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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.

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.

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 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.

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 &deg, C. We also compared developmental rate of A. grahami in three groups of constant versus fluctuating temperatures: 8 &deg, C vs. 6&ndash, 12 &deg, C, 12 &deg, C vs. 10&ndash, 16 &deg, C, and 16 &deg, C vs. 14&ndash, 20 &deg, C. Our data showed that A. grahami is cold tolerant with a mean (&plusmn, SE) development threshold temperature (D0) of 3.41 &plusmn, 0.48 &deg, C and a thermal summation constant (K) of 8125.2 &plusmn, 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.

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.

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 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.

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.

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...

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.

Calliphora vomitoria is a significant insect which belongs to the Calliphoridae family. In this study, the mitochondrial genome of C. vomitoria was completely sequenced for species identification. The entire mitogenome was 16,134 bp in length, composing of 13 protein-encoding genes, 22 transfer RNA genes and two ribosomal RNA genes, and then the array of the genes was similar to the other insects have discovered. The overall base compositions of A, G, C and T were 39.40%, 9.37%, 13.08% and 37.13% respectively. What is more, phylogenetic analyses tree indicated that entire mitochondria genome sequences of C. vomitoria had high degree of identification among the species listed in. We hope that the results from the present study will provide useful dipteran mitochondrial genomes information for the further studies on genetic structure and phylogenetic analyses of C. vomitoria in the species identifications.