Directed Research Project
Many recent studies were conducted concerning bacterial succession in decomposing animal carrion in terrestrial system. However, not much is known about the microorganisms involved in aquatic decomposition. To the best of our knowledge, there are currently no published studies which estimate minimum postmortem submersion interval PMSImin based on eukaryotic community succession in aquatic system. The main goal of this study was to determine the eukaryotic community succession associated with porcine skeletal remains in a lentic environment, and to derive a statistical model for PMSImin prediction. Henleys Lake in Crozet, VA was the location chosen for this research. Fresh pig bones (rib N = 100, scapula N = 100) were placed in cages (10 x 10 inch2), attached to a floatation device, and submerged together with waterproof loggers, and a Yellow Springs, OH Sonde. Every 250 Accumulated Degree Days (ADD), one cage containing 5 rib and 5 scapula samples was collected over a total research period of 5,200 ADDs (559 Days). Water samples were also collected every 250 ADD and filtered using a cellulose membrane filtration system. DNA extraction was performed using the Invitrogen ChargeSwitch® gDNA Plant Kit Protocol. Variable region nine (V9) of the 18S rDNA was amplified and sequenced using a dual-index strategy on the MiSeq FGX sequencing platform. Sequenced data were quality controlled and analyzed via the MiSeq SOP in Mothur version 1.42.3 and in R v3.6.0. Hierarchical classification of good quality sequences was performed using SILVA119 reference database. A phylogenetic approach was utilized for α-and β-diversity estimation. For relative abundance and diversity estimations, sequences were subsampled at a threshold of 5048 reads. Permutational multivariate analysis of variance (PERMANOVA) revealed a significant difference in eukaryotic community structure among sample types (p = 0.000999) and ADD (p ≤ 0.01099). NMDS ordinations revealed distinct clustering of samples associated with each ADD. Among the top ten most influential PMSI predictor taxa, six were shared between both bone types, of which Arthropoda_unclassified was the most influential taxa. Two random forest models were generated for rib and scapula, which predicted PMSImin with an error rate of ± 101 days (936.89 ADD) and ± 61 days (563.77 ADD), respectively. Scapula was found to be a better predictor than Rib. Future directions include comparison of 18S rDNA data between lentic and lotic bodies of water. There is also potential for this 18S rDNA data to be combined with 16S rDNA data to increase accuracy of PMSImin prediction models. In conclusion, this study suggests that eukaryotic succession is capable of predicting long termPMSI in lentic systems.
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VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
Available for download on Sunday, May 04, 2025