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Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMI

Lhasa is located on the Qinghai–Tibet Plateau, with an altitude of 3,650 m, and a unique geography. Its climate is dry and cold all year round. Forensic entomological studies of the region are scarce. In this study, the diversity and seasonality of necrophagous flies in eight counties among Lhasa region were determined, and succession of necrophagous flies colonizing on rabbits in the Chengguan area of Lhasa was studied, so as to provide reference data for estimating postmortem interval (PMI) and location of death. In total, 22 species of necrophagous flies, belonging to six families were identified in Lhasa. Protophormia terraenovae (Robineau-Desvoidy, 1830) (Diptera: Calliphoridae) was the dominant species throughout the year, the diversity index (4.5834) indicated that the study on necrophagous flies in the Lhasa region is representative.

The reproduction strategies of invertebrates, oviparity, ovoviviparity and viviparity, always reflect the relationship between individuals and their surroundings. There is plasticity in the reproductive strategies of sarcosaphagous flies as they adjust to rapidly changing circumstances. The transition from oviparity to ovoviviparity or viviparity involves numerous changes in physiology, morphology and immunology. Demonstrating these processes can make the application of entomology work in forensic practice more reliable. This essay reviews means of reproduction in sarcosaphagous flies and identifies related features. It is shown that not only the reproduction traits, such as fast location of carrion, and uncommon number of ovaries and oogenesis, but also some morphological features are related to viviparity. In general, viviparous flies have larger adult bodies but smaller eggs and chorions. Moreover, the length of terminalia and the shape of the vagina also varies among those three modes. Reproductive plasticity is a bridge between the three reproductive modes, and it can greatly influence the inference of the post-mortem interval (PMImin).

Brittleness index is an important mechanical index to evaluate the fracturability of conventional oil and gas reservoirs. However, the brittleness index of an organic coal reservoir is more complex. In this study, based on array sonic logging and density logging data, coal brittleness index is calculated using an elastic parameter method in the Mabidong coalbed methane (CBM) block in southern Qinshui Basin. In combination with coal-body structure observation, the maceral analysis, and proximate data of coal cores, a comprehensive study of geological influencing factors of coal brittleness index has been carried out. According to coal fragmentation degree, the coal-body structure of coal seams can be divided into primary, cataclastic, and granulate structure. The average interval of brittleness index of primary, cataclastic, and granulate structural coals is 63.3-71.48, 73.01-74.85, and 77.41-82.77, respectively. The results indicate that generally the order of brittleness index is primary structural coal < cataclastic structural coal < granulate structural coal. Young’s modulus and the brittleness index have a good positive correlation. Poisson’s ratio and the brittleness index are negatively correlated in No. 3 coal seam but are positive correlated in No. 15 coal seam. The vitrinite content is positively correlated with brittleness index, and the inertinite content is negatively correlated. For the primary and cataclastic structure coal, the ash and volatile is positively correlated with the brittleness index. The correlation of brittleness index, macerals, and coal quality parameters in the primary structure coal is better than that of the cataclastic and granulate structure coal. The research results are helpful to guide the coal brittleness index and coal-body structure prediction in fracturing of CBM wells.

The subfamily Sarcophaginae is extremely diverse in morphology, habit and geographical distribution, and usually considered to be of significant ecological, medical, and forensic significance. In the present study, 18 mitochondrial genomes (mitogenomes) of sarcophagid flies were first obtained. The rearrangement and orientation of genes were identical with that of ancestral insects. The degrees of compositional heterogeneity in the datasets were extremely low. Furthermore, 13 protein-coding genes were evolving under purifying selection. The phylogenic relationship of the genus-group taxa Boettcheria + (Sarcophaga + (Peckia + (Ravinia + Oxysarcodexia))) was strongly supported. Four subgenera were recovered as monophyletic (Liopygia, Liosarcophaga, Pierretia, Heteronychia) in addition to Parasarcophaga as polyphyletic. The sister-relationships between S. dux and S. aegyptiaca, S. pingi and S. kawayuensis were recovered, respectively. Moreover, the molecular phylogenetic relationships among the subgenera Helicophagella, Kozlovea, Kramerea, Pandelleisca, Phallocheira, Pseudothyrsocnema, Sinonipponia and Seniorwhitea were rarely put forward prior to this study. This study provides insight into the population genetics, molecular biology, and phylogeny for the subfamily Sarcophaginae, especially for the subgeneric classification of Sarcophaga. However, compared with the enormous species diversity of flesh flies, the available mitogenomes are still limited for recovering the phylogeny of Sarcophaginae.

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In forensic pathology, traumatic brain injury (TBI) is a frequently encountered cause of death. Unfortunately, the statistic autopsy data, risk investigation about injury patterns, and circumstances of TBI are still sparse. Estimates of survival time post-TBI and postmortem diagnosis of TBI are especially important implications in forensic medicine. Neurogranin (Ng) and myelin basic protein (MBP) represent potential biomarkers of TBI. The present study analyzed retrospectively the forensic autopsy records of TBI cases at a university center of medico-legal investigation from 2008 to 2020. Immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to investigate the expression changes of Ng and MBP in the cortical brain injury adjacent tissues and serum, respectively, from cases of TBI at autopsy with different survival times post-TBI. The results show that the major mechanism of death of TBI is assault, and accident was the major manner of death. Ng and MBP are mainly expressed in the cortical nerve cells and the myelin sheath, respectively. The serum levels of Ng and MBP in each TBI group were higher compared with those in the controls. The brain cortical levels of Ng and MBP decreased at first and then steadily increased with extended survival time post-TBI. The immunopositive ratios and serum concentration of Ng and MBP have shown significant differences among control group and all TBI group (p 0.001). Collectively, the immunohistochemical analyses of Ng and MBP in human brain tissues may be useful to determine the survival time after TBI, and Ng and MBP level in the human blood specimens could be considered as a postmortem diagnostic tools of TBI in forensic practice.

Age determination of necrophagous flies serves as an important tool for postmortem interval (PMI) estimation in forensic investigations. Drugs or toxins in cadavers may alter the developmental time of larvae, and lead to deviation in PMI estimation. Methamphetamine (MA), as one of the most abused psychostimulant drugs in Asia and North America, is often involved in forensic entomotoxicological cases. This study investigated the effects of MA (0, 45, 90, and 180 ng/mg) on the developmental rate, morphology, and survival of Aldrichina grahami (Aldrich, 1930). The results showed that 1) the developmental time to reach the pupal instar was statistically slower for the larvae reared on rabbit mince containing MA than for the control; 2) the mean length of the larvae exposed to MA concentrations was longer than those of the control; 3) the mean weight of the pupae exposed to the highest concentration of MA was significantly lighter than those of the control; 4) the GC–MS method can detect the content of MA in A. grahami immatures and empty puparia (EP).

Muscidae, commonly known as house flies and their close relatives, is one of the dipteran insects of recognized medical, veterinary, and ecological importance. Mitochondrial genomes (Mitogenomes) have been widely used for exploring phylogenetic analysis and taxonomic diagnosis due to the difficulty in distinguishing them morphologically. In this study, our complete mitogenomes of muscid flies were sequenced and aligned, which ranged from 15,117 bp (Synthesiomyia nudiseta) to 16,089 bp (Musca sorbens) in length, and contained a typical circular molecule comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a non-coding control region. The order and orientation of genes were identical with that from the ancestral insects. The phylogenetic analysis based on the mitochondrial genes indicated that the subfamily relationships within Muscidae were reconstructed as (Mydaeinae (Muscinae (Reinwardtiinae + Azeliinae))). Similar tree topologies were recovered from both Maximum Likelihood (ML) and Bayesian Inference (BI) analysis. Furthermore, we compared the phylogenetic analyses that were constructed using internal transcribed spacer 2 (ITS2), elongation factor-1α (EF-1α), 13 PCGs and 13 PCGs + ITS2 + EF-1α, respectively. Combined analysis of nuclear gene partitions improved support and resolution for resulting topologies but the positions of branches were obviously inconsistent due to limited species. More mitogenomes should be sequenced representing various taxonomic levels, especially close related species, which will enhance our understanding of phylogenetic relationships among muscids.

Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world, is of medical and forensic importance. Traditional methods of inferring developmental age rely on the life stage of insects and morphological changes. However, once the larvae reach the pupal and adult stage, morphological changes would become barely visible, so that the classic method would be invalid. Here, we studied the cuticular hydrocarbon profile of S. peregrina of the whole life cycle from larval stage to adult stage by GC–MS. Sixty-three compounds with carbon chain length ranging from 8 to 36 were detected, which could be categorized into four classes: n-alkanes, branched alkanes, alkenes, and unknowns. As developmental increased, branched alkanes dominant, and the content of high-molecular-weight hydrocarbons is variable, especially for 2-methyl C19, DiMethyl C21, docosane (C22), and tricosane (C23). This study shows that the composition of CHC could be used to determine the developmental age of S. peregrina and aid in postmortem interval estimations in forensic science.

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.

Sarcophaga brevicornis (Diptera: Sarcophagidae) was of utmost forensic importance due to their wide distribution, ubiquitous, and synanthropic nature. The complete mitochondrial genome (mitogenome) of S. brevicornis was first sequenced and assembled in this study. The length of circular mitogenome was 15,152 bp, which showed in a typical arthropod genome, including 13 protein-coding genes (13 PCGs), two ribosomal RNA (two rRNA) genes, 22 transfer RNA (22 tRNA) genes, and an AT-rich region. Its nucleotide composition was A 39.0%, C 12.7%, G 10.6%, and T 37.7%. Furthermore, phylogenetic relationships of S. brevicornis and the published sarcophagid species were evaluated based on 13 PCGs. The results indicated that S. brevicornis was clearly separated from the other sarcophagid species, but it was closed to the species of Sarcophaga similis. This study provided a significant database reference for genetic structure and phylogenetic analysis of Sarcophagidae.

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.

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 dux (Diptera: Sarcophagidae) is a necrophagous flesh fly species with potential forensic value for estimating minimum postmortem interval (PMImin). The basic developmental data and precise age estimates of the pupae are significant for PMImin estimation in forensic investigations. In the present study, we investigated the development data of that species at seven constant temperatures varying from 16 °C to 34 °C, including body length changes of the larve, developmental duration and accumulated degree hours of the preadults. Several reference genes for relative quantification of the differentially expressed genes (DEGs) were firstly selected and evaluated in the pupae of different ages under different temperatures. The DEGs of the insects during the pupal period at different constant temperatures (34, 25 and 16 °C) were further analyzed for more precise age estimation. The results showed that the developmental durations of the preadults at 16, 19, 22, 25, 28, 31 and 34 °C were 1478.6 ± 18.3 h, 726.1 ± 15.8 h, 538.5 ± 0.9 h, 394.1 ± 9.5 h, 375.6 ± 10.8 h, 284.1 ± 7.3 h, and 252.5 ± 6.1 h, respectively. The developmental threshold temperature the flies was 12.27 ± 0.35 °C, and the thermal summation constant was 5341.71 ± 249.29° hours. The most reliable reference genes during the pupal period at different temperatures were found: GST1 and 18S rRNA for the 34 °C group, GST1 and RPL49 for 25 °C, and 18S rRNA and 28S rRNA for 16 °C. The four differential expression genes (Hsp60, A-alpha, ARP, and RPL8) have the potential to be used for more precise age estimation of pupal S. dux. This work provides important basic developmental data and a more precise age estimation method for pupal S. dux, and improves the value of this species for PMImin estimation in forensic investigations.

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.

BackgroundSarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly with potential forensic value in estimating minimum postmortem interval (PMImin). The basic developmental data and precise intrapuparial age estimates are significant for PMImin estimationof application entomological data in legal medicine.MethodsThe development parameters of S. dux at seven constant temperatures from 16°C to 34°C were investigated by rearing using pig lung in the artificial climate box. The appropriate reference genes and intrapuparial differentially expressed genes (DEGs) of S. dux at constant temperatures 34, 25 and 16°C were selected and analyzed using RT-qPCR for more precisely age estimations. ResultsThe developmental durations of S. dux at 16, 19, 22, 25, 28, 31 and 34°C from larviposition to adult eclosion were 1478.6±18.3, 726.1±15.8, 538.5±0.9, 394.1±9.5, 375.6±10.8, 284.1±7.3, and 252.5±6.1 h, respectively. The thermal summation constant of S. dux was 5341.71±249.29 degree hours, and the developmental threshold temperature was 12.266±0.35°C. The most reliable reference genes under intrapuparial and different temperature in our study were: GST1 and 18S rRNA for 34°C group, GST1 and RPL49 for 25 °C group, and 18S rRNA and 28S rRNA for 16 °C group，the four differential expression genes (Hsp60, A-alpha, ARP, RPL8) can be used to more precisely intrapuparial age estimation of S. dux. ConclusionsThe basic developmental data of S. dux at constant temperatures, such as body length changing of the larva period, accumulated degree hours and duration of development, to establish developmental models that can be used to estimate the PMImin. The selection and evaluation of appropriate reference genes at different experimental conditions are primary with RT-qPCR. The differentially expressed gene can contribute to more accuracy age estimations of S. dux intrapuparial. The result from this study can make contributes to the use of S.dux for estimating PMImin.

Sarcophaga peregrina is considered to be of great ecological, medical and forensic significance, and has unusual biological characteristics such as an ovoviviparous reproductive pattern and adaptation to feed on carrion. The availability of a high-quality genome will help to further reveal the mechanisms underlying these charcateristics. Here we present a de novo-assembled genome at chromosome scale for S. peregrina. The final assembled genome was 560.31 Mb with contig N50 of 3.84 Mb. Hi-C scaffolding reliably anchored six pseudochromosomes, accounting for 97.76% of the assembled genome. Moreover, 45.70% of repeat elements were identified in the genome. A total of 14,476 protein-coding genes were functionally annotated, accounting for 92.14% of all predicted genes. Phylogenetic analysis indicated that S. peregrina and S. bullata diverged ~ 7.14 million years ago. Comparative genomic analysis revealed expanded and positively selected genes related to biological features that aid in clarifying its ovoviviparous reproduction and carrion-feeding adaptations, such as lipid metabolism, olfactory receptor activity, antioxidant enzymes, proteolysis and serine-type endopeptidase activity. Protein-coding genes associated with ovoviparity, such as yolk proteins, transferrin and acid sphingomyelinase, were identified. This study provides a valuable genomic resource for S. peregrina, and sheds insight into further revealing the underlying molecular mechanisms of adaptive evolution.

Sarcophaga peregrina is an important flesh fly species for estimating the minimum postmortem interval (PMImin) in forensic entomology. The accurate determination of the developmental age is a crucial task for using necrophagous sarcophagids to estimate PMImin. During larval development, the age determination is straight forward by the morphological changes and variation of length, weight, and width; however, the age estimation of sarcophagid intrapuparial is more difficult due to anatomical and morphological changes not being visible. The analysis of differentially expressed genes (DEGs) during sarcophagid metamorphosis is a potential method for age estimation of intrapuparial. In the present study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the differential gene expression level of S. peregrina intrapuparial in different constant temperatures (35°C, 25°C, and 15°C). In addition, the appropriate reference genes of S. peregrina were selected in the intrapuparial and at different temperatures to obtain reliable and valid gene expression profiles. The results indicated that two candidate genes (18S rRNA and 28S rRNA) were the most reliable reference genes, and four DEGs (Hsp90, A-alpha, AFP, AFBP) have the potential to be used to more accuracy estimate the age of S. peregrina intrapuparial.

The flesh flies (Diptera: Sarcophagidae) are significant in forensic investigations. The mitochondrial genome (mitogeome) has been widely used as genetic markers for phylogenetic analysis and species identification. To further understand the mitogenome-level features in Sarcophagidae, the complete mitogenome of Sarcophaga formosensis (Kirneret Lopes, 1961) (Diptera: Sarcophagidae) and Sarcophaga misera (Walker, 1849) (Diptera: Sarcophagidae) was firstly sequenced, annotated, and compared with other 13 Sarcophagidae species. The result indicated that the gene arrangement, gene content, base composition, and codon usage were conserved in the ancestral arthropod. Evolutionary rate of the mitogenome fragments revealed that the nonsynonymous and synonymous substitution rates (Ka and Ks) ratio was less than 1.00, indicating these variable sites under strong purifying selection. Almost all transfer RNA genes (tRNAs) have typical clover-leaf structures within these sarcophagid mitogenomes, except tRNA-Ser (AGN) is lack of the dihydrouridine arm. This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the Sarcophagidae. Phylogenetic analyses containing the interspecific distances from different regions in these species provided us new insights into the application of these effective genetic markers for species identification of flesh flies.

Some representatives of flesh flies visiting/colonizing the decomposed remains demonstrated their values in estimating the minimal postmortem interval (PMImin) since death. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. Although mitochondrial genes have been proposed as a potential DNA barcode for the species-level identification of sarcophagids, some defects still remain such as the substantial memory and processing time taken for homologous comparisons online. Moreover, species identification is mainly achieved by Sanger sequencing based on PCR with genus-specific primers. In the present study we characterized 24 single nucleotide polymorphisms (SNPs) as robust markers of genetic variation for identifying different sarcophagids based on available cytochrome c oxidase I (COI) data and verified them through pyrosequencing (PSQ) technology to establish a SNP-based genotyping system. The system provides a preliminary foundation for developing a rapid, reliable, and high-throughput assay so as to efficiently and accurately identify the sarcophgid flies. Furthermore, the PSQ approach is proved to be faster, more cost-effective as well as more sensitive and specific than custom Sanger sequencing.

Muscid Flies (Diptera: Muscidae) are of great forensic importance due to their wide distribution, ubiquitous and synanthropic nature. They are frequently neglected as they tend to arrive at the corpses later than the flesh flies and blow flies. Moreover, the lack of species-level identification also hinders investigation of medicolegal purposes. To overcome the difficulty of morphological identification, molecular method has gained relevance. Cytochrome c oxidase subunit I (COI) gene has been widely utilized. Nonetheless, to achieve correct identification of an unknown sample, it is important to survey certain muscid taxa from its geographic distribution range. Accordingly, the aim of this study is to contribute more geographically specific. We sequenced the COI gene of 51 muscid specimens of 12 species, and added all correct sequences available in GenBank to yield a total data set of 125 COI sequences from 33 muscid species to evaluate the COI gene as a molecular diagnostic tool. The interspecific distances were extremely high (4.7-19.8%) in either the standard barcoding fragment (658 bp) or the long COI sequence (1,019-1,535 bp), demonstrating that these two genetic markers were nearly identical in the species identification. However, the intraspecific distances of the long COI sequences were significantly higher than the barcoding region for the conspecific species that geographical locations vary greatly. Therefore, genetic diversity presented in this study provides a reference for species identification of muscid flies. Nevertheless, further investigation and data from more muscid species are required to enhance the efficacy of species-level identification using COI gene as a genetic marker.

Chrysomya nigripes (Diptera: Calliphoridae) is a blow fly species of forensic importance. Here we demonstrated the complete mitochondrial genome of this species for the first time. Phylogenetic analyses indicated that entire mitochondrial genome sequences can provide more useful information for distinguishing C. nigripes from the other species.

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.

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 tim...

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 leng...

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...