The Study of Forensically Important Insects Recovered from Human Corpses in Taiwan.

A study of entomological specimens recovered from 117 human corpses in 114 forensic cases was conducted in Taiwan between 2011 and 2018. The comparisons and discussions of the entomological data were based on the locations (indoor vs. outdoor), environments (urban vs. suburban), season and decomposition stages of corpses. In the study, both morphology and DNA-based comparison methods were used to facilitate species identification. In total, nine families and twenty-two species were thus identified. The two most abundant fly species recovered from human corpses were Chrysomya megacephala (35.1%, 1735 out of 4949) and Chrysomya rufifacies (21.7%, 1072 out of 4949). As for case frequency, both the two were also the most common fly species (both 40%, 46 out of 114), particularly in outdoor cases (also both 74%, 25 out of 34). We found that Chrysomya pinguis and Lucilia porphyrina appeared in low temperature scenes in this study. Synthesiomyia nudiseta was the most common species detected on indoor (36%, 29 out of 80 cases) and urban (41%, 22 out of 54 cases) corpses. Sarcophagidae were strongly associated with urban environments (35%, 19 out of 54 cases), and Parasarcophaga (Liosarcophaga) dux, Liopygia ruficornis and Boettcherisca peregrina were the most frequent sarcophagid species collected from corpses. Hydrotaea spinigera was often found on corpses immersed in water (60%, three out of five cases) with advanced decay or remains stages. Megaselia scalaris was closely correlated with indoor cases (24%, 19 out of 80). In addition, Piophila megastigmata was collected from a corpse in the remains stage and the data represent the first report in Taiwan.

Application of forensic entomology to postmortem interval determination of a burned human corpse: a homicide case report from southern Taiwan.

Determining the postmortem interval (PMI) is strongly impacted by several variables, which consequently results in inaccuracy in the estimation of PMI used in court trials. A PMI experiment was conducted in Kaohsiung County by disposing a burned pig corpse in the woods. One month later, unexpectedly and interestingly, a homicide case, very similar to this mock study, occurred at a distance of 6 km away from the experimental site. The female victim had been killed and burned. The maggots collected from the victim were identified to be Chrysomya megacephala by morphologic observation and were then confirmed by mitochondrial DNA sequence. A PMI of 50 hours was concluded for the burned human body, based on the information of the maggots from the pig corpse. The murderer was eventually arrested and confessed to the crime. According to his statement, the elapsed time since death was calculated to have been 46 hours. In this case, the PMI was estimated successfully and it was almost precise. It would appear that the more similar the surrounding environment between the mock study and the actual case, the more precise can be the PMI estimation.

Mitochondrial DNA sequence alterations observed between blood and buccal cells within the same individuals having betel quid (BQ)-chewing habit.

There are hundreds of millions of betel quid (BQ) lovers widely spreading around the world. Compositions in BQ may generate reactive oxygen species, which would induce DNA damage. However, oral epithelial cells as well as blood have often been used as reference samples in comparison with the mitochondrial DNA (mtDNA) sequence of hairs. The main purpose of this study was to investigate the extent of mtDNA sequence variation in regular BQ-chewers' oral epithelial cells, and thus to evaluate the forensic availability of the buccal cells from BQ-chewers using the mtDNA markers. The hypervariable segments I and II in the D-loop control region of mtDNA between paired samples of blood and buccal scrape cells from 75 non-BQ-chewers (to be a control group), 60 BQ-chewers, and 67 oral cancerous patients were DNA sequenced and compared. Among the three groups, the alteration rates of 1.3% (1 out of 75), 10% (6 out of 60), and 61% (41 out of 67) were identified from the control, BQ-chewers, and the cancerous group, respectively. In the cancerous group, as expected, high rate of DNA alteration between blood and buccal samples was found. In the BQ-chewers, one and five individuals had the length and point alterations, respectively. Interestingly, most of point alteration sites, e.g., mtDNA positions 153, 16189, 16093 identified from BQ-chewers, were also observed in previous literatures. As for the control subjects, one case with point alteration, and none with length alteration, was identified. For all the three groups, not only the oral cells but also the normal blood samples exhibited high frequency (>55%) of length heteroplasmy at poly-(C)n track. Statistical analyses revealed that significance was observed between the severity of mtDNA alteration in BQ-chewers' oral epithelial cells and the history of BQ-chewing (p = 0.02), with a tendency of positive association. Based on the guidelines by Carracedo et al., we suggest that the interpretation of mtDNA variations between criminal evidences and the oral epithelial cells (as a reference or known sample) from BQ-chewers should be performed with particular caution using the PCR-based mtDNA sequencing. Our findings would be valuable in mtDNA analysis of hair evidence, especially for those countries where the habit of BQ-chewing is popular.

Evaluation of the DNA stability of forensic markers used in betel-quid chewers' oral swab samples and oral cancerous specimens: implications for forensic application.

Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. BQ-chewing is also a risk factor for oral diseases and/or cancers. Archived medical oral-specimens can be used to identify specific individuals under adverse conditions, although STR markers are known to be unstable in various tumor tissues. This study evaluates the DNA stability of forensic marker systems in BQ-chewers' oral epithelial cells, and in archived clinical specimens of oral cancer patients. The genotypes of oral and paired peripheral blood samples in 200 subjects were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTR markers (including 9 STR loci and the Amelogenin gene). The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed withboth DQA1/PM and STR marker systems. In the 100 oral cancer patients, one discordant result at D7S8 was found in the 600DQA1/PM-marker loci, and 25 allelic alterations with expansion or contraction were detected in the 900 STR loci. The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers' oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.

Allelic alterations at the STR markers in the buccal tissue cells of oral cancer patients and the oral epithelial cells of healthy betel quid-chewers: an evaluation of forensic applicability.

Although cancerous specimens are usually not used in forensic DNA typing, they might be forcibly employed under certain instances. On the other hand, though the oral epithelial samples have been applied to forensic identification, the great popularity of betel quid (BQ)-chewing in Taiwan, which is known to be a risk factor leading to an oral cancer, makes this application questionable. The DNA stability of nine short tandem repeat (STR) markers (the AmpFlSTR kit) was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy BQ-chewers and the archived clinical specimens from oral cancer patients. The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, as well as their paired peripheral blood samples, and a group of 100 non-BQ-chewers were used for the control. In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalance, and allelic alterations including the expansion, the contraction and the un-classified type (i.e. can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (five patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found from 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, five had the contraction, and three were the un-classified type. Further, among these 17 patients, three were found to acquire multiple allelic alterations at multiple loci. In the group of 100 unrelated healthy BQ-chewers, two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, chi(2), and Scheffe tests indicated that the means of allelic imbalance at the nine STR loci of the oral cancerous group revealed a significant difference from those in the control group. Our results suggest that oral cancer tissues cannot be used as references for forensic purposes using the PCR-based STR systems, whereas the oral swabs from healthy BQ-chewers can be employed, but should be done with caution.