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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).

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.

Blow flies are among the most important insects in forensic entomology casework. Identification of blow fly species can be a time consuming and difficult task, especially at their early development stages. Present DNA-based technologies provide a promising identification method for these forensically important calliphorids. The cytochrome oxidase subunit I (COI) sequence has been applied as a suitable DNA marker in calliphorid identification for many years; however, limitation exists in using short sequence to determine genetically close species. In this study, COI long sequences were utilized in species-level identification. Seventy-two specimens were collected from 27 locations across 22 Chinese provinces, and unambiguously determined as 16 species under seven genera of Calliphoridae. Analysis of long mitochondrial COI sequence (1,021-1,382 bp) data from forensically relevant blow flies collected in the inland region of China provided a reliable marker for accurate identification. Our data provide genetic diversity and reference for global forensic-related blow fly species identification, and conductive meaning on future utilization of Chinese calliphorids used in forensic entomological practice.

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.

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.

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...