Prediction of minimum postmortem submersion interval (PMSImin) based on eukaryotic community succession on skeletal remains recovered from a lentic environment.

Although recent studies explored using microbial succession during decomposition to estimate the postmortem interval (PMI) and postmortem submersion interval (PMSI), there is currently no published research using aquatic eukaryotic community succession to estimate the minimum postmortem submersion interval (PMSImin). The goals of this study were to determine whether eukaryotic community succession occurs on porcine skeletal remains in a lentic environment, and, if so, to develop a statistical model for PMSImin prediction. Fresh porcine bones (rib N = 100, scapula N = 100) were placed in cages (10'' x 10'') attached to floatation devices and submerged in a fresh water lake (Crozet, VA), using waterproof loggers and a YSI Sonde to record temperature and water quality variables, respectively. In addition to baseline samples, one cage, containing five ribs and five scapulae, and water samples (500 mL) were collected approximately every 250 accumulated degree days (ADD). Nineteen sample cohorts were collected over a period of 5200 ADD (579 Days). Variable region nine (V9) of the 18S ribosomal DNA (rDNA) was amplified and sequenced using a dual-index strategy on the MiSeq FGx sequencing platform. Resulting sequences underwent quality control parameters and analysis in mothur v 1.42.3, R v 3.5.3, and R v 3.6.0. Permutational multivariate analysis of variance (PERMANOVA) revealed a significant difference in phylogenetic ß-diversity among ribs, scapulae and water (p = 0.001) and among ADD (p ≤ 0.011), which was supported by distinct clustering of samples associated with each ADD in UniFrac distance based non-metric multidimensional scaling (NMDS) ordinations. Using similarity percentage (SIMPER) analysis of class and family level taxa, differences observed between bone types were attributed to Peronosporomycetes_cl, Eukaryota_unclassified, and Intramacronucleata (e.g., Armophorida), however these differences were not statistically significant. Alpha diversity revealed a non-linear increase in phylogenetic diversity with an increase in ADD. Random forest models for ribs and scapulae predicted PMSImin with an error rate within±104 days (937 ADD) and±63 days (564 ADD), respectively. In conclusion, this study suggests that eukaryotic succession is capable of predicting long term PMSImin in lentic systems.

Postmortem submersion interval (PMSI) estimation from the microbiome of sus scrofa bone in a freshwater lake.

While many studies have developed microbial succession-based models for the prediction of postmortem interval (PMI) in terrestrial systems, similar well-replicated long-term decomposition studies are lacking for aquatic systems. Therefore, this study sought to identify temporal changes in bacterial community structure associated with porcine skeletal remains (n = 198) for an extended period in a fresh water lake. Every ca. 250 ADD, one cage, containing 5 ribs and 5 scapulae, was removed from the lake for a total of nineteen collections. Water was also sampled at each interval. Variable region 4 (V4) of 16S rDNA was amplified and sequenced for all collected samples using Illumina MiSeq FGx Sequencing platform; resulting data were analyzed with the mothur (v1.39.5) and R (v3.6.0). Bacterial communities associated with ribs differed significantly from those associated with scapulae. This difference was mainly attributed to Clostridia, Holophagae, and Spirochaete relative abundances. For each bone type, α-diversity increased with ADD; similarly, ß-diversity bacterial community structure changed significantly with ADD and were explained using environmental parameters and inferred functional pathways. Models developed using 24 rib and 34 scapula family-level taxa allowed the prediction of PMSI with root mean square error of 522.97 ADD (~57 days) and 333.8 ADD (~37 days), respectively.

Development of a quantitative PCR-based method for studying temporal DNA degradation in waterlogged bone.

While several studies have examined temporal DNA degradation in bones collected from terrestrial environments, studies on temporal DNA degradation in bones collected from aquatic environments are limited and mostly based on case studies. The objective of this study was to assess the impact of long-term submersion, aquatic environment, bone type and DNA extraction method on DNA quality and quantity. Bone samples (scapulae and ribs), collected every ~1000 ADD from a freshwater lake and river, underwent DNA extraction via ChargeSwitch® gDNA Plant Kit and organic phenol-chloroform methods, and DNA quantitation using both TaqMan and SYBR Green-based quantitative PCR (qPCR) methods. Results suggest that in both bone types, quality of recovered DNA (i.e., degradation index) declined significantly with increase in submersion time. Among two bone types, quality of recovered DNA from scapulae declined faster than rib samples. There was no significant difference in recovered DNA quantity between bone types, DNA extraction methods, or locations but various interactions between these variables showed significant difference. Overall, it can be concluded that DNA can be extracted from waterlogged bone in sufficient quantity to generate an STR profile up to 4000 ADD.

Postmortem submersion interval (PMSI) estimation from the microbiome of Sus scrofa bone in a freshwater river.

Due to inherent differences between terrestrial and aquatic systems, methods for estimating the postmortem interval (PMI) are not directly applicable to remains recovered from water. Recent studies have explored the use of microbial succession for estimating the postmortem submersion interval (PMSI); however, a non-disturbed, highly replicated and long-term aquatic decomposition study in a freshwater river has not been performed. In this study, porcine skeletal remains (N = 200) were submerged in a freshwater river from November 2017-2018 (6322 accumulated degree days (ADD)/353 days) to identify changes and successional patterns in bacterial communities. One cage (e.g., 5 ribs and 5 scapulae) was collected approximately every 250 ADD for twenty-four collections; baseline samples never exposed to water acted as controls. Variable region 4 (V4) of 16S rDNA, was amplified and sequenced via the Illumina MiSeq FGx sequencing platform. Resulting sequences were analyzed using mothur (v1.39.5) and R (v3.6.0). The abundances of bacterial communities differed significantly between sample types. These differences in relative abundance were attributed to Clostridia, Holophagae and Gammaproteobacteria. Phylogenetic diversity increased with ADD for each bone type; comparably, ß-diversity bacterial community structure ordinated chronologically, which was explained with environmental parameters and inferred functional pathways. Models fit using rib samples provided a tighter prediction interval than scapulae, with a prediction of PMSI with root mean square error of within 472.31 (∼27 days) and 498.47 (∼29 days), respectively.

Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study.

When deaths occur in water, soft tissue decomposes after a temperature-dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol-chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.