Post-mortem interval determinations using insects collected from illegally hunted and dehorned rhinoceros in the Republic of South Africa from 2014 to 2021.

Wildlife forensic science is a growing research field globally with application in criminal cases of illegal hunting requiring an estimate of time of death based on insect fauna. The techniques and procedures of forensic entomology acquired over the last 40 years, used in legal cases relating to human remains, can be adapted to decomposing wildlife. Research on carrion utilising the rate of development of insect immatures provides a biological clock from which a minimum post-mortem interval (minPMI) can be derived. The following study concerns 19 rhinoceros that were illegally killed and dehorned in the Republic of South Africa between 2014 and 2021. The paper details 74 samples of insect evidence collected from these rhinoceros remains from which an accurate estimate of their PMI was calculated. The specimens comprised 18 species from 12 families belonging to three insect orders. Many Dipteran and Coleopteran species were found on and around each carcass. The species of fly larvae (family Calliphoridae) used in each case to estimate the PMI are as follows: Chrysomya marginalis (Wiedemann) (13 cases), Chrysomya chloropyga (Wiedemann) (2 cases), Chrysomya albiceps (Wiedemann) (1 case) and Chrysomya megacephala (Fabricius) (1 case). Two species of Coleoptera from the family Dermestidae and Silphidae involved Dermestes maculatus (DeGeer) and Thanatophilus micans (Fabricius), respectively, also were involved in one PMI estimation each. The paper highlights opportunities for improving our global understanding of gaps in procedures and training related to wildlife criminal cases.

A nondestructive technique for the sex identification of third instar Cochliomyia macellaria larvae.

Forensic entomology plays an important role in medicolegal investigations by using insects, primarily flies, to estimate the time of colonization. This estimation relies on the development of the flies found at the (death) scene and can be affected (and sometimes corrected) by external factors, such as temperature and humidity, and internal factors, such as species and sex. This study leverages infrared (IR) spectroscopy combined with machine learning models-Partial Least Squares Discriminant Analysis (PLS-DA) and eXtreme Gradient Boosting trees Discriminant Analysis (XGBDA)-to differentiate between male and female Cochliomyia macellaria larvae, commonly found on human remains. Significant vibrational differences were detected in the infrared spectra of third instar C. macellaria larvae, with distinct peaks showing variations in relative absorbance between sexes, suggesting differences in biochemical compositions such as cuticular proteins and lipids. The application of PLS-DA and XGBDA yielded high classification accuracies of about 94% and 96%, respectively, with female spectra consistently having higher sensitivity than males. This non-destructive approach offers the potential to refine supplemental post-mortem interval estimations significantly, enhancing the accuracy of forensic analyses.

Development and age estimation of the intrapuparial period of Phormia regina (Diptera: Calliphoridae) for postmortem interval estimation.

Phormia regina (Meigen, 1826; Diptera: Calliphoridae) is a Holarctic species that rapidly colonizes carcasses and has been used as an indicator for determining the minimum postmortem interval. However, studies using morphological methods to estimate the intrapuparial age of P. regina are lacking. In this study, morphological changes within the puparium were observed under a stereomicroscope at 7 constant temperatures ranging from 16 °C to 34 °C. The intrapuparial period was categorized into 12 substages. Morphological indicators, including compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, were recorded in detail. The observed morphological changes were divided into 6-10 substages, and the duration of each substage was also recorded in detail. The results of this study provide primary data for using the intrapuparial morphology of P. regina when pupae are collected at a crime scene and estimating the minimum postmortem interval.

Age estimation of Phormia regina pupae based on ATR-FTIR and chemometrics.

Accurate postmortem interval estimation is vital in the investigation of homicides, suicides, and accidental deaths. It is key in narrowing suspect lists, improving crime-solving efficiency, and offering solace to bereaved families. The intra-puparial period, comprising about half of a fly's developmental cycle, presents challenges for morphological age estimation. External changes are limited to color shifts and the appearance of respiratory horns on the puparium only within several hours after pupariation, while detailed internal development analysis often requires invasive methods like removing the puparium, which can be damaging. Additionally, these techniques usually depend on a forensic entomologist's expertise, which lead to subjective biases. This study employed attenuated total reflection-fourier transform infrared spectroscopy, a rapid, non-destructive method for analyzing proteins, chitosan, and chitin in puparia. Data showed a consistent reduction in the concentration of the amide I band within the puparium during the intra-puparial development at five constant temperatures (19 °C, 22 °C, 25 °C, 28 °C and 31 °C). This trend in the spectral data effectively distinguishes pupae at various stages of intra-puparial development, facilitating precise age estimation, which is critical for the estimation of the minimum postmortem interval (PMImin). Finally, this work combined the total reflection-fourier transform infrared spectroscopy with chemometric analysis and successfully developed a partial least squares discriminant analysis model and a random forest model, with accuracies of 88 % and 81 %, respectively. These models enable the non-invasive age estimation of P. regina in its intra-puparial period, a stage traditionally difficult to assess morphologically, thus laying the groundwork for PMImin estimation using fly pupae.

First description of immature stages and notes on the biology of the blow fly, Calliphora lopesi Mello, 1962.

Several species of the worldwide distributed genus Calliphora Robineau-Desvoidy (Insecta, Diptera, Calliphoridae) are medically important vectors and agents of myiasis. Furthermore, these flies are relevant in forensics because they are found in corpses. Information regarding the taxonomy, bionomics and distribution of Calliphora species endemic to South America, including Calliphora lopesi Mello, is scarce. To expand knowledge on C. lopesi, this study presents descriptions of eggs, larvae, puparia and developmental data at 14, 17, 20, 23 and 26 ± 1°C for the first time. Adult flies were collected from the field and kept in the laboratory to obtain samples for morphological and biological studies. Immatures were examined using light and scanning electron microscopy. To assess the growth rate, 10 specimens from each temperature group were randomly removed from the diet and weighed every 24 h from larval hatching until pupation. The minimum developmental threshold, thermal constant and linear development-rate equations were calculated for each stage. Considering weight gain records and survival rates, the optimum temperature for the development of C. lopesi ranges from 23 to 26°C. A key to third-instar larvae of known Neotropical species of Calliphora was also provided to assist in identification. The information provided in this study should be useful in expanding knowledge about Neotropical Calliphoridae species of forensic importance.

Várias espécies do gênero Calliphora Robineau­Desvoidy (Insecta, Diptera, Calliphoridae), distribuídas mundialmente, são vetores e agentes causadoras de miíases clinicamente importantes. Além disso, tais moscas são relevantes no âmbito forense porque são encontradas em cadáveres. Informações sobre a taxonomia, bionomia e distribuição de espécies de Calliphora endêmicas da América do Sul, incluindo Calliphora lopesi Mello, ainda são escassas. Para ampliar o conhecimento sobre a espécie C. lopesi, este estudo apresenta pela primeira vez as descrições de ovos, larvas e pupários, assim como dados sobre o desenvolvimento de imaturos a 14, 17, 20, 23 e 26 ± 1°C. Moscas adultas foram coletadas no campo e mantidas em laboratório para obtenção de amostras para estudos morfológicos e biológicos. Os imaturos foram examinados utilizando microscopia óptica e eletrônica de varredura. Para avaliar a taxa de crescimento, 10 espécimes de cada grupo de temperatura foram retirados aleatoriamente do substrato em que se alimentavam e pesados a cada 24 horas, desde a eclosão das larvas até a pupariação. O limiar mínimo de desenvolvimento, a constante térmica e as equações lineares da taxa de desenvolvimento foram calculados para cada estágio. Levando em consideração os registros de ganho de peso e taxas de sobrevivência, a temperatura ótima para o desenvolvimento de C. lopesi varia de 23 a 26°C. Uma chave para larvas de terceiro estádio de espécies neotropicais conhecidas de Calliphora também está sendo disponibilizada para auxiliar na identificação. Espera­se que as informações fornecidas neste estudo possam ser úteis para ampliar o conhecimento sobre espécies Neotropicais de Calliphoridae de importância forense.

Identification of Diptera Puparia in Forensic and Archeo-Funerary Contexts.

Diptera identification is fundamental in forensic entomology as well as in funerary archeoentomology, where the challenge is exacerbated by the presence of immature stages such as larvae and puparia. In these two developmental stages, specimens possess a very limited number of diagnostic features, and for puparia, there is also a lack of identification tools such as descriptions and identification keys. Morphological analysis, DNA-based techniques, and cuticular chemical analyses all show good potential for species identification; however, they also have some limitations. DNA-based identification is primarily hindered by the incompleteness of genetic databases and the presence of PCR inhibitors often co-extracted from the puparial cuticle. Chemical analysis of the cuticle is showing promising results, but this approach is also limited by the insufficient profile database and requires specific, expensive equipment, as well as trained personnel. Additionally, to ensure the repeatability of the analysis-a critical aspect in forensic investigations-and to preserve precious and unique specimens from museum collections, non-invasive protocols and techniques must be prioritized for species identification.

Forensic case reports of Dermestes maculatus (Coleoptera: Dermestidae) in Santa Catarina state, Southern Brazil.

Only a few cases describing the activity of dermestids on human corpses have been reported in the literature. Dermestes maculatus is a cosmopolitan beetle associated with carcasses at different decomposition stages, usually colonizing skeletonized and mummified remains. In this study, we presented two forensic case reports of D. maculatus associated with human corpses in Santa Catarina State, southern Brazil. In the first case, a human corpse in an advanced stage of decomposition was found hanged in an outdoor urban area. In the other one, a mummified cadaver was found inside of a closed house. In this last case, larvae of D. maculatus were essential to estimate the minimum postmortem interval (PMI) for the first time in Scientific Police of Santa Catarina. Our records highlight the significance of necrophagous beetles in ecological succession and in estimating PMI in cases involving human remains and corpses in advanced stage of decomposition.

Variations in cuticular hydrocarbons of Calliphora vicina (Diptera: Calliphoridae) empty puparia: Insights for estimating late postmortem intervals.

Necrophagous flies, particularly blowflies, serve as vital indicators in forensic entomology and ecological studies, contributing to minimum postmortem interval estimations and environmental monitoring. The study investigates variations in the predominant cuticular hydrocarbons (CHCs) viz. n-C25, n-C27, n-C28, and n-C29 of empty puparia of Calliphora vicina Robineau-Desvoidy, 1830, (Diptera: Calliphoridae) across diverse environmental conditions, including burial, above-ground and indoor settings, over 90 days. Notable trends include a significant decrease in n-C25 concentrations in buried and above-ground conditions over time, while n-C27 concentrations decline in buried and above-ground conditions but remain stable indoors. Burial conditions show significant declines in n-C27 and n-C29 concentrations over time, indicating environmental influences. Conversely, above-ground conditions exhibit uniform declines in all hydrocarbons. Indoor conditions remain relatively stable, with weak correlations between weathering time and CHC concentrations. Additionally, machine learning techniques, specifically Extreme Gradient Boosting (XGBoost), are employed for age estimation of empty puparia, yielding accurate predictions across different outdoor and indoor conditions. These findings highlight the subtle responses of CHC profiles to environmental stimuli, underscoring the importance of considering environmental factors in forensic entomology and ecological research. The study advances the understanding of insect remnant degradation processes and their forensic implications. Furthermore, integrating machine learning with entomological expertise offers standardized methodologies for age determination, enhancing the reliability of entomological evidence in legal contexts and paving the way for future research and development.

A methodological approach to age estimation of the intra-puparial period of the forensically relevant blow fly Calliphora vicina via Fourier transform infrared spectroscopy.

Estimating the age of immature blow flies is of great importance for forensic entomology. However, no gold-standard technique for an accurate determination of the intra-puparial age has yet been established. Fourier transform infrared (FTIR) spectroscopy is a method to (bio-)chemically characterise material based on the absorbance of electromagnetic energy by functional groups of molecules. In recent years, it also has become a powerful tool in forensic and life sciences, as it is a fast and cost-effective way to characterise all kinds of material and biological traces. This study is the first to collect developmental reference data on the changes in absorption spectra during the intra-puparial period of the forensically important blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Calliphora vicina was reared at constant 20°C and 25°C and specimens were killed every other day throughout their intra-puparial development. In order to investigate which part yields the highest detectable differences in absorption spectra throughout the intra-puparial development, each specimen was divided into two different subsamples: the pupal body and the former cuticle of the third instar, that is, the puparium. Absorption spectra were collected with a FTIR spectrometer coupled to an attenuated total reflection (ATR) unit. Classification accuracies of different wavenumber regions with two machine learning models, i.e., random forests (RF) and support vector machines (SVMs), were tested. The best age predictions for both temperature settings and machine learning models were obtained by using the full spectral range from 3700 to 600 cm-1. While SVMs resulted in better accuracies for C. vicina reared at 20°C, RFs performed almost as good as SVMs for data obtained from 25°C. In terms of sample type, the pupal body gave smoother spectra and usually better classification accuracies than the puparia. This study shows that FTIR spectroscopy is a promising technique in forensic entomology to support the estimation of the minimum post-mortem interval (PMImin), by estimating the age of a given insect specimen.

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.

Novel use of a servosphere to study apodous insects: Investigation of blow fly post-feeding larval dispersal.

Blow flies (Diptera: Calliphoridae) are arguably the most important providers of an estimate of minimum post-mortem interval in forensic investigations. They usually undergo a post-feeding dispersal from the body. While previous studies have looked at dispersal of groups of larvae, recording the dispersal activity of individual larvae has not previously been demonstrated. A servosphere was used here to record the speed, directionality and phototaxis of individual post-feeding larvae of two species of blow fly on a smooth plastic surface over time. The servosphere rotates to compensate for the movement of an insect placed at its apex, thereby enabling its unimpeded locomotion in any direction to be studied and behavioural changes to external stimuli recorded. To our knowledge, the servosphere has not previously been used to study apodous insects. The objective of our study was to compare dispersal behaviour of Calliphora vicina Robineau-Desvoidy and Protophormia terraenovae (Robineau-Desvoidy), both common primary colonisers of human and animal cadavers, but showing different post-feeding dispersal strategies. Larvae of C. vicina generally disperse from the body while those of P. terraenovae remain on or close to the body. Our aims were to study (1) changes in dispersal speed over a 1-h period; (2) changes in dispersal speed once a day for 4 days, between the end of feeding and onset of pupariation; and (3) response of dispersing larvae to light. We demonstrated that (1) the movement of three C. vicina larvae tracked for 1 continuous hour on 1 day slowed from an average of 3 to <1.7 mms-1; (2) the average speed of 20 larvae of C. vicina (4.08 mms-1) recorded for 5 min once per day over a 4-day period between onset of dispersal and pupariation was significantly greater than that of P. terraenovae (2.36 mms-1; p < 0.0001), but that speed of both species increased slightly over the 4 days; (3) the responses of larvae of C. vicina to changes in light direction from the four cardinal directions of the compass, showed that they exhibited a strong negative phototactic response within 5 s, turning to move at approximately 180° away from the new light position. While conducted to observe larval calliphorid post-feeding behaviour, the results of this proof of concept study show that apodous insects can be studied on a servosphere to produce both qualitative and quantitative data.

Intrapuparial stage aging and PMI estimation based on the developmental transcriptomes of forensically important Aldrichina grahami (Diptera: Calliphoridae) gene expression.

Background: The expression profiles of differentially expressed genes (DEGs) during pupal development have been demonstrated to be vital in age estimation of forensic entomological study. Here, using forensically important Aldrichina grahami (Diptera: Calliphoridae), we aimed to explore the potential of intrapuparial stage aging and postmortem interval (PMI) estimation based on characterization of successive developmental transcriptomes and gene expression patterns. Methods: We collected A. grahami pupae at 11 successive intrapuparial stages at 20 °C and used the RNA-seq technique to build the transcriptome profiles of their intrapuparial stages. The DEGs were identified during the different intrapuparial stages using comparative transcriptome analysis. The selected marker DEGs were classified and clustered for intrapuparial stage aging and PMI estimation and then further verified for transcriptome data validation. Ultimately, we categorized the overall gene expression levels as the dependent variable and the age of intrapuparial A. grahami as the independent variable to conduct nonlinear regression analysis. Results: We redefined the intrapuparial stages of A. grahami into five key successive substages (I, II, III, IV, and V), based on the overall gene expression patterns during pupal development. We screened 99 specific time-dependent expressed genes (stage-specific DEGs) to determine the different intrapuparial stages based on comparison of the gene expression levels during the 11 developmental intrapuparial stages of A. grahami. We observed that 55 DEGs showed persistent upregulation during the development of intrapuparial A. grahami. We then selected four DEGs (act79b, act88f, up and ninac) which presented consistent upregulation using RT-qPCR (quantitative real-time PCR) analysis, along with consideration of the maximum fold changes during the pupal development. We conducted nonlinear regression analysis to simulate the calculations of the relationships between the expression levels of the four selected DEGs and the developmental time of intrapuparial A. grahami and constructed fitting curves. The curves demonstrated that act79b and ninac showed continuous relatively increasing levels. Conclusions: This study redefined the intrapuparial stages of A. grahami based on expression profiles of developmental transcriptomes for the first time. The stage-specific DEGs and those with consistent tendencies of expression were found to have potential in age estimation of intrapuparial A. grahami and could be supplementary to a more accurate prediction of PMI.

Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae): A potentially forgotten forensically important species in Malaysia.

Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) has been collected in human forensic case work. This species is endemic in Malaysia but is not one of the most common species and is often found in outdoor cases. However, it is hypothesized that the presence of L. cuprina larvae may provide useful information in forensic case studies in Malaysia where this species has previously been ignored. This review will discuss the current background knowledge on L. cuprina, particularly when it comes to other forensic cases in Malaysia. General biology as well as key information for forensic work such as geographical distribution and developmental data will be reviewed. Finally, we discuss the potential for L. cuprina to provide beneficial and unique forensic insight into indoor cases with refuse.

Influence of Soil Type and Moisture on Pupal Development of Chrysomya rufifacies (Macquart) at Two Different Temperatures.

The present study investigates the developmental process of Chrysomya rufifacies (Macquart) pupae and their dependency on soil composition, moisture levels, and temperature changes. This research holds implications for forensic and veterinary applications, providing crucial insights for estimating minimum postmortem intervals and managing myiasis-causing flies in diverse environments. Specifically, the study explores the impact of five moisture content levels in loam and sandy soils (0%, 20%, 40%, 60%, and 80%) on the pupal development of Ch. rufifacies under two distinct constant temperature regimes (24 ± 1 °C and 30 ± 1 °C). A significant correlation was observed between soil type and temperature regarding the time required to complete the pupal stages; however, moisture had no significant impact. Larvae exhibited varying survival rates across the two temperatures and five moisture levels in the two types of soils, particularly under extremely lower moisture conditions (0%) at 30 ± 1 °C, failing to progress to the pupariation stage. Additionally, growth parameters such as pupal length and width of the fully formed puparia were significantly impacted by temperature, soil type, and moisture level. Adult head width was systematically measured across different moisture levels and soil types, revealing distinct temperature-dependent responses. Furthermore, a sex-specific analysis highlighted that female Ch. rufifacies consistently displayed larger head widths and higher emergence rates compared to their male counterparts. This research enhances our understanding of the intricate interrelationship among three environmental variables: soil type, moisture level, and temperature, elucidating their collective impact on the pupation processes of dipterans.

Spatio-Temporal Influence on the Distribution of Forensically Relevant Blowflies (Diptera: Calliphoridae) in Gyeongsangnam-do, South Korea.

The study of blowfly (Diptera: Calliphoridae) biodiversity and distribution is crucial for forensic investigations. Abiotic and biotic factors, such as season and habitat type, have a significant impact on blowfly populations. However, only a few forensic entomology studies have been conducted in South Korea, particularly in the Gyeongsangnam-do region. To address this, an extensive year-long survey was conducted to analyze the compositions, habitat preferences, distribution, and seasonal abundance of forensically relevant blowflies in urban and forested habitats of Gyeongsangnam-do, with sampling conducted twice a month using mouse carcass-baited traps set for 48 h each time. A total of 3470 adult blowflies were recorded, encompassing five genera and 13 species, with a noted absence of specimens during the winter months. The predominant species was Lucilia porphyrina, accounting for 37.2% of the total sample, followed by Chrysomya pinguis (27.6%), Lucilia sericata (7.6%), and Lucilia illustris (7.1%). The species composition was consistent across all surveyed regions; however, seasonal variation in species diversity was evident, with a peak in spring and a decline in summer. Notably, certain species exhibited clear preferences for either urban (Calliphora calliphoroides and L. sericata) or forested habitats (L. porphyrina and Ch. pinguis). This pioneering study elucidates the diverse blowfly communities in Gyeongsangnam-do, highlighting significant seasonal and habitat-dependent variations. These findings enrich our understanding of blowfly ecology in this region, offering valuable insights for forensic applications and underscoring the necessity for ongoing entomological surveillance and research.

Development Modeling of Phormia regina (Diptera: Calliphoridae).

A series of experiments were conducted on Phormia regina, a forensically important blow fly species, that met the requirements needed to create statistically valid development models. Experiments were conducted over 11 temperatures (7.5 to 32.5 °C, at 2.5 °C intervals) with a 16:8 L:D cycle. Experimental units contained 20 eggs, 10 g of beef liver, and 2.5 cm of sand. Each life stage (egg to adult) had five sampling times. Each sampling time was replicated four times for a total of 20 measurements per life stage. For each sampling time, the cups were pulled from the chambers, and the stage of each maggot was documented morphologically through posterior spiracular slits and cephalopharyngeal development. Data were normally distributed with the later larval (L3m) and pupation stages having the most variation within and transitioning between stages, particularly between 12.5 °C and 20.0 °C. The biological minimum was between 10.0 °C and 12.5 °C, with little egg development and no egg emergence at 7.5 °C and no maturation past L1 at 10.0 °C. Phormia regina did not display increased mortality associated with the upper temperature of 32.5 °C. The development data generated illustrate the advantages of large data sets in modeling blow fly development and the need for curvilinear models in describing development at environmental temperatures near the biological minima and maxima.

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.

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.

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.