Metagenomic amplicon sequencing as a rapid and high-throughput tool for aquatic biodiversity surveys

Abstract

Healthy aquatic systems are essential for life on this planet, and provide a variety of goods and services for humans such as drinking water, food production, waste disposal, and climate regulation. Anthropogenic impacts such as over exploitation of aquatic resources, introductions of invasive species, pollution, and climate change pose great risks for the health and sustainability of these ecosystems. Because of these risks, extensive and detailed biological surveys are regularly required to monitor and manage aquatic ecosystem health. Traditional survey approaches, including morphological-based identification and counting of organisms, are time consuming, costly, and dependent upon highly skilled taxonomic experts. Recently developed molecular methods, where DNA mixtures present in environmental samples are sequenced and taxonomically identified using genomic markers, are rapid and cost-effective, and may substantially improve biological surveys and ultimately aquatic system management. In particular, metagenomics amplicon sequencing of environmental DNA (eDNA) can characterize hundreds to thousands of species within a single sample in a timely and cost effective manner, and allow hundreds of samples to be sequenced in a single reaction. This PhD thesis aims to develop and refine eDNA amplicon sequencing approaches in order to examine current global problems in aquatic ecosystems. Specifically, this thesis includes a review of eDNA amplicon sequencing protocols and provides recommendations for sampling aquatic environments, laboratory procedures, and bioinformatics processes (Chapter 2). Following this, chapter three utilized eDNA sequencing to monitor invasive and threatened fish species in a sensitive and ecologically important river system, comparing results from fyke net- and eDNA-based surveys to analyze the accuracy and effectiveness of eDNA amplicon sequencing approaches (Chapter 3). In Chapter four, I examine the introduction and distribution of harmful algal taxa in high-risk marine locations across the entire continent of Australia, characterising a variety of harmful algal species associated with international and domestic shipping activities to potentially manage and mitigate the spread of these species. The use of historical port sediment samples within this study provides valuable temporal information to establish a baseline for the biodiversity distribution of harmful algae, essential for several international conventions focused on limiting the transmission of these harmful microorganisms. In chapters five and six, eDNA sequencing was utilised to identify bacterial taxa within drinking water distribution systems (DWDSs). Chapter five focuses on comparing current commonly used compliance measures with metagenomic approaches while screening for key indicator and pathogenic species throughout two full-scale DWDSs. Chapter six utilized this novel sequencing approach to analyze the efficiency of different water treatment procedures, while focusing on limiting biofilm formation in DWDSs. Overall, the thesis develops and demonstrates the practical applications of metagenomic eDNA sequencing on three distinctive taxonomic groups: vertebrates, eukaryotic microbes, and bacteria. The methods developed throughout provide critical advances for environmental monitoring organisations, including governmental departments, shipping, fishing and aquaculture industries, and water quality corporations. Further, this thesis and the future use of these molecular approaches greatly extends the knowledge of both bacterial and eukaryotic microbial communities in natural and man-made aquatic environments, improving industry efficiency and mitigating public health crises.