Author ORCID Identifier

https://orcid.org/0000-0002-6476-8014

Defense Date

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Integrative Life Sciences

First Advisor

Tal Simmons, PhD

Second Advisor

Baneshwar Singh, PhD

Third Advisor

Jenise Swall, PhD

Fourth Advisor

Rima Franklin, PhD

Fifth Advisor

Jamie Fredericks, PhD

Abstract

Following water-related deaths, soft tissue on victims’ remains may be entirely absent after a temperature dependent period of time. Due to their physical and chemical components, which protect them from environmental deterioration and biological attack, bones preserve DNA for longer periods of time. Therefore, the ability to both identify an individual and estimate the postmortem submersion interval (PMSI) may depend on DNA obtained from skeletal remains. The goals of the dissertation were to examine differences in both the degradation of skeletal DNA and the microbiome inhabiting bone in freshwater aquatic environments (e.g., Henley Lake and the James River). At each location, five porcine ribs and scapulae were placed in 10’’ x 10’’ cages and submerged. Every ca. 250 accumulated degree days (ADD), one cage was collected for a total of nineteen collections at the lake (November 2016 to June 2018) and twenty-four collections at the river (November 2017 to November 2018). In concurrence with bone samples, water samples were also collected from the level of submersion.

To evaluate microbial changes, all samples underwent 16S rDNA variable region 4 (V4) library preparation and Illumina MiSeq sequencing. Site-specific analyses indicated significant differences among water, rib and scapula samples. Overall, alpha-diversity increased with time at both sites, but was higher at the James River. Furthermore, beta-diversity within each site ordinated chronologically along one axis and separated according to location along the second axis. Relative abundances were used to both identify taxa that differed between samples and to generate random forest models for estimating PMSI. Models fit using 34 scapula family-level taxa provided a tighter prediction of PMSI with root mean square error of 333.8 ADD (~37 days) at Henley Lake when compared to ribs; meanwhile, ribs outperformed scapulae at the James River, predicting PMSI with root mean square error of within 472.31 ADD (~27 days).

To evaluate how bone DNA was impacted by prolonged submersion, a subset of samples, including those collected every ca. 1000 ADD from both sites, underwent extraction using both organic phenol-chloroform and ChargeSwitch® gDNA Plant Kit before being amplified and separated via the ABI 7500 and Prism® 3130 genetic analyzer, respectively. Findings suggest that peak presence and percent complete profiles were significantly different among ADD and between bone types. Likelihood of obtaining successful STR profile decreases with ADD and target fragment size of a STR locus. Results suggest that, regardless of extraction method and submersion site, ribs should be prioritized as they preserve recoverable DNA for longer periods of time and generate more complete STR profile in aquatic environment . Overall, this dissertation confirms that bacterial succession on skeletal remains can be used to predict long-term PMSI in both freshwater lakes and rivers. Along with microorganisms, temperature and water have been reported to fragment or damage DNA, which was observed with submersion time, as bone type and ADD impacted STR profile development.

Rights

© Claire Cartozzo

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-30-2020

Available for download on Tuesday, April 29, 2025

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