DNA-based Methods for Studying the Diet of Marine Predators

Diets of large marine predators have been extensively studied to assess interactions with fisheries, monitor links between diet and reproductive success, and understand trophic interactions in marine ecosystems. Since marine species can rarely be observed foraging directly, most studies rely on the identification of prey remains in stomach contents or faeces to determine the prey items being consumed. While this approach has provided a wealth of information, it has several limitations resulting primarily from difficulties identifying digested prey and from biased recovery of remains due to differential digestion. My thesis explores the use of molecular genetic methods in dietary studies of large marine predators. DNA-based identification techniques have been used in several diet studies, but the methods and applications are still in the early stages of development. Through a number of studies, I investigated the ability to recover genetic data from various dietary samples using a range of genetic techniques. A) Genetic screening for prey in the gut contents from a giant squid - I assessed the use of polymerase chain reaction (PCR)-based methods for isolation of prey DNA from an Architeuthis gut content sample. A taxonomically informative molecular marker was selected and a screening method developed using denaturing gradient gel electrophoresis. The methodology was used to identify prey from otherwise unidentifiable hard-part remains and the amorphous slurry component of the squid gut sample. The techniques developed here provided a framework for later chapters. B) Analysis of prey DNA in faeces of captive sea lions Part I: DNA detection, distribution and signal persistence - A feeding trial with captive Steller sea lions (Eumetopias jubatus) was carried out to investigate the use of genetic faecal analysis as a tool to study diet. I used group-specific PCR detection to determine: (i) the reliability of prey DNA recovery, (ii) the distribution of prey DNA within faeces and (iii) the persistence of the genetic signal after a prey item was removed from the diet. The proportions of prey DNA in several samples were also determined using a clone library approach to determine if DNA quantification could provide semi-quantitative diet composition data. Results show that the prey DNA could be reliably detected in sea lion faeces and the genetic signal could persist in samples up to 48 hours after ingestion. Proportions of prey DNA isolated from faeces were roughly proportional to the mass of the prey items consumed. Part II: DNA quantification - Quantitative real-time PCR was used to further investigate if quantitative diet composition data could be obtained through quantification of the DNA present in faeces. I quantified the relative amounts of DNA in three fish species being fed to captive sea lions, then determined the amount of DNA recovered from these prey items in the sea lions - faeces. The results indicate that diet composition estimates based on the relative amounts of DNA in faeces can be biased due to the differential survival of DNA from different fish species; however, these biases may be less than those commonly observed in the conventional analysis of prey hard remains. C) Quantification of damage in DNA recovered from faecal samples - I developed a general method to quantify the frequency of DNA damage present in specific gene regions. The technique was applied to assess the amount of DNA damage in predator and prey DNA recovered from sea lion faeces. The estimated frequency of DNA damage was always higher for the prey DNA than for the predator DNA within a faecal sample. The findings have implications for marker development and comparison of results obtained in future DNA-based diet studies. D) Studying seabird diet through genetic analysis of faeces - I investigated the diet of macaroni penguins (Eudyptes chrysolophus) through conventional analysis of stomach contents and through the analysis of prey DNA extracted from faeces. Genetic data was obtained from faecal samples using PCR tests to determine the presence or absence of DNA from potential diet items and also using a clone library approach. Approximately half of the faecal samples tested positive for one or more of the prey groups targeted with PCR tests. Euphausiid DNA was most commonly detected in early stages of chick rearing and DNA from a myctophid fish was prevalent in faeces collected later; this trend mirrored the data obtained from the stomach contents. Analysis of prey sequences in 'universal'clone libraries revealed a highly biased recovery of sequences from fish prey; this bias is most likely caused by the use of degenerate primers with a higher binding affinity for fish DNA template compared to DNA from other prey groups. Results obtained from the genetic and traditional approaches are compared, and potential future applications of the genetic techniques to studying seabird diet are discussed. This series of studies has contributed significantly to our understanding of the strengths and the limitations of DNA-based diet analysis. The work identifies situations where genetic methods can be successfully applied to study the diet of marine predators and provides guidance for future studies in this emerging field

DNA-Based Diet Analysis for Any Predator

Background: Prey DNA from diet samples can be used as a dietary marker; yet current methods for prey detection require a priori diet knowledge and/or are designed ad hoc, limiting their scope. I present a general approach to detect diverse prey in the feces or gut contents of predators. Methodology/Principal Findings: In the example outlined, I take advantage of the restriction site for the endonuclease Pac I which is present in 16S mtDNA of most Odontoceti mammals, but absent from most other relevant non-mammalian chordates and invertebrates. Thus in DNA extracted from feces of these mammalian predators Pac I will cleave and exclude predator DNA from a small region targeted by novel universal primers, while most prey DNA remain intact allowing prey selective PCR. The method was optimized using scat samples from captive bottlenose dolphins (Tursiops truncatus) fed a diet of 6–10 prey species from three phlya. Up to five prey from two phyla were detected in a single scat and all but one minor prey item (2% of the overall diet) were detected across all samples. The same method was applied to scat samples from free-ranging bottlenose dolphins; up to seven prey taxa were detected in a single scat and 13 prey taxa from eight teleost families were identified in total. Conclusions/Significance: Data and further examples are provided to facilitate rapid transfer of this approach to any predator. This methodology should prove useful to zoologists using DNA-based diet techniques in a wide variety of study systems

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology- and DNA-based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta-diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer-binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well-developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment

Studying seabird diet through genetic analysis of faeces: a case study on Macaroni Penguins (Eudyptes chrysolophus)

Determination of seabird diet usually relies on the analysis of stomach-content remains obtained through stomach flushing; this technique is both invasive and logistically difficult. We evaluate the usefulness of DNA-based faecal analysis in a dietary study on chick-rearing macaroni penguins (Eudyptes chrysolophus) at Heard Island. Conventional stomach-content data was also collected, allowing comparison of the approaches. Methodology/Principal Findings. Preyspecific PCR tests were used to detect dietary DNA in faecal samples and amplified prey DNA was cloned and sequenced. Of the 88 faecal samples collected, 39 contained detectable DNA from one or more of the prey groups targeted with PCR tests. Euphausiid DNA was most commonly detected in the early (guard) stage of chick-rearing, and detection of DNA from the myctophid fish Krefftichthys anderssoni and amphipods became more common in samples collected in the later (cre`che) stage. These trends followed those observed in the penguins’ stomach contents. In euphausiid-specific clone libraries the proportion of sequences from the two dominant euphausiid prey species (Euphausia vallentini and Thysanoessa macrura) changed over the sampling period; again, this reflected the trend in the stomach content data. Analysis of prey sequences in universal clone libraries revealed a higher diversity of fish prey than identified in the stomachs, but non-fish prey were not well represented. Conclusions/Significance. The present study is one of the first to examine the full breadth of a predator’s diet using DNA based faecal analysis. We discuss methodological difficulties encountered and suggest possible refinements. Overall, the ability of the DNA-based approach to detect temporal variation in the diet of macaroni penguins indicates this non-invasive method will be generally useful for monitoring population-level dietary trends in seabirds

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research

Quantifying prey availability using the foraging plasticity of a marine predator, the little penguin

Detecting changes in marine food webs is challenging, but top predators can provide information on lower trophic levels. However, many commonly measured predator responses can be decoupled from prey availability by plasticity in predator foraging effort. This can be overcome by directly measuring foraging effort and success and integrating these into a measure of foraging efficiency analogous to the catch per unit effort (CPUE) index employed by fisheries. We extended existing CPUE methods so that they would be applicable to the study of generalist foragers, which introduce another layer of complexity through dietary plasticity. Using this method, we inferred species‐specific patterns in prey availability and estimated taxon‐specific biomass consumption. We recorded foraging trip duration and body mass change of breeding little penguins Eudyptula minor and combined these with diet composition identified via non‐invasive faecal DNA metabarcoding to derive CPUE indices for individual prey taxa. We captured weekly patterns of availability of key fish prey in the penguins’ diet and identified a major prey shift from sardine Sardinops sagax to red cod Pseudophycis bachus between years. In each year, predation on a dominant fish species (~150 g/day) was replaced by greater diversity of fish in the diet as the breeding season progressed. We estimated that the colony extracted ~1,300 tonnes of biomass from their coastal ecosystem over two breeding seasons, including 219 tonnes of the commercially important sardine and 215 tonnes of red cod. This enhanced pCPUE is applicable to most central‐placed foragers and offers a valuable alternative to existing metrics. Informed prey‐species biomass estimates extracted by apex and meso predators will be a useful input for mass‐balance ecosystem models and for informing ecosystem‐based management. A free Plain Language Summary can be found within the Supporting Information of this article

Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

BACKGROUND: Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ) can be estimated by determining the rate of decline. RESULTS: The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λ(predator )= 0.0106 per nucleotide; mean λ(prey )= 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. CONCLUSION: We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay