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Forensic entomology serves a vital role in criminal investigations, contributing to the determination of time of colonization (TOC), minimum post-mortem interval (minPMI), and other forensic evidence related to the decomposition process. This study aimed to identify Calliphoridae species attracted to carcasses in two distinct locations in Algeria and to understand their role in forensic investigations. Two sites in Constantine were selected: a forest in Jebel Ouahch and an urban area in Chaabat Erssas. Two dog carcasses, an adult and a puppy, were monitored for decomposition stages. The puppy carcass reached the dry stage faster than the adult carcass, with decomposition rates varying between sites. A total of 594 Calliphoridae flies were collected, including Calliphora vicina, Calliphora vomitoria, Calliphora subalpina, Lucilia sericata, Lucilia illustris, Lucilia silvarum, and Chrysomya albiceps. These species were present in both locations, with Ch. albiceps being most prevalent, especially on the puppy carcass. Initial blowfly colonization occurred within four hours, with L. sericata and Ch. albiceps particularly abundant. This study underscores the importance of accurate species identification and environmental reconstruction in improving the precision of forensic estimates. Continued research on species phenology and the impacts of climate change will further enhance the applicability of forensic entomology in Algeria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT The use of monitoring data to build prediction models for the abundance and activity of necrophagous blow flies is common practice in forensic entomology, but its advantages and disadvantages are still debated. A frequently asked question is the transferability of such species prediction models. So far no study has examined whether the assumption of low transferability of these data between cities and seasons holds true. In the present study, we evaluate whether models calibrated with a specific training data set from a specific place and time can be transferred to other data sets for different time periods and locations. We developed models using five different algorithms to predict the activity and abundance of four forensically relevant blow fly species (Calliphora vicina Robineau‐Desvoidy, Lucilia ampullacea Villeneuve, Lucilia caesar (Linnaeus), Lucilia sericata (Meigen)). The training data set was obtained from a single city, and the transferability of the models was evaluated using monitoring data from this and three other cities. The geographic transferability of the models was confirmed for all algorithms, but only for two species, C. vicina and L. sericata, and for two of the four cities. Lucilia caesar and L. ampullacea were rare in the test data set, and their species‐specific adaptation to environmental parameters was not captured by the models. Cities that did not work differed from the training data set in terms of climate and habitat features. To build generalised predictive models of blow fly abundance and activity, we need training data sets based on monitoring data from different regions, seasons and years to cover a wide range of environmental conditions. This is essential for describing and predicting natural variability. [ABSTRACT FROM AUTHOR]

Simple Summary: Flies are often associated with urban waste, myiasis and decomposing bodies. Helicobia aurescens is found in carcasses and could be useful to forensic entomologists in the determination of the postmortem interval. However, a few studies are available regarding the morphology and biology of this species. We describe aspects of the life cycle at two temperatures (27 ± 1 °C and 29 ± 1 °C) and analyze the morphological characteristics of the eggs, larvae, and puparia of H. aurescens using scanning electron microscopy (SEM). A lower temperature affected the flesh fly development, increasing the total development time. The immature morphology was very similar to those of the others sarcophagids. No hatching lines or median areas were detected on the eggshells. The first-instar larva is very similar to those of the other species. The anterior spiracles have six or seven ramifications aligned regularly at the second instar. But, on the third instar, these structures have eight ramifications in a regular row and are located dorsolaterally. The puparia are similar to that of the third-instar larvae. Helicobia aurescens is a flesh fly associated with pig and rat carcasses. This study aims to describe the life cycle at two temperatures (27 ± 1 °C and 29 ± 1 °C) and analyze the morphological characteristics of the eggs, larvae, and puparia of H. aurescens using scanning electron microscopy (SEM). Temperature is an abiotic factor that greatly influences the development of insects. The larval development of H. aurescens lasts longer at 27 ± 1 °C than it does at 29 ± 1 °C, affecting the growth of newly hatched larvae into adults. The females larviposited three times more at 27 °C than they did at 29 °C, and the number of days laying larvae was also greater. At 27 °C, they laid larvae for 38 days, whereas, at 29 °C, the females larviposited for 21 days. No hatching lines or median areas were detected on the exochorion with SEM, as in the other sarcophagid species. The first-instar larva is very similar to those of the other species. The second instar has anterior spiracles present on the first thoracic segment, with six or seven ramifications aligned regularly. On the third instar, these structures have eight ramifications in a regular row and are located dorsolaterally. The puparium morphology is similar to that of the third-instar larvae. [ABSTRACT FROM AUTHOR]

Background: The fleshfly Sarcophaga dux (Diptera;Sarcophagidae) is found in Indian Sub continent is of limited forensic significance as its poorly known larval development relationship with temperature and relative humidity. Objective: The objective of this study was to determine the effect of the temperature and relative humidity in the larval development of this fly in two seasons, Summer and Rain, so that the results of this study may enabling the forensic use of this fly in the estimation of Post Mortem Interval(PMI). Material and Method: The present study was carried out in the outer premises of the Regional Forensic Science Laboratory Building, Jagdalpur, Bastardist,Chhattishgarh, India. The fresh meat of male goat was used for rearing of larvae. The effect of temperature and relative humidity on the larval developmental time of this fly in two seasons, Rainy and Summer were studied in normal condition. Statistical analysis of variance was performed and standard significance level of P<0.05 was taken as standard. Results: The results of this study shown that in Rainy season it was completed in 264 h (11 days) when the average temperature ranged from 21.8°C to 24.5°C and the average humidity ranged from 79.5% to 95.5% where as in Summer it was completed in 528 hrs (22 days), when the temperature ranged from 26.5°C to 35.1°C and the humidity ranged from 34% to 67.5%. The P value was found to be P<<.05 in Temperature variation; andP<<0.05 in Humidity variation in different stages(1st instar larvae, 2nd instar larvae, 3rd instar larvae, Prepupae, Pupae and Adult). Conclusion: In the present study we have found that fluctuating temperature and relative humidity of outer condition in two seasons, Rainy and Summer effect the life cycle duration of the fly from the larvi position up to adult eclosion and statistical analysis has proved it to significant rejecting null hypothesis. This data can be further used in the estimation of Post Mortem Interval (PMI) in Homicide and Suspicious cases of both human and wild life poaching death cases. [ABSTRACT FROM AUTHOR]

Blow flies (Diptera: Calliphoridae) can be used in forensic entomology to provide information, including an estimate of the time of colonization and minimum postmortem interval, based on insect development. This study examined the development of third instar Calliphora terraenovae Macquart in four substrates: pine shavings, soil, sand, and clay. Pupation time, survival to adult, and wing vein length were measured to examine the substrate influence. There was a significant difference in the time to pupation (F3,36 = 11.87, p < 0.0001) and the number of flies that eclosed (F3,36 = 4.716, p = 0.007) among the substrates. Blow flies pupated faster in pine shavings and eclosed as adult flies faster in sand than in other substrates. Adults eclosed in the sand in 21.9 days, followed by pine shavings (22.8), clay (24.2), and soil (26.6). Although overall survivorship was low (10–46%), the greatest number of flies eclosed in sand, and the fewest in clay. Understanding the factors that impact blow fly development can help forensic entomologists improve rearing protocols and apply this information to death investigations, especially in cases with buried remains.

A female dog had allegedly been alive one day before its death. The veterinary office thought about accusing the dog’s owner for animal cruelty and commissioned forensic entomological expertise for the calculation of the time of insect colonization on the dog’s body. The statement of the dog’s owner was proven false by us on the grounds of (a) the advanced state of decomposition and (b) the minimal developmental time of fly larvae found on the dog’s body. The darkening and deformation of the fly maggots as well as insufficient temperature data made case work trickier than usual. We worked through the case by creating forensic entomological temperature scenarios. The court used the entomological evidence and issued a penalty order.

Different taxa of insects have been reported to modify the skeletal remains of vertebrates during feeding or the creation of their larval and pupal chambers. Anecdotal reports on the modification of skeletal remains by ants exist but are rare. In this paper, a case of modification of bone due to the feeding activity of the ant, Crematogaster cf. liengmei (Hymenoptera: Formicidae), is reported. In November 2022, a bone was encountered incidentally in the vicinity of the Table Mountain National Park. The bone had been colonized by several individuals of Crematogaster cf. liengmei. Several ants were observed feeding on and removing bone particles and soft tissue remnants. A closer observation revealed striae/furrows on the epiphyseal ends of the bone. A 60-kg pig cadaver used in a winter decomposition trial in the same area was similarly colonized by this ant species, resulting in soft-tissue modification. Due to the similarity in the striae/furrows observed on the bone and the bitemarks inflicted by the ants on the right ear of the pig cadaver in the vicinity, it was concluded that the striae/furrows seen on the bone were created by the ants. Our observations show that Crematogaster cf. liengmei is an important taphonomic bio-agent that can contribute to the modification of skeletal remains in terrestrial environments. [ABSTRACT FROM AUTHOR]

Simple Summary: As an important group of species in forensic entomology, sarcosaprophagous beetles can provide valuable clues to the postmortem interval of cadavers, especially in the late stages of decay. However, compared with flies, these species have not received much attention, and their potential value in forensic science is yet to be fully exploited. Therefore, in order to attract people's attention to them and to increase their use in forensic practice, we conducted a bibliometric analysis of the literature on sarcosaprophagous beetles, statistically analyzed the beetles mentioned in successions, cases, and other experiments in relation to cadavers, briefly introduced each family of sarcosaprophagous beetles, and discussed the value of their immature species identification and forensic postmortem interval estimation. In forensic entomology, research focused on sarcosaprophagous flies, but the sarcosaprophagous beetles, as important "forensic indicator species" of late-stage PMI in cadaver decomposition, received less attention. To increase attention on, and use and understanding of, sarcosaprophagous beetles in forensic entomology, this paper presents a bibliometric analysis of the available relevant literature. The occurrence frequency of beetle families and species from succession studies, actual cases, and experiments were calculated and illustrated using graphs. As a result, a total of 14 families and 1077 species associated with carcasses were collected, with Staphylinidae being the most frequently recorded among the families, and Necrobia rufipes (Fabricius, 1781) (Coleoptera: Cleridae) being the most frequently recorded species. In addition, a brief introduction of the cadaver-related beetles of each family is given, and research on the species identification of the immature stages, age estimation of the immature stages, and estimation of the arrival time of sarcosaprophagous beetles are discussed and prospected. This work will aid in the increased use of sarcosaprophagous beetles in forensic science practice in the future. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Blow flies (Diptera: Calliphoridae) can be used in forensic entomology to provide information, including an estimate of the time of colonization and minimum postmortem interval, based on insect development. This study examined the development of third instar Calliphora terraenovae Macquart in four substrates: pine shavings, soil, sand, and clay. Pupation time, survival to adult, and wing vein length were measured to examine the substrate influence. There was a significant difference in the time to pupation (F3,36 = 11.87, p < 0.0001) and the number of flies that eclosed (F3,36 = 4.716, p = 0.007) among the substrates. Blow flies pupated faster in pine shavings and eclosed as adult flies faster in sand than in other substrates. Adults eclosed in the sand in 21.9 days, followed by pine shavings (22.8), clay (24.2), and soil (26.6). Although overall survivorship was low (10–46%), the greatest number of flies eclosed in sand, and the fewest in clay. Understanding the factors that impact blow fly development can help forensic entomologists improve rearing protocols and apply this information to death investigations, especially in cases with buried remains. [ABSTRACT FROM AUTHOR]

A female dog had allegedly been alive one day before its death. The veterinary office thought about accusing the dog's owner for animal cruelty and commissioned forensic entomological expertise for the calculation of the time of insect colonization on the dog's body. The statement of the dog's owner was proven false by us on the grounds of (a) the advanced state of decomposition and (b) the minimal developmental time of fly larvae found on the dog's body. The darkening and deformation of the fly maggots as well as insufficient temperature data made case work trickier than usual. We worked through the case by creating forensic entomological temperature scenarios. The court used the entomological evidence and issued a penalty order. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Coleopterans constitute the second largest order of insects of forensic importance, after the dipterans, which scavenger species are fundamental participants in the process of cadaveric decomposition in terrestrial ecosystems. This study aims to: (i) investigate whether coleopterans are indeed more attracted to the final stages of decomposition in the Cerrado, as observed in other biomes; (ii) identify which species colonize carcasses; and (iii) compile a list of forensically relevant beetles for the biome. We performed the experiments in two protected areas between June 2017 and January 2019, in months representative of the dry and rainy seasons. We conducted collections of beetles using 72 pig carcasses throughout all stages of decomposition. A total of 3,006 beetles were identified into 66 taxa from 12 families. Beetles were most abundant in the final stages of decomposition, particularly during the skeletonization stage, which accounted for approximately 45% of the collected individuals. Considering the most abundant families, Scarabaeidae showed no preference in attractiveness amongst the stages of decomposition, whilst Staphylinidae, Silphidae, Dermestidae, and Histeridae were more attracted by the two latter stages of the process. Two species, Dermestes maculatus (De Geer, 1774) and Oxelytrum discicolle (Brullé, 1840), were observed colonizing the carcasses. This work is pioneering for the Cerrado, uses a large number of carcasses and therefore contributes to a better understanding of ecological processes in the area by approaching the ecology of decomposition of carcasses and scavenger beetles. A list of the species in the region and their utility to forensic entomology is also discussed. [ABSTRACT FROM AUTHOR]

Blowflies and beetles are among the invertebrate species that are attracted to dead bodies and can provide valid evidence in cases of murder or suspicious death investigation. An important consideration is the quality of the evidence which partly depends on the accurate identification of invertebrates. The morphological identification of blowflies has some limitations, especially in relation to damaged samples, or immature stages of the flies. DNA-based techniques such as the polymerase chain reaction (PCR) and sequence analysis have been applied to overcome the shortfalls of the classical identification methods. The present study combined the use of classical and PCR methods to identify invertebrates associated with pig carrions in Nigeria. Adult invertebrates were collected on and around pig carcasses in Nigeria. A total of 416 invertebrates belonging to Diptera; Calliphoridae (n = 160), Sarcophagidae (n = 26) and Muscidae (n = 72) while Coleopterans; Dermestidae (n = 52), Histeridae (n = 63), Cleridae (n = 25) and Silphidae (n = 18) families collected from pig carrions in Nigeria were identified. The cytochrome oxidase 1 (cox1) gene from selected specimens was amplified and sequenced. Nucleotide sequence analysis and phylogenetic trees confirmed the presence of dipterans and coleopterans of forensic importance in Nigeria. To the best of our knowledge, this is the first report to use the mitochondrial cox1 gene coupled with sequence analysis to identify and characterize arthropods of forensic importance in Nigeria. This study provides reliable baseline data for further use in forensic entomology within the country and other regions where these arthropod species exist. [ABSTRACT FROM AUTHOR]

Simple Summary: In forensic entomology, insects are used to estimate the time since death and to answer other investigative questions, such as determining if the body was moved postmortem or identifying the season of death. In funerary archeoentomology, insects provide information related to the season of death, body transfer, and funerary practices. To obtain this information, the correct identification of specimens collected from the scene is fundamental. In old cases and archeological contexts, the majority of entomological evidence samples found are fly puparia, barrel-like structures formed from the third-instar larval cuticle where metamorphosis takes place. Despite recent publications of identification keys for certain fly larvae and adults, there is still a lack of tools for puparium identification. This problem is exacerbated by the scarcity of diagnostic features present on the puparia. This paper critically reviews the techniques available for puparium identification in addition to traditional morphological analysis. It describes DNA-based approaches and chemical analyses of cuticular lipids and hydrocarbons, highlighting the importance of using non-invasive methods to guarantee the repeatability of the analysis—a key element in forensic science—or to preserve precious and unique specimens from museum collections. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The advantages demonstrated by infrared spectroscopy (IR) in forensic science and entomology have aroused the interest of forensic entomologists and it has become a powerful analytical tool in the field of forensic entomology (FE) as it yields reliable results in insect identification, gut content analysis, distribution analyses, and developmental time estimations. In this paper, we summarize the current challenges and future perspectives of IR in three main research areas of FE, namely postmortem interval, carrion, and identification. With data from the past three decades (1998-2023), we report on the potential of IR to improve the efficiency of litigation cases and the scientific validity of FE laboratory research. Results show that although IR still has a long way to go and its accessibility needs to be improved, it possesses the potential to become a reliable research technique in FE. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The decomposition of a body, and the associated gaseous and liquid discharges emanating from it, attract gravid female blow flies which lay their eggs in or on the body. After the eggs have hatched, the emerging larvae start feeding on the body. As decomposition progresses, the blow fly larvae often migrate away, typically in a random manner in search of favourable conditions for pupation. In this paper, we report on a rarely described phenomenon of unidirectional mass migration of blow fly larvae and postulate on the factors which may drive this process. A decomposition trial utilising a 60-kg pig carcass, deployed in the summer months in Table Mountain National Park, Cape Town, South Africa, was conducted in 2022. On the fifth day of the trial, simultaneous unidirectional mass dispersal of blow fly larvae was observed. The larvae moved downhill in a southeasterly direction, following the flow of decomposition fluids oozing out from the pig carcass. The 'larval migration stream' had a length of approximately 1.5 m with a width of 40 cm, tapering to 17 cm at the terminal point. The larval migration stream consisted of the larvae of Chrysomya albiceps and Chrysomya chloropyga. This study demonstrates the importance of understanding the timing and pattern of dispersal of post-feeding blow fly larvae in each geographical region. This is crucial as the minimum post-mortem interval can be miscalculated if older immature insects dispersing from the corpse are not considered and collected during crime scene investigations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Research into the community composition and succession of blowflies can provide valuable insights into the timing and environmental conditions of colonization events, enhancing the accuracy of forensic analyses. Blowfly species diversity and distribution are influenced by various environmental factors such as season, habitat, and geographical location, which in turn affect their behavior and colonization patterns. The researchers surveyed blowfly populations across four distinct regions over the course of a year, examining how factors like season, habitat, and location influenced the blowfly community. The findings showed significant seasonal variations, emphasizing the impact of environmental conditions on blowfly behavior and colonization patterns. This research provides important regional data on forensically relevant blowfly species, which can help improve the accuracy of estimating the time and circumstances of death during criminal investigations. By enhancing our understanding of blowfly ecology, this work also contributes to broader scientific knowledge about decomposition processes and the role of insects in ecosystem functioning, ultimately benefiting crime scene investigations and advancing the field of forensic science. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Simple Summary: The blow fly Phomia regina is arguably the most important forensic indicator species in the United States of America. Typically, the development rate of forensically important insects is used to estimate the postmortem interval in situations with decomposing bodies. Consequently, this study examined the development of P. regina across 11 temperatures. Evidence demonstrated that maggots have a normal distribution in their transitions between stages. The large data set allowed for the development of a comprehensive degree-day model across all life stages. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Chrysomya rufifacies (Macquart), commonly known as the hairy maggot blow fly, is important in forensic and medical contexts because it colonizes carcasses and is a causative agent of myiasis in animals and humans. Studying the development of its pupae is crucial for understanding the time since death in forensic investigations and for containing the spread of myiasis. This study investigated how temperature, soil type, and moisture affect the development of Ch. rufifacies pupae in Western Australia. 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. [ABSTRACT FROM AUTHOR]

• • At 19 − 34 °C, D. maculatus can complete the entire immature stage of development. • • The lower and upper developmental thresholds were 15.28 and 34.03 °C, respectively. • • The survival rate was the highest at 22 °C and the lowest at 34 °C. • • Body length, the widths of head capsule and pronotum were modeled over time. Necrophagous beetles are sometimes used to estimate the minimum postmortem interval (PMI min) in the decay and remains stages of a corpse. Among these, the Dermestidae is one of the most common groups used and therefore has important research and application value. In this study, the developmental events of Dermestes maculatus de Geer, 1774, were recorded at six constant temperatures, and isomorphen diagrams were established. The thermobiological parameters were estimated using linear and non-linear models, and morphological indicators such as larval body length were measured. The results showed that the developmental duration of the whole immature stage decreased from 66.13 ± 8.58 days at 19 °C to 21.9 ± 2.01 days at 34 °C. The survival rate of the immature stages, especially the egg stage, varies greatly with temperature, with the lowest survival observed at 34 °C and the highest at 22 °C. The lower developmental threshold, the intrinsic optimum temperature, and the upper lethal developmental threshold obtained by the curvilinear Optim SSI models were 15.28 °C, 28.36 °C, and 34.03 °C, respectively. The body length, head capsule width, and pronotum width showed obvious growth patterns with larval developmental duration, which were characterized by equations and isomegalen diagrams. This study provides important basic data for the application of D. maculatus to estimate the PMI min in forensic entomology in the Yangtze River Delta region of China. [ABSTRACT FROM AUTHOR]

• Lucilia cuprina (Diptera: Calliphoridae) are associated with human decomposition. • L. cuprina have been recorded in very few cases within Malaysia. • This species has been collected in a recent indoor forensic case in Malaysia. • Further research on L. cuprina 's development and behavior in casework is necessary. 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. [ABSTRACT FROM AUTHOR]

ABSTRACT This study investigated the effects of burning on the richness, abundance, and community structure of necrophagous flies in southern Brazil. Flies were collected over a period of seven days in the field, and were subsequently observed for 60 days in the laboratory. There were three bovine liver treatments: unburnt, burnt, and gasoline-treated (control). A total of 2,501 flies, representing 14 families and 41 species, were identified, including Chrysomya albiceps, Fannia subgroup pusio, Synthesiomyia nudiseta, and Fannia femoralis. While the abundance of flies was similar across the three treatments, exhibiting a quadratic distribution, species richness displayed a quadratic trend with a significant temporal shift; peak richness occurred 20 hours earlier in the unburnt liver than in the burnt or the control treatments. Both total abundance and patterns of community composition in our analysis indicated important factors to take into account. Total abundances were similar between the burnt and unburnt treatments, demonstrating a uniform ecological response to burning, irrespective of the condition of the liver. In contrast, the community composition of these treatments showed a significant divergence from the control treatment, which was distinctively dominated by Drosophilidae and Phoridae. These findings are crucial for accurate postmortem Interval (PMI) estimations in forensic entomology, particularly in cases involving burnt remains, and highlight the significant impact of fire on necrophagous fly communities.

ABSTRACT Sarcophagidae flies have great forensic importance, since different instars of their larval development are used in the determination of the postmortem interval (PMI) of corpses. However, for many species, intrapuparial developmental data are scarce. Here we analyzed the intrapuparial development of the sarcophagid Peckia pexata and describe the chronological and morphological changes that occur during metamorphosis. We reared approximately 330 specimens originating from the Colombian Amazon in the laboratory. Prepupae were identified by size reduction and coloration change. During the first five days of the study, four individuals were sampled every three hours, then every six hours until adult emergence. Specimens were fixed in 96% alcohol, then immersed in Carnoy solution for 24 hours and in formic acid (5%) for 48 hours for dissection and analysis of morphological changes. Four morphological phases of intrapuparial development of P. pexata were observed: 1) larva pupa apolysis, which lasted, in average, 9 h; 2) cryptocephalic pupa, 6 h; 3) phanerocephalic pupa, 6.5 h; and 4) pharate adult (transparent eyes, 48 h; yellow eyes, 216 h; pink eyes, 35 h; and red eyes, 52 h). The pharate adult was fully formed at 340 h and adult emergence occurred at 372.5 h or 15.5 days. We also described the formation of the prepupa, the pupariation and the final metamorphosis for the imago and compared it with the known events for Peckia intermutans, Peckia lambens (Sarcophagidae) and Lucilia eximia (Calliphoridae).

Forensic entomology is the field of research that applies the biology of insects to criminal investigations. This study, which used rabbits as experimental models, was carried out in Al-Riaydh, Saudi Arabia, to gather data on forensically important insects and their succession and decomposition on decaying buried carcasses at two depths during two periods: the first period lasted from January 28-May 30, 2021 (winter-spring), and the second period was from September 5, 2021, to January 4, 2022 (summer-onset winter). Decomposition was seen in three stages during the first period (bloated, decay, and drying) and two stages during the second period (decay and drying). Temperature, humidity, and depth of burial influenced the stages of carcass decomposition and abundance of insects. Eleven species from the Diptera order, encompassing five families (Calliphoridae, Sarcophagidae, Muscidae, Phoridae, and Ulidiidae), were identified. Additionally, four species from the Coleoptera order were recorded, representing four families (Dermestidae, Tenebrionidae, Histeridae, and Nitidulidae). During the first period of the study, all species were identified at a depth of 20 cm (16 species), while, at a depth of 40 cm, nine types of them were detected. In the second period, only one species of beetle was observed at 20 cm, whereas there were only four fly species present. Furthermore, at a depth of 40 cm, only one species of fly was discovered throughout the entire period. The identified insect species can function as reliable forensic indicators, aiding in PMI estimation and enhancing the overall precision of forensic investigations. These results indicate that these insects are significant in carcass decay and therefore forensically important. [ABSTRACT FROM AUTHOR]

Accurate insect identification is critical to the estimation of time of colonization (TOC) and post-mortem interval (PMI) in medicolegal death investigations. DNA testing is advantageous because it enables the identification of immature specimens that may not be identified based on morphology alone. We describe here a simplified DNA barcoding method for identifying relevant species that may be implemented by forensic genetics laboratories. A cytochrome oxidase (COI) fragment is analyzed after PCR amplification with a single primer set. The method is effective for many species commonly encountered in death investigations in the USA: members of blowfly genera Calliphora, Chrysomya, Cochliomyia, Lucilia, and Phormia; members of the flesh fly genera Blaesoxipha, Oxysarcodexia, Ravinia, and Sarcophaga; and the scuttle fly Megaselia scalaris. We tested the method on specimens with verified identifications and used it to build a collection of reference sequences from specimens collected in Harris County, Texas. We show here the correct identification of larvae, pupae, and pupal exuviae from the medicolegal casework. [ABSTRACT FROM AUTHOR]

Identification of many forensically important insects has been achieved using morphological keys with only a few relying on molecular techniques. Yet, information regarding the molecular identification of Dipteran flies from sub-Saharan Africa is scarce. Therefore, this study used the COI gene to analyze fly specimens collected from decomposing pig (Sus scrofa) carcasses in Iwo South-Western Nigeria. Several batches of eggs from carcasses were reared to adults and preserved in 75% ethanol for subsequent identification using molecular techniques in triplicate. A total of 450 bp sequences was obtained from BLAST analysis of the samples from the population, which has led to the identification of four families consisting of five species with the following breakdown: Sarcophaga africa (95.4%), Chrysomya putoria (99.1%), Chrysomya inclinata (98.3%), Tricogena rubricosa (89.6%), and Chirosia flavipennis (88.8%). The phylogenetic analysis identified Chrysomya chloropyga (AY139694) as the same as C. putoria and S. africa as the same as Sarcophaga cruentata, suggesting them as sister species. This study concluded that T. rubricosa and C. flavipennis are implicated in carrion decomposition, which provides crucial insights for forensic investigations. [ABSTRACT FROM AUTHOR]

Phormia regina (Meigen, 1826) (Diptera: Calliphoridae) can colonize carcasses quickly, and its immature stages are reliable entomological evidence for the estimation of the minimum postmortem interval (PMImin). There are discrepancies in the developmental data from previous studies on P. regina, and the related PMImin indicators need to be refined. We investigated the accuracy of forensic entomological evidence using development durations, growth accumulated degree hours, and larval body length variations of P. regina at seven constant temperatures ranging from 16 to 34 °C. We also established development models such as the isomorphen diagram, thermal summation model, isomegalen diagram, and body length simulation equation to assist with PMImin estimation. The developmental duration of P. regina from egg to adult at 16, 19, 22, 25, 28, 31, and 34 °C was 840.8 ± 42.8 h, 580.1 ± 10.1 h, 390.4 ± 8.7 h, 316.8 ± 9.4 h, 291.4 ± 21.2 h, 238.4 ± 2.8 h, and 222.5 ± 5.2 h, respectively. The lower threshold temperature TL was 9.97 ± 0.50 °C, while the thermal constant K was 5052.7 ± 229 degree days. The lower developmental thresholds, intrinsic optimum temperature, and upper lethal developmental threshold obtained by the Optim SSI models were 13.15, 21.20, and 36.86 °C, respectively. This study aims to provide developmental models for P. regina aimed at common case-site temperatures in the northern provinces of China, which can be used for accurate PMImin estimation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the field of forensic entomology, Chrysomya rufifacies (Macquart 1843) (Diptera: Calliphoridae) has proven to be an important species for postmortem interval (PMI) assessment in criminal investigations. The developmental patterns of C. rufifacies exhibit temperature-dependent variations that are geographically specific, and these variations are influenced by seasonal changes. Hence, understanding the geographically specific development patterns of C. rufifacies in response to temperature and seasonal changes is crucial for improving the accuracy of PMI predictions. In the present study, we examined the developmental rates of C. rufifacies across a temperature gradient (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C). Primary objective of present study was to gather precise developmental data essential for calculating the post-mortem interval (PMI) within varying temperature ranges. Furthermore, we investigated the influence of seasonal variations, encompassing the summer, rainy and winter seasons, on the growth and developmental patterns of C. rufifacies. The results of present studies show that temperature significantly affects the growth and development of C. rufifacies. The length of developmental stages and morphological parameters varied seasonally, with longer duration and colder temperatures in winter and shorter duration and warmer temperatures in summer. The finding that faster development was directly correlated with higher temperatures highlighted the significance of temperature in affecting an insect's life cycle. Our research highlights the necessity of gathering information on evolution trends that are specific to a given area in order to accurately calculate PMI in forensic entomological investigations. Understanding the interactions between temperature, seasonal variation, and morphological traits is critical for establishing baseline data for PMI estimates. The accuracy of PMI estimates will ultimately increase thanks to this information, and forensic entomologists will have even more opportunities to contribute to forensic investigations. [ABSTRACT FROM AUTHOR]

The decomposition of a body, and the associated gaseous and liquid discharges emanating from it, attract gravid female blow flies which lay their eggs in or on the body. After the eggs have hatched, the emerging larvae start feeding on the body. As decomposition progresses, the blow fly larvae often migrate away, typically in a random manner in search of favourable conditions for pupation. In this paper, we report on a rarely described phenomenon of unidirectional mass migration of blow fly larvae and postulate on the factors which may drive this process. A decomposition trial utilising a 60-kg pig carcass, deployed in the summer months in Table Mountain National Park, Cape Town, South Africa, was conducted in 2022. On the fifth day of the trial, simultaneous unidirectional mass dispersal of blow fly larvae was observed. The larvae moved downhill in a southeasterly direction, following the flow of decomposition fluids oozing out from the pig carcass. The ‘larval migration stream’ had a length of approximately 1.5 m with a width of 40 cm, tapering to 17 cm at the terminal point. The larval migration stream consisted of the larvae of Chrysomya albiceps and Chrysomya chloropyga. This study demonstrates the importance of understanding the timing and pattern of dispersal of post-feeding blow fly larvae in each geographical region. This is crucial as the minimum post-mortem interval can be miscalculated if older immature insects dispersing from the corpse are not considered and collected during crime scene investigations. Significance: • Understanding the timing and pattern of dispersal of blow fly larvae from decomposing vertebrate remains in each geographical region is important in time-since-death estimations when using entomological evidence. • In the summer season of the Western Cape Province, Chrysomya albiceps and Chrysomya chloropyga are among the blow fly species of forensic importance as they deposit their eggs on vertebrate remains. • When a decomposing body is found within the Table Mountain region, we suggest that crime scene investigators should examine the downhill area of the scene for larval/pupal entomological evidence that can assist in time and season of death estimations. Supplementary video 1: https://doi.org/10.6084/m9.figshare.26028766.v1 Supplementary video 2: https://doi.org/10.6084/m9.figshare.26028805.v2

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.