphyrina (Walker). Based on the posterior spiracles geometry, the cross-validation revealed a relatively high percentage of correct classification in most species, ranking from 86% to 100%. The results of this study confirmed that the geometric morphometric (GM) analysis of posterior spiracles might be utilized as a larva identification tool. Therefore, this GM method represents one way of overcoming difficulties with the identification of blow fly larvae and can support further studies of these flies.

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

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

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.

Lucilia silvarum Meigen (Diptera: Calliphoridae) Is a Primary Colonizer of Domestic Cats (Felis catus).

Lucilia silvarum Meigen (Diptera: Calliphoridae) is widespread throughout North America and Europe. Described in 1826, this blow fly was quickly associated with myiasis in amphibians, and to date has rarely been reported in carrion. There is limited data regarding the time of colonization of animals with fur and the interpretation of this data is difficult due to variation in the animal models used. During an examination of initial insect colonization of cats (Felis catus) with light and dark fur, twelve domestic short-haired cats were placed in cages 15.2 m apart in a grassy field in West Lafayette, Indiana, USA. Eggs from initial oviposition events were collected and reared to identify the colonizing species. Three species of Lucilia (Diptera: Calliphoridae), including L. silvarum, colonized the cats on the initial day of placement. In this study, L. silvarum was the primary colonizer of cats, and this may be the first study where a large number of L. silvarum were collected. Further studies should include development studies on L. silvarum to understand its life history and aid in time of colonization estimations. More work regarding the colonization of furred mammals is needed to further examine L. silvarum as a primary colonizer.

New geographic location data on the occurrence and abundance of carrion insects of forensic interest.

The world's forensic entomologists have much in common. They face similar research challenges, apply the same scientific methodology, study the same kinds of evidence, and access global research databases. Nevertheless, some regional heterogeneity inevitably exists. For most countries, and particularly those that have complex and diverse ecosystems, the current priority is to stimulate use of forensic entomology by establishing open access databases with time series data using standardised protocols for occurrence, abundance, distribution, niche preferences, life cycle, and identification characteristics for the key regional species that may be encountered in forensic entomology cases. Even in countries where forensic entomology is routinely used as a tool in reconstructing the history of corpses found on crime scenes in accordance with principles found in the rapidly developing body of literature, there is still much to learn about forensically useful insects. Examples of regional gaps include the taxonomy of lesser-known carrion insect species, seasonal occurrence of carrion species in the country's various geographical regions, and rates of development of the local species modeled in the many site situations of forensic interest. The first published study of carrion insects found in Athens, Greece published in this journal is an example of entomologists in a region taking the necessary first step towards establishing baseline data about native and introduced species and hence, physiological and behavioural responses to local environmental conditions, including life cycles and likelihood of occurrence or absence in the region.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

First record of Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae) colonization on a human corpse.

A partially skeletonized human corpse was found in bushes in Selangor, Malaysia in June 2020. Entomological evidence was collected during the autopsy and sent to the Department of Medical Microbiology and Parasitology, Faculty of Medicine, Universiti Teknologi MARA (UiTM) for minimum postmortem interval (PMImin) analysis. Standard protocols were applied when processing preserved and live insect specimens of both larval and pupal stages. Entomological evidence revealed that the corpse was colonized by Chrysomya nigripes Aubertin, 1932 (Diptera: Calliphoridae) and Diamesus osculans (Vigors, 1825) (Coleoptera: Silphidae). Chrysomya nigripes was chosen as the PMImin indicator as this fly species is an earlier colonizer compared to D. osculans beetle larvae which their presence is the indicative of late stage of decomposition. For the present case, the pupae of C. nigripes were the oldest insect evidence collected and based on the available developmental data, the estimated minimum PMI was established between 9 and 12 days. It is noteworthy to highlight that this is the first record of D. osculans colonization on a human corpse.