Age determination of Chrysomya megacephala (Diptera: Calliphoridae) using lifespan patterns, gene expression, and pteridine concentration under constant and variable temperatures.

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), is a blowfly species widely studied in medical, veterinary, and entomological research. Our study examined the impact of constant (15, 20, 25, 30, and 35 °C) and variable (ranging from 21.0 to 25.4 °C, with an average of 23.31 °C) temperatures on the development and larval body length of C. megacephala. Additionally, we analyzed the age of the adult C. megacephala through pteridine content and related metabolic genes analysis. Our findings revealed three distinct growth patterns: isomorphen diagram, isomegalen diagram, and thermal accumulated models. At constant temperatures of 15, 20, 25, 30, and 35 °C, egg-hatching times were 44.5 ± 8.9, 26.7 ± 4.6, 12.6 ± 1.1, 11.0 ± 1.0, and 9.9 ± 1.9 h, respectively, while it was 15.3 ± 5.9 h at variable temperatures. The total development times from oviposition to adult eclosion in C. megacephala required 858.1 ± 69.2, 362.3 ± 5.9, 289.6 ± 17.8, 207.3 ± 9.3, and 184.7 ± 12.1 h at constant temperatures of 15, 20, 25, 30, and 35 °C, respectively. This duration was extended to 282.0 ± 64.1 h under variable temperatures. However, no significant differences were found in hatching times and the total developmental durations between 25 °C and variable temperatures. A developmental threshold temperature (D0) of 9.90 ± 0.77 °C and a thermal summation constant (K) of 4244.0 ± 347.0° hours were ascertained. Pteridine content patterns varied significantly across constant temperatures, but not between 25 °C and variable temperatures. Sex and temperature were identified as the primary factors influencing pteridine levels in the head of C. megacephala. Gene expression associated with pteridine metabolism decreased following adult eclosion, matching with increased pteridine concentration. Further investigations are needed to explore the use of pteridine cofactors for age-grading adult necrophagous flies. These findings provide valuable insights into the lifespan of C. megacephala, thereby offering valuable groundwork for forthcoming investigations and PMImin determination.

Molecular identification and functional analysis of chitinase genes reveal their importance in the metamorphosis of Sarcophaga peregrina (Diptera: Sarcophagidae).

Chitinases play a crucial role in insect metamorphosis by facilitating chitin degradation. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a typical holometabolous insect and an important hygiene pest that causes myiasis in humans and other mammals and acts as a vector for various parasitic agents, including bacteria, viruses, and parasites. Enhancing the understanding of the metamorphosis in this species has significance for vector control. In this study, we identified a total of 12 chitinase genes in S. peregrina using bioinformatic analysis methods. Based on transcriptome data, SpIDGF2 and SpCht10 were selected for further functional investigation. The down-regulation of these genes by RNA interference led to developmental delays, disruptions in molting, and differences in cuticle composition during the pupal stage. These findings underscore the pivotal role of chitinase genes in the metamorphic process and offer valuable insights for effective control strategies.

Effects of heat tolerance on the gut microbiota of Sarcophaga peregrina (Diptera: Sarcophagidae) and impacts on the life history traits.

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.

Analysis of the Influence of Changing and Fixed Temperatures on the Growth and Pteridine Content in the Head of Adults Sarcophaga crassipalpis (Diptera: Sarcophagidae).

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.

Temporal Changes of the Oral and Fecal Microbiota after Mild Traumatic Brain Injury in Rats by 16S rRNA Sequencing.

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.

Genome-wide analysis of long noncoding RNAs and their association in regulating the metamorphosis of the Sarcophaga peregrina (Diptera: Sarcophagidae).

BACKGROUND: The flesh fly, Sarcophaga peregrina (Diptera: Sarcophagidae), is an important hygiene pest, that causes myiasis in humans and other mammals, typically livestock, and as a vector for various parasitic agents, including bacteria, viruses, and parasites. The role of long non-coding RNAs (lncRNAs) in regulating gene expression during metamorphosis of the flesh fly has not been well established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed genome-wide identification and characterization of lncRNAs from the early pupal stage (1-days pupae), mid-term pupal stage (5-days pupae), and late pupal stage (9-days pupae) of S. peregrina by RNA-seq, and a total of 6921 lncRNAs transcripts were identified. RT-qPCR and enrichment analyses revealed the differentially expressed lncRNAs (DE lncRNAs) that might be associated with insect metamorphosis development. Furthermore, functional analysis revealed that the DE lncRNA (SP_lnc5000) could potentially be involved in regulating the metamorphosis of S. peregrina. RNA interference of SP_lnc5000 caused reduced expression of metamorphosis-related genes in 20-hydroxyecdysone (20E) signaling (Br-c, Ftz-F1), cuticle tanning pathway (TH, DOPA), and chitin related pathway (Cht5). Injection of dsSP_lnc5000 in 3rd instar larvae of S. peregrina resulted in deformed pupae, stagnation of pupal-adult metamorphosis, and a decrease in development time of pupal, pupariation rates and eclosion rates. Hematoxylin-eosin staining (H&E), scanning electron microscope (SEM) observation and cuticle hydrocarbons (CHCs) analysis indicated that SP_lnc5000 had crucial roles in the metamorphosis developmental by modulating pupal cuticular development. CONCLUSIONS/SIGNIFICANCE: We established that the lncRNA SP_lnc5000 potentially regulates the metamorphosis of S. peregrina by putatively affecting the structure and composition of the pupal cuticle. This study enhances our understanding of lncRNAs as regulators of metamorphosis in S. peregrina, and provide valuable insights into the identification of potential targets for vector control and the development of effective strategies for controlling the spread of myiasis and parasitic diseases.

Development of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) and Intra-Puparial Age Estimation Utilizing Multiple Methods at Constant and Fluctuating Temperatures.

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.

Pupal Age Estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) at Different Constant Temperatures Utilizing ATR-FTIR Spectroscopy and Cuticular Hydrocarbons.

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.

Multimethod combination for age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) with implications for estimation of the postmortem interval.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMImin). Here, the developmental patterns of S. peregrina were investigated at 5 constant temperatures (15-35 °C). Morphological changes at different developmental stages of the pupa were observed at 4 constant temperatures (15-30 °C) by removing the puparium and staining the pupa with hematoxylin and eosin. Furthermore, differentially expressed genes (DEGs) were analyzed at 25 °C in the intrapuparial period to estimate the age of S. peregrina during the intrapuparial stage. S. peregrina completed development from larviposition to adult eclosion at 15 °C, 20 °C, 25 °C, and 30 °C; the developmental durations were 1090.3 ± 30.6 h, 566.6 ± 21.9 h, 404.6 ± 13.01 h, and 280.3 ± 4.5 h, respectively, while the development could not be completed at 35 °C. The intrapuparial period of S. peregrina was divided into 12 sub-stages on the basis of the overall external morphological changes; 6 sub-stages on the basis of individual morphological structures such as the compound eyes, antennae, thorax, legs, wings, and abdomen; and 10 sub-stages on the basis of internal morphological changes detected using histological analysis. The period of each sub-stage or structure that appeared was determined. Moreover, we found that 6 genes (NDUFS2, CPAMD8, NDUFV2, Hsp27, Hsp23, and TPP) with differential expression can be used for the precise age estimation of S. peregrina during the intrapuparial period. This study provided basic developmental data for the use of S. peregrina in PMImin estimation, and we successfully estimated PMImin in a real forensic case by using a multimethod combination.

Predicting the Weathering Time by the Empty Puparium of Sarcophaga peregrina (Diptera: Sarcophagidae) with the ANN Models.

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.

Temporal Expression Profiles Reveal Potential Targets during Postembryonic Development of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae).

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.

The complete mitochondrial genome of Sarcophaga caerulescens (Diptera: Sarcophagidae).

Sarcophaga caerulescens (Zetterstedt 1838) (Diptera: Sarcophagidae) belongs to Sarcophagidae, which is closely associated with human life in ecological habits and has a clear environmental preference. Sarcophaga caerulescens can be better correlated with migration and postmortem interval (PMI) inference in forensic practice. In this study, we reported the complete mitochondrial genome (mitogenome) of S. caerulescens. The length of this mitogenome was 15,720 bp in total (GenBank accession No. MW551788), containing 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. Its nucleotide composition was A (39.7%), C (14.1%), G (9.4%), and T (36.9%). The phylogenetic relationships indicated that the species of S. caerulescens was closely related to S. similis. This study provides the mitochondrial data of S. caerulescens for further study of mitochondrial genome and enriches our understanding of the phylogenetic relationship of sarcophagid flies.

Characterization and Comparative Analysis of Mitochondrial Genomes Among the Calliphoridae (Insecta: Diptera: Oestroidea) and Phylogenetic Implications.

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.

Analysis of the risk of traumatic brain injury and evaluation neurogranin and myelin basic protein as potential biomarkers of traumatic brain injury in postmortem examination.

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.

A Study of Cuticular Hydrocarbons of All Life Stages in Sarcophaga peregrina (Diptera: Sarcophagidae).

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.

A Preliminary Study of Necrophagous Flies (Insecta: Diptera) in the Lhasa Region (Qinghai-Tibet Plateau), China.

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.

High-throughput sequencing to evaluate the effects of methamphetamine on the succession of the bacterial community to estimate the postmortem interval.

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 2 374 209 high-quality sequences and 2 937 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.

Chromosome-level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies.

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.

Development of Sarcophaga dux (diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of the pupae.

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.

Comparative analysis of mitochondrial genomes among the subfamily Sarcophaginae (Diptera: Sarcophagidae) and phylogenetic implications.

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.