The data chapters of this thesis (Chapter 2 – Chapter 6) were written as separate scientific papers that have either been published, submitted, or are in the process of being published. These papers include research on a diverse group of marine predators and each focuses on distinct questions; the thesis is tied together by the common theme of developing a DNA-based approach for studying diet.
Chapter 2 details the analysis of the gut contents from a giant squid (Architeuthis sp.). This chapter differs from the rest of the thesis by focusing on DNA-based analysis of material from stomach contents, rather than on faecal samples. Considering only one sample was available, the study is quite detailed. However, very little is known about the diet of giant squids and previous dietary studies on squids commonly report significant numbers of unidentifiable remains, indicating that the development of a DNA-based approach for identification of their prey would be useful. In addition, the prey species likely to be consumed by the giant squid (fish, squid and crustaceans) were the same groups that I was interested in detecting in dietary samples from other predators. Thus, this study provided an opportunity to design suitable PCR primers to look at these prey groups and allowed
Chapter 3 outlines a feeding trial carried out with captive Steller sea lions (Eumetopias jubatus) to investigate the feasibility of using DNA-based analysis of the soft matrix of faeces to study pinniped diet. Based on previous diet work (both
molecular and conventional) it was apparent that controlled feeding experiments with captive animals would be required to validate the DNA-based approach. No previous controlled feeding trials investigating DNA-based diet methods had been carried out with mammals. In field-collected pinniped faeces, prey DNA had been isolated from hard parts, but not from the soft-matrix of faeces (Purcell et al. 2000). Since it is known that digestion bias effects recovery of hard-parts, I wanted to focus on prey DNA extracted from soft matrix of faeces. The feeding trial was designed to addresses a number of fundamental questions:
• Can prey DNA be reliably detected in the soft matrix of sea lion faecal samples? • Can DNA from prey items fed as a small proportion of the diet be detected? • How is prey DNA distributed in faeces?
• How long after ingestion does a signal from prey DNA persist in samples? • How long does prey DNA survive in unpreserved samples?
• Are the relative amounts of DNA recovered from prey species proportional to their mass in the diet?2
• What is the quality of the prey DNA recovered?3
Chapter 4 contains a detailed analysis of the amount of prey DNA present in the sea lion faecal samples. Using quantitative real-time PCR (qPCR), I further investigated results from the previous chapter that showed (based on analysis of PCR clone libraries) that the proportions of fish DNA in faeces from captive sea lions were roughly proportional to the mass of the prey items consumed. The use of qPCR allowed more samples to be analysed and provided an independent method of measuring the amount of prey DNA present in the samples.
Chapter 5 focuses on measuring the quality of DNA recovered in faecal samples. The concept of DNA quality is often discussed, and usually refers to the level of DNA degradation in a sample – but there is no way of measuring this in many situations. I present a general method to quantify DNA damage present in specific DNA sequences. The approach is applied to determine the quality of DNA originating
2
This question is further evaluated in the next data chapter.
3
from predator (sea lion) and prey (herring) in the faecal samples collected during the captive feeding trial.
Chapter 6 presents results from a field-based study on the diet of macaroni penguins (Eudyptes chrysolophus). Dietary information was collected using DNA- based analysis of faecal material as well as through conventional stomach content analysis. Comparisons are made between the traditional and genetic data, and future prospects for using a DNA-based approach to study seabird diet are discussed.
The thesis concludes with a review of some concurrently published DNA- based diet studies on pinnipeds, a general discussion and suggestions for future directions of research in this field. The appendices include: (I) an evaluation of the strengths and weaknesses of nested-PCR; (II) details of PCR primers used in the thesis; (III) summary of qPCR data collected in Chapter 4; (IV) detailed results from the macaroni penguin faecal DNA clone library analysis (Chapter 6).
For chapters that have been published (or submitted) citation and co-
authorship details are included at the end of this section. In all cases, I was the senior author, and conducted the laboratory work, analysis of data and writing of the papers. My co-authors contributed significantly to the initiation of the projects, sample collection, advice on analysis, and/or by critically reviewing the papers in preparation for publication. The published papers have been modified to integrate the chapters into this thesis; however, each chapter is essentially self-contained and there may be some repetition in content throughout the thesis.