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Drug and toxin-related deaths are common worldwide, making it essential to detect the postmortem concentration of various toxic substances at different stages of decomposition in a corpse. Indeed, determining the postmortem interval (PMI) and cause of death in an advanced stage of decomposed corpses has been a significant challenge in forensic investigations. Notably, the presence of drugs or toxins can have a significant impact on the microbial profile, potentially altering the succession of microbial communities and subsequent production of volatile organic compounds (VOCs), which, in turn, affect insect colonization patterns. This review aims to highlight the importance of investigating the interactions between drugs or toxins, microbial succession, VOC profiles, and insect behavior, which can provide valuable insights into forensic investigations as well as the ecological consequences of toxins occurring in decomposition. Overall, the detection of drugs and other toxins at different stages of decomposition can yield more precise forensic evidence, thereby enhancing the accuracy of PMI estimation and determination of the cause of death in decomposed remains.

Abstract Background Heat tolerance is a distinct abiotic factor affecting the distribution and abundance of insects. Gut microbiota can contribute to host fitness, thereby increasing resistance to abiotic stress conditions. In this study, Sarcophaga peregrina is closely associated with human life in ecological habits and shows remarkable adaptability to daily and seasonal temperature fluctuations. To date, the role of gut microbiota in S. peregrina response to heat stress and its influence on the host phenotypic variability remain poorly studied. Methods We exposed S. peregrina to heat stress at 40 °C for 3 h every day throughout the developmental stages from newly hatched larva to adult, after which gut DNA was extracted from third-instar larvae, early pupal stage, late pupal stage, and newly emerged adults, respectively. Then, 16S rRNA microbial community analyses were performed. Results Firstly, we analyzed whether heat stress could have an impact on the life history traits of S. peregrina and showed that the growth rate of larvae was higher and the developmental time was significantly shorter after heat stress. We then proposed the role of the gut microbiota in the heat tolerance of S. peregrina, which indicated that the bacterial abundance and community structure changed significantly after heat tolerance. In particular, the relative abundance of Wohlfahrtiimonas and Ignatzschineria was higher in the third-instar larval larvae; the former increased and the latter decreased significantly after heat stress. To further explore the effect of disturbing the microbial community on thermotolerant phenotype, newly hatched larvae were fed with amikacin under heat stress, which indicated that the larval length and the whole developmental cycle was significantly shorter. Conclusion This study indicated that Wohlfahrtiimonas and Ignatzschineria should play an important role in the post-feeding stage under heat stress, but further study is still needed. In general, heat tolerance can affect the gut microbial community structure, which in turn affects the fitness of the host. Graphical Abstract

In forensic medical examinations, estimating the postmortem interval (PMI) is an important factor. Methamphetamine (MA) is a synthetic stimulant that is commonly abused, and estimation of the PMI after MA abuse has become one of the main tasks in forensic investigation. Microorganisms play a vital role in carrion decomposition. Analysing the bacterial succession patterns can be used as a forensic tool to estimate the PMI. The present study aimed to analyse bacterial succession changes during the decomposition of MA to estimate the PMI. We analysed bacterial communities in rabbits treated with three different concentrations of MA (0, 22.5, and 90 mg/kg) under the natural conditions of 20 °C and 70% humidity by sequencing 16S rRNA gene amplicons using the Illumina MiSeq system. We obtained 2 774 209 high-quality sequences and 2937 operational taxonomic units (OTUs). The relative abundances of the bacterial communities varied markedly in response to different MA concentrations. Interestingly, in response to the different concentrations of MA, Bacteroidetes became disparate in the rectum in the late PMI. Increased numbers of bacterial taxa were identified in the rectum and buccal cavity samples, except at the highest concentration of MA in the rectum samples when PMI was 0–h, than were present in live rabbits. Meanwhile, the PMI correlated significantly with bacterial succession at different taxonomic levels. Our results suggested that bacterial community succession could be used as a “microbial clock” to estimate the PMI in cases of MA-related death; however, further study is required to gain a deeper understanding of this concept.KeypointsAs a forensic tool, microbiome succession patterns have great potential for PMI estimation.At different taxonomic levels, the variation in the succession of microorganisms’ under three different concentrations of MA during the processes of decomposition is significantly related to PMI for estimating MA abuse.These findings provide a new idea for the estimation of PMI of MA abused by changes in microorganisms.

The intestinal bacteria of insects are crucial to the growth and development of the host. It has been found that various physiological processes of insects, such as immune response, metabolism, reproductive ability, and growth and development, involve the gastrointestinal flora. However, many external factors affect the composition of insects’ intestinal microorganisms, such as the type of dietary substrate. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is of great significance in medicine and forensic science. In this study, we investigated the effects of ciprofloxacin on the growth and gut microbiota of S. peregrina. The results demonstrated that the maximum body length of larvae was not affected by ciprofloxacin, while the growth rate of body length quickened as the concentration of the drug increased. The weight of the pupa and adult was reduced significantly due to the effect of ciprofloxacin. After analyzing the gut microbiota composition of S. peregrina in different drug groups, it was indicated that Ignatzschineria, Providencia, Wohlfahrtiimonas, Proteus, Myroides, and Bacteroides play important roles in the growth of S. peregrina. However, they still need to be further studied. In general, ciprofloxacin can affect the gut microbial community structure, which in turn affects the fitness of the host.

Forensic entomology offers unique advantages for the minimum postmortem interval (PMImin) estimation of decomposed corpses in forensic investigations. Accurate species identification and up-to-date locality information are essential. Hainan Island has a tropical rainforest climate and a vast territory. In this study, the community structure of necrophagous flies on Hainan Island was investigated in detail according to geographical environment. The results showed that the dominant species included C. megacephala, S. peregrina, C. rufifacies, S. misera, H. ligurriens, S. sericea, S. cinerea, S. dux, C. pinguis, and M. domestica. Furthermore, C. rufifacies and C. villeneuvi were found only in the high-altitude areas of Wuzhi Mountain, while S. cinerea was distributed only in coastal areas; the latter is a representative species of Hainan Island and has not been reported before. Furthermore, a GenBank database of forensically important flies was established, whilst a high-resolution melt (HRM) curve analysis was applied to identify the common species of Hainan Island for the first time. This study enriches the database of forensically important flies in tropical rainforest regions.

Flesh flies (Diptera: Sarcophagidae) are regarded as significant in medical and veterinary entomology, and their development models can be utilized as considerable markers to ascertain the minimum postmortem interval (PMImin). In this research, we explored the growth cycle and larval body length of Sarcophaga crassipalpis Macquart 1839 (Diptera: Sarcophagidae) reared under variable temperatures ranging from 15.7 to 31.1 °C, with an average of 24.55 °C and relative humidity ranges from 31.4 to 82.8% and at six fixed temperatures of 15, 20, 25, 30, 32, and then 35 °C. Moreover, pteridine from the head was used to assess adult age grading. Our results allowed us to provide three development models: the isomorphen chart, the isomegalen chart, and the thermal summation models. The time taken for S. crassipalpis to complete its development from larviposition to adult emergence at constant temperatures of 15, 20, 25, 30, 32, and 35 °C was 1256.3 ± 124.2, 698.6 ± 15.1, 481.8 ± 35.7, 366.0 ± 13.5, and 295.8 ± 20.5 h, respectively, except 35 °C, where all pupae were unable to attain adulthood. They lasted 485.8 ± 5.4 h under variable temperatures. The minimum developmental limit (D0) temperature and the thermal summation constant (K) of S. crassipalpis were 9.31 ± 0.55 °C and 7290.0 ± 388.4 degree hours, respectively. The increase in pteridine content exhibited variations across different temperatures. There was quite a considerable distinction in the pteridine contents of male and female S. crassipalpis at 15 °C (p = 0.0075) and 25 °C (p = 0.0213). At 32 °C and variable temperatures, the pteridine content between female and male S. crassipalpis was not statistically divergent. However, temperature and gender remain the main factors influencing the pteridine content in the head of S. crassipalpis. We aim to provide detailed developmental data on S. crassipalpis that can be used as a valuable resource for future research and PMI estimation.

A mild traumatic brain injury (mTBI) can increase the risk of neurodegenerative-related disease, and serious long-term outcomes are often overlooked. In forensic science, the accurate identification of mTBIs can directly affect the application of evidence in practice cases. Recent research has revealed that the oral cavity and fecal microbiota play a fundamental role in deeply interconnecting the gut and brain injury. Therefore, we investigated the relationship between the temporal changes of the oral cavity and fecal bacterial communities with damage identification and post-injury time estimation after mTBI. In this study, we analyzed the oral cavity and fecal bacterial communities in mTBI rats under 12 different post-injury times (sham, 0 h, 2 h, 6 h, 12 h, 24 h, 2 d, 3 d, 5 d, 7 d, 10 d, and 14 d post-injury) using 16S rRNA sequencing technology. The sequence results revealed bacteria belonging to 36 phyla, 82 classes, 211 orders, 360 families, 751 genera, and 1398 species. Compared to the sham group, the relative abundance of the bacterial communities varied markedly in the post-injury groups. Importantly, our data demonstrated that Fusobacteria, Prevotellaceae, Ruminococcaceae, and Lactobacillaceae might be the potential candidates for mTBI identification, and 2 h post-injury was a critical time point to explore the temporal changes of mTBI injury-time estimation. The results also provide new ideas for mTBI treatment in the clinic.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) has the potential to estimate the minimum postmortem interval (PMImin). Development data and intra-puparial age estimation are significant for PMImin estimation. Previous research has focused on constant temperatures, although fluctuating temperatures are a more real scenario at a crime scene. The current study examined the growth patterns of S. peregrina under constant (25.75 °C) and fluctuating temperatures (18–36 °C; 22–30 °C). Furthermore, differentially expressed genes, attenuated total reflectance Fourier-transform infrared spectroscopy, and cuticular hydrocarbons of S. peregrina during the intra-puparial period were used to estimate age. The results indicated that S. peregrina at fluctuating temperatures took longer to develop and had a lower pupariation rate, eclosion rate, and pupal weight than the group at constant temperatures did. Moreover, we found that six DEG expression profiles and ATR-FTIR technology, CHCs detection methods, and chemometrics can potentially estimate the intra-puparial age of S. peregrina at both constant and fluctuating temperatures. The findings of the study support the use of S. peregrina for PMImin estimation and encourage the use of entomological evidence in forensic practice.

The Calliphoridae (blowflies) are significant for forensic science, veterinary management, medical science, and economic issues. However, the phylogenetic relationships within this family are poorly understood and controversial, and the status of the Calliphoridae has been a crucial problem for understanding the evolutionary relationships of the Oestroidea these years. In the present study, seven mitochondrial genomes (mitogenomes), including six calliphorid species and one Polleniidae species, were sequenced and annotated. Then a comparative mitochondrial genomic analysis among the Calliphoridae is presented. Additionally, the phylogenetic relationship of the Calliphoridae within the larger context of the other Oestroidea was reconstructed based on the mitogenomic datasets using maximum likelihood (ML) and Bayesian methods (BI). The results suggest that the gene arrangement, codon usage, and base composition are conserved within the calliphorid species. The phylogenetic analysis based on the mitogenomic dataset recovered the Calliphoridae as monophyletic and inferred the following topology within Oestroidea: (Oestridae (Sarcophagidae (Calliphoridae + (Polleniidae + (Mesembrinellidae + Tachinidae))))). Although the number of exemplar species is limited, further studies are required. Within the Calliphoridae, the Chrysomyinae were recovered as sister taxon to Luciliinae + Calliphorinae. Our analyses indicated that mitogenomic data have the potential for illuminating the phylogenetic relationships in the Oestroidea as well as for the classification of the Calliphoridae.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly that has potential value in estimating the PMImin. The precise pupal age estimation has great implications for PMImin estimation. During larval development, the age determination is straightforward by the morphological changes and variation of length and weight, however, the pupal age estimation is more difficult due to anatomical and morphological changes not being visible. Thus, it is necessary to find new techniques and methods that can be implemented by standard experiments for accurate pupal age estimation. In this study, we first investigated the potential of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and cuticular hydrocarbons (CHCs) for the age estimations of S. peregrina pupae at different constant temperatures (20 °C, 25 °C, and 30 °C). The orthogonal projections latent structure discrimination analysis (OPLS-DA) classification model was used to distinguish the pupae samples of different developmental ages. Then, a multivariate statistical regression model, partial least squares (PLS), was established with the spectroscopic and hydrocarbon data for pupal age estimations. We identified 37 CHCs with a carbon chain length between 11 and 35 in the pupae of S. peregrina. The results of the OPLS-DA model show a significant separation between different developmental ages of pupae (R2X > 0.928, R2Y > 0.899, Q2 > 0.863). The PLS model had a satisfactory prediction with a good fit between the actual and predicted ages of the pupae (R2 > 0.927, RMSECV < 1.268). The results demonstrate that the variation tendencies of spectroscopy and hydrocarbons were time-dependent, and ATR-FTIR and CHCs may be optimal for the age estimations of pupae of forensically important flies with implications for PMImin estimation in forensic practice.

Empty puparium are frequently collected at crime scenes and may provide valuable evidence in cases with a long postmortem interval (PMI). Here, we collected the puparium of Sarcophaga peregrina (Diptera: Sarcophagidae) (Robineau-Desvoidy, 1830) for 120 days at three temperatures (10 °C, 25 °C, and 40 °C) with the aim to estimate the weathering time of empty puparium. The CHC profiles were analyzed by gas chromatography-mass spectrometry (GC-MS). The partial least squares (PLS), support vector regression (SVR), and artificial neural network (ANN) models were used to estimate the weathering time. This identified 49 CHCs with a carbon chain length between 10 and 33 in empty puparium. The three models demonstrate that the variation tendency of hydrocarbon could be used to estimate the weathering time, while the ANN models show the best predictive ability among these three models. This work indicated that puparial hydrocarbon weathering has certain regularity with weathering time and can gain insight into estimating PMI in forensic investigations.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) is a species of medical and forensic importance. In order to investigate the molecular mechanism during postembryonic development and identify specific genes that may serve as potential targets, transcriptome analysis was used to investigate its gene expression dynamics from the larval to pupal stages, based on our previous de novo-assembled genome of S. peregrina. Totals of 2457, 3656, 3764, and 2554 differentially expressed genes were identified. The specific genes encoding the structural constituent of cuticle were significantly differentially expressed, suggesting that degradation and synthesis of cuticle-related proteins might actively occur during metamorphosis. Molting (20-hydroxyecdysone, 20E) and juvenile (JH) hormone pathways were significantly enriched, and gene expression levels changed in a dynamic pattern during the developmental stages. In addition, the genes in the oxidative phosphorylation pathway were significantly expressed at a high level during the larval stage, and down-regulated from the wandering to pupal stages. Weighted gene co-expression correlation network analysis (WGCNA) further demonstrated the potential regulation mechanism of tyrosine metabolism in the process of puparium tanning. Moreover, 10 consistently up-regulated genes were further validated by qRT-PCR. The utility of the models was then examined in a blind study, indicating the ability to predict larval development. The developmental, stage-specific gene profiles suggest novel molecular markers for age prediction of forensically important flies.

Forensic entomology could provide valuable data for the minimum postmortem interval (PMImin) estimation and other relevant information, such as causes and circumstances of death. Some representatives of flesh flies are one of the dominant necrophagous insects during early stages of decomposition, demonstrating unique biological characteristics compared with other necrophagous flies. Moreover, they lead to global health concerns as carriers of various pathogenic micro-organisms, and dominantly result in the traumatic myiasis. Thus, sarcophagid flies are considered important in decomposition processes for PMImin estimation. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. The aim of this paper is to provide a brief review on the species, distribution and biological habit of forensically important sarcophagid flies. In addition, the relation between traumatic myiasis and flesh flies, molecular identification methods and developmental pattern of flesh flies are summarized.

Sarcophaga gracilior Chen, 1975 (Diptera: Sarcophagidae) plays a significant role in epidemiology and medicine. In this study, we first report the complete mitochondrial genome (mitogenome) of S. gracilior. This mitogenome was 15,534 bp in length (GenBank No. MW531675), comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The arrangement of genes was identical to that of ancestral metazoan. Nucleotide composition revealed a strong A + T bias, accounting for 76.7% (A 39.6%, G 9.3%, C 14.0%, T 37.1%). The phylogenetic relationships indicated that the species of S. gracilior emerged as sister to Sarcophaga melanura. This study provides important mitochondrial data for further studying evolutionary relationships and species identification of flesh flies.

Sarcophila mongolica Chao & Zhang, 1988 (Diptera: Sarcophagidae) is considered to be of ecological and medical significance. In this study, we report the mitochondrial genome (mitogenome) of S. mongolica. This mitogenome was composed of 15,936 bp in length (GenBank accession no. MT845211), comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The arrangement of genes was identical to that of ancestral metazoan. Nucleotide composition revealed a strong A + T bias, accounting for 75.40% (A 38.2%, G 9.7%, C 14.9%, and T 37.2%). Phylogenetic analysis indicated that S. mongolica was obviously separated from the other flesh flies. This mitogenome provides important genetic data for further understanding of the evolutionary relationship within Sarcophagid flies.

Sarcophaga tuberosa has certain sanitary and epidemiological significance and takes proteins from decaying matter. In this study, we first sequenced and analyzed the complete mitochondrial genome (mitogenome) of S. tuberosa. The length of mitogenome was 15,173 bp, consisting of A (39.5%), G (9.4%), T (37.0%), and C (14.1%), and the order and orientation of genes were identical with that from the mittogenomes of flesh flies previously reported. Phylogenetic analyses showed that S. tuberosa was clustered separately, but which was closed to the species of Sarcophaga portschinskyi. This study provided better support for further understanding of phylogenetic relationships and enriched the mitogenome database of flesh flies.

Sarcophaga pterygota (Diptera: Sarcophagidae) plays a crucial role in medical and veterinary management. The complete mitochondrial genome (mitogenome) of S. pterygota was first sequenced and annotated. The circle DNA is composed of 13 protein-coding genes (13 PCGs), 2 ribosomal RNA (2 rRNAs), 22 transfer RNA (22 tRNAs) and a AT-rich region. It shows that arrangement of the genes is similar with the classical metazoan. The size of mitogenome is 15,236 bp containing A (40.0%), G (9.3%), T (36.6%), and C (14.1%). Moreover, phylogenetic analysis reveals that the branch of S. pterygota is clustered separately. This study enriches the mitogenome database of flesh flies and represents progress for analyzing of phylogenetic relationships.

Sarcophaga caudagalli belonging to the Sarcophagidae is significant for medical and veterinary management. The complete mitochondrial genome (mitogenome) of S. caudagalli was first sequenced and annotated. The circular molecule was 15,029 bp in length, containing 37 genes (13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and an A + T-rich region). Phylogenetic analysis showed that the clade of S. caudagalli was clustered separately.

Necrobia ruficollis is a common beetle found on decaying corpse of forensic studies. The complete mitogenome of N. ruficollis was reported in the present study. The mitogenome was 14,443 bp in length totally, with a base composition of 39.4% for A, 10.3% for G, 37.9% for T, and 12.4% for C. The AT content bias was 77.3%. Twenty-two tRNA genes, 13 protein-coding genes (PCGs), and two rRNA genes were anotated. Most of tRNAs could folded into the typical clover-leaf structure, excepte tRNA-Ser (AGN). The result of phylogenetic analyses exhibited that N. ruficollis belong to the family Cleridae.

Hydrotaea dentipes (Diptera: Muscidae) is one of the forensically important fly species and distributed worldwide. We describe the complete mitochondrial genome of the H. dentipes for the first time. It is a circular molecule of 16,070 bp in size, consisting of A (40.10%), G (9.20%), T (38.50%), and C (12.30%). The order and structure of the genes are similar to other Muscidae species. The mitochondrial genome (mitogenome) is composed of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), two ribosomal RNA genes (rRNA), and a non-coding AT-rich region. Most PCGs used the canonical putative codon to start and stop.

Seniorwhitea reciproca belongs to the Sarcophaga genus, Sarcophagidae family and Diptera order. Here, we report the complete mitochondrial genome of S. reciproca. The circular molecule is 15,973 bp in length, consisting of A (39.80%), G (9.27%), T (36.44%), and C (14.49%), and it is composed of 22 tRNA genes, 13 protein-coding genes (PCGs), two rRNA genes, and a non-coding AT-rich region. 12 PCGs start with ATN, particularly, COI gene starting with TCG, and most PCGs use the typical stop codon TAA. Besides, the phylogenetic analyses upon mitogenomics would provide us further insights on the taxonomy and phylogeny of Sarcophagidae.

Sarcophaga ruficornis (Pape ) plays a significant role in medicolegal application for postmortem interval (PMI) estimation. The mitochondrial genome (mitogenome) has been broadly applied to phylogenetic analyses and species identification as a genetic marker. The complete mitogenome of S. ruficornis was first sequenced and analyzed. This study would strengthen our understanding of its phylogenetic relationship and contribute to taxonomic diagnosis.

Sarcophaga (Liosarcophaga) dux Thomson (Diptera: Sarcophagidae) is a flesh fly species of medical and forensic importance in many parts of the world, both as a fly with the necrophagous habits found on human corpses and a vector of disease or parasitic, myiasis-producing agent. The complete mitochondria gene (mitogenome) of S. dux was first sequenced, annotated and phylogenetic analyses with other 10 species of the sarcophagidae in this study. This work improves the databases of sarcophagidae species, and provides support for further study of mitochondrial genome architecture and phylogenetics.

Graphomya rufitibia (Diptera: Muscidae) was distributed worldwide. In this study, the complete mitogenome of G. rufitibia was sequenced and annotated, and the full-length was a 15,374 bp fragment, consisting of A (40.64%), G (8.96%), T (36.80%), and C (13.59%). The mitogenome is composed of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding AT-rich region. Most PCGs used the canonical putative codon to start and stop, 21 tRNAs are folded into the classic clover-leaf structure, with the one exception of tRNA-Ser (AGN). A total length of 11,182 bp encoding 3727 amino acids, and account for 72.7% of the whole mitogenome. Phylogenetic analyses showed that G. rufitibia belongs to family Mydaeinae.

Necrophagous fly species are commonly used as forensic tools to estimate the minimum postmortem interval (PMImin). Many researchers raised necrophagous flies under constant temperature regimes to collect their developmental data. However, in most forensic cases, the ambient temperature fluctuates. In order to investigate a forensically important fly developmental mode (the Isomegalen diagram, Isomorphen diagram and Thermal summation models) and make comparisons of the developmental rate between constant temperatures and fluctuating temperatures, we used Aldrichina grahami (Diptera: Calliphoridae) to investigate the life history parameters at eight constant temperatures ranging from 8 to 36 °C. We also compared developmental rate of A. grahami in three groups of constant versus fluctuating temperatures: 8 °C vs. 6−12 °C, 12 °C vs. 10−16 °C, and 16 °C vs. 14−20 °C. Our data showed that A. grahami is cold tolerant with a mean (±SE) development threshold temperature (D0) of 3.41 ± 0.48 °C and a thermal summation constant (K) of 8125.2 ± 288.4-degree hours. The three groups subjected to fluctuating temperatures took longer to develop compared to those developing in constant temperatures when simulated in a model. These results not only provide detailed developmental data for the use of A. grahami in the estimation of the PMI, but also indicate that ambient temperature fluctuation must be taken into consideration for the use of all necrophagous fly species.