Optimization of environmental DNA collection and extraction techniques for early detection of aquatic invasive species

Environmental DNA (eDNA) refers to genetic material released by organisms into their surrounding environment. The use of eDNA as a surveillance method has gained popularity as a less labor-intensive method for detecting both invasive and rare aquatic species, as well as assessing biodiversity more broadly. However, the efficacy of eDNA as a tool for species detection depends on factors including sampling strategy in relation to the life history of the target organisms as well as subsequent laboratory methods used to process the samples. My thesis includes two studies exploring factors relating to how specific eDNA methods affect species detectability with the goal of optimizing eDNA protocols. In the first study titled Environmental DNA storage and extraction method affects detectability for multiple aquatic invasive species, my collaborators and I compared the performance of two eDNA storage and extraction methods in detecting three common aquatic invasive species across five bodies of water. One method involved storing samples in 95% ethanol and extracting DNA using a Qiagen DNeasy Powersoil Pro Kit, whereas the other method used cetyl trimethylammonium bromide (CTAB) for storage and a phenol-chloroform-isoamyl (PCI) procedure for DNA extraction. We also investigated the effect of eDNA extract volume (1 µl relative to 3 µl) in qPCR reactions on eDNA detectability for the ethanol-Qiagen method. The CTAB-PCI method yielded significantly more positive detections, across all three species, compared to the commonly used ethanol-Qiagen method. Moreover, we found that using 1 µl of DNA extract in qPCR reactions was equally effective as using 3 µl in terms of eDNA detectability. In the second study titled Does crayfish molting affect environmental DNA detectability?, my collaborators and I used marbled crayfish (Procambarus virginalis) to determine how molting, a key life history event for many aquatic species, influences eDNA detectability across eDNA particle sizes. We collected water samples from aquariums at time intervals that coincided with pre- and post-molt and filtered our samples using a sequential filtration technique. In contrast to our a priori hypothesis, we found that molting had a weak negative effect on eDNA detectability. However, eDNA detectability was significantly influenced by filter pore size, with >5.0 µm showing the highest detectability. Additionally, the time spent in the tank also had a strong effect on eDNA detectability, with eDNA detectability increasing throughout our study. Both studies produced findings that are useful for the practical application of eDNA for aquatic species detection. However, challenges arose, including limited sample sizes in the molting study, data exclusion due to contamination and DNA extract shortage in the storage and extraction methods study, and low eDNA copy number requiring adjustments to our analyses. These complications highlight the inherent research complexities of eDNA studies.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Natural Res & Env Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Garcia, Samantha M.

Contributors dc:contributor

• Larson, Eric R
• Fournier, Auriel MV
• Davis, Mark A

• Edna
• Biological Invasions
• Molecular Methods

Statement dc:rights

• Copyright 2023 Samantha Garcia

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122260

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122260