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DNA-based Methods for Studying the Diet of Marine Predators


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Deagle, BE 2006 , 'DNA-based Methods for Studying the Diet of Marine Predators', PhD thesis, University of Tasmania.

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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.

Item Type: Thesis - PhD
Authors/Creators:Deagle, BE
Keywords: marine, fish, diet, DNA
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