Geographical Distribution and Multimethod Species Identification of Forensically Important Necrophagous Flies on Hainan Island.

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.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

OBJECTIVES: To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. METHODS: The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. RESULTS: When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). CONCLUSIONS: The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

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.

Characterized Gene Repertoires and Functional Gene Reference for Forensic Entomology: Genomic and Developmental Transcriptomic Analysis of Aldrichina grahami (Diptera: Calliphoridae).

Many flies of Diptera are common entomological evidence employed in forensic investigation. Exploring the existence of inter- and intra-species genomic differences of forensically relevant insects is of great importance. Aldrichina grahami is a common blow fly species of forensic importance. The present study characterized the gene repertoires of A. grahami, and provides insights into issues related to forensic entomology, such as necrophagous behavior, gene family features, and developmental patterns. Gene families were clustered and classified according to their function in different aspects of the necrophagous lifestyle, generating several gene repertoires. The genes under positive selection pressure and evolutionary changes were screen and identified. Moreover, genes that exhibited potential prediction value in the post mortem interval (PMI) estimation and development of immature stages were subjected to analysis based on the developmental transcriptome. Related insect species were compared at the genomic level to reveal the genes associated with necrophagous behaviors. The expression of selected genes in separated repositories was verified using qPCR. This work was conducted using a high-quality chromosome-level genome assembly of A. grahami and its developmental transcriptome. The findings will facilitate future research on A. grahami and the other forensically important species.

The complete mitochondrial genome of Sarcophila mongolica (Diptera: Sarcophagidae).

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.

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.

The evaluation of forensic characteristics and the phylogenetic analysis of the Ong Be language-speaking population based on Y-STR.

The Ong Be language, an important branch of the Tai-Kadai language family, is one of the most distinctive languages on Hainan Island. Ong Be language speakers, who have lived in the Lingao district of Hainan Island for generations, were classified as Han Chinese in the early days of the establishment of the People's Republic of China but have distinct differences from the Han Chinese in language, lifestyle, customs and values and particularly in appearances and features. Currently, Y-chromosomal short tandem repeat (STR) haplotypes have been widely used in genetic applications, such as human forensics, historical investigations and genealogical research. In this study, 487 unrelated male individuals residing in the Lingao district volunteered, and their Y-STR haplotypes were investigated using the Yfiler and Yfiler Plus with 17 and 27 Y-STR loci, respectively. Furthermore, we combined our population data on the Lingao people with existing datasets from Asian nations (East, South and Southeast Asia) to explore the genetic variance and relationships with Han Chinese from different administrative regions in Northern and Southern China and Chinese ethnic minorities officially recognized by the PRC. Population comparisons demonstrated that the Lingao people had distant relationships with Asian nations at the national level and had relatively close genetic and linguistic relationships with Hainan Li and Guizhou Gelao, both of whom belong to the Tai-Kadai language family. The present results increase our understanding of the genetic relationships between the Lingao people and other groups, and further research in genetics and other areas is still needed to trace the origin of the Lingao people.

Critical role of insulin­like growth factor binding protein­5 in methamphetamine­induced apoptosis in cardiomyocytes.

Methamphetamine (MA) is a highly abused amphetamine­like psychostimulant. At present, the mechanisms underlying MA­induced cardiotoxicity are poorly understood. The cardiotoxic effects have yet not been clearly elucidated with respect to the apoptotic pathway. Insulin­like growth factor binding protein­5 (IGFBP5) is important for cell growth control and the induction of apoptosis. The aim of the present study was to analyze whether IGFBP5 is involved in MA­induced apoptosis as a novel target. MA­induced apoptosis was observed in neonatal rat ventricular myocytes (NRVMs) in a concentration­dependent manner using a terminal deoxyribonucleotide transferase­mediated dUTP nick end­labeling assay. Using reverse transcription polymerase chain reaction and western blotting, MA was demonstrated to induce concentration­dependent increases in the expression of IGFBP5. Silencing IGFBP5 with small interfering RNA significantly reduced apoptosis and suppressed the expression of caspase­3 in NRVMs following treatment with MA. To the best of our knowledge, the present study provided the first evidence suggesting that IGFBP5 is a potential therapeutic target in MA­induced apoptosis in vitro, providing a foundation for future in vivo studies.

Remodeling of ion channel expression may contribute to electrophysiological consequences caused by methamphetamine in vitro and in vivo.

Methamphetamine (MA) is a psychostimulant. MA may induce numerous cardiotoxic effects, leading to cardiac arrhythmias, heart failure, eventually leading to sudden cardiac death. The deleterious effects of methamphetamine work in tandem to disrupt the coordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. Remodeling of ion channels is an important mechanism of arrhythmia. Although arrhythmogenic remodeling involves alterations in ion channel expression, it is yet unknown whether MA induced electrical remodeling by affecting gene expression, and whether the changes in protein expression are paralleled by alterations in mRNA expression. Our study focused on the expression of ion channels which were correlated to the electrical remodeling caused by MA. We used RT-PCR and western blot to assess of the transcript and translate levels of ion channel subunits, including Ito: kv1.4, kv1.7, kv3.4, kv4.2; IK1: kir2.1, kir2.2, kir2.3, kir2.4; and ICa-l: Ca(2+)α1, Ca(2+)ß, respectively. The reversible effect of these changes after MA withdrawal was also evaluated. MA caused decrease in mRNA and protein levels in all ion channel subunits in vitro and also in vivo, is at this work. The kv3.4 and all 4 subunits of Kir2.0 family showed significant decrease than the other genes. Most of the channel subunit expression started to reverse after MA withdrawal for 4 weeks and significantly reverse in all of the channel subunits after MA withdrawal for 8 weeks. We found that CACNA1C and Kir2.0 family showed lower recoverability than the others after MA withdrawal for 8 weeks. The reduction of the ion channel expression levels may be the molecular mechanism that mediates the electrical remodeling caused by methamphetamine.