The use of DNA barcodes in tracking and conservation of sea turtles
A.N. Blasingame
Biology Department, Colorado State University, Fort Collins, CO 80523, USA
ablasing@rams.colostate.edu
Tracking and conservation of sea turtles
Abstract
Sea turtles are in danger of extinction and currently new techniques are being developed
and utilized in order to further the conservation of these species. DNA markers are used in
species identity. Researchers have begun to use DNA barcodes to identify meat and
unrecognizable samples. Here DNA markers are used in order to track the harvesting of sea
turtles through the sale of eggs and meat in various meat markets around the world. Certain
species will be present in higher concentrations in certain locations. The variance in species
demonstrates where each species is most threatened. Conservationists will then be able to
regulate the harvesting of each species in the area where it is in the highest demand.
Keywords
Sea turtles; DNA markers; conservation; sea turtle eggs; sea turtle meat.
Introduction
DNA barcodes have been formed from the mitochondrial cytochrome c oxidase subunit 1
gene (Eaton et al., 2009). This gene can be used to differentiate among species. A database has
been created containing primers specific for several taxonomic groups including birds (Kerr et
al., 2007) and sharks (Martin 1991). Researchers have used DNA barcodes to determine the
species that meat has come from in Central Africa and South America as a means to monitor
poaching and commercial trade (Eaton et al., 2009). Recently, barcodes have been created for
five of the seven species of sea turtles (Vargas, Araujo, Santos, 2009). Barcodes are useful in
taxonomy, conservation as well as other fields in development.
Despite the potential of DNA barcodes there are still many drawbacks associated with the
use of these techniques. One concern is that DNA barcodes are not an adequate source to be used
alone in taxonomy to discover a species (Rubinoff, Cameron, Will, 2006 and DeSalle 2006). It is
still generally accepted that DNA barcodes are useful for identifying species after taxonomy has
been established. Since, the purpose of this research is to identify and not define species, the use
of DNA barcodes will be adequate for the intentions of this study.
Currently, seven species of marine turtles are recognized. These species are the
leatherback turtle Dermochelys coriacea, green turtle Chelonia mydas, flatback turtle Natator
depressus, loggerhead turtle Caretta caretta, olive ridley turtle Lepidochelys olivacea, Kemp’s
ridley turtle Lepidochelys kempi, and the hawksbill turtle Eretmochelys imbricata (Plotkin,
2007). Sea turtles are a threatened species and many populations are in decline. Due to the slow
reproductive rate and migratory lifestyle of these species it is hard to keep track of them. It has
been documented that one of the main causes for the decline in sea turtle populations is the
consumption of turtle eggs by many cultures (Thorbjarnarson et al., 2000). Another reason for
the decline in sea turtle populations may be the harvesting of turtles at unsustainable rates due to
the demand for their meat (Koch et al., 2005). These factors contribute to the threat to sea turtles.
This proposed study will expand the current research to encompass all seven sea turtle
species. Cytochrome c oxidase subunits 1 barcodes for each species will be developed and used
to determine the identity of eggs and meat in areas where turtle eggs and meat are eaten or
trafficked. This information will be used to determine which species are threatened in certain
areas to enable conservation of sea turtles. These barcodes could also be used to identify remains
and hybrids between two species but these topics are beyond the scope of our discussion. The
intent is to focus on the use of barcodes to identify the species of sea turtle eggs and meat.
Materials and Methods
This study will require samples taken from meat and amniotic fluid from eggs in
locations where sea turtle meat and eggs are trafficked and eaten from around the world. Many
of these locations are in Asia, South America, and Australia. At least 27 samples of both eggs
and meat for each species will be obtained. The identity of each sample will be tentatively
determined based on information provided by local vendors. These samples will be stored in
70% ethanol and kept frozen. After collection DNA will be extracted from the samples using
previously described methods for DNA extraction in sea turtle samples (Lara-Ruiz, 2006). DNA
markers have been created for five of the seven species of sea turtles (Vargas et al., 2009). The
markers from these species will be used in comparison to the DNA extracted from the samples
and as a model in order to develop markers for the other two species. For the flatback and
Kemp’s ridley turtles DNA barcodes must be generated using the procedure developed for the
other five species (Vargas et al., 2009). The DNA extracted from the eggs and meat samples will
be compared to the barcodes for each of the five species of sea turtles. These comparisons are
going to be accomplished by denaturing the extracted DNA and adding the barcode primer for
each species individually to a solution of single stranded DNA. The DNA will then be annealed
so that the primer will bind to the DNA from the samples. Then aragose gel and ethidium
bromide will be used to determine if the DNA is bound to a primer. DNA hybridized to a primer
will result in a band that has not traveled as far as unbound DNA. The brightest bands will be
accepted as indication that a species specific primer is bound to the sample. If the DNA is not
bound, then a barcode from another species will be applied to the sample. The process will be
repeated until a species specific primer binds to the DNA from each egg and meat sample.
Expected Results
After hybridization, it should be clear whether the barcode primer has annealed to the
sample DNA. Bright bands are expected to be found in the aragose gel run for each sample.
These bands will be a different weight than unbound DNA and will produce a band closer to the
wells. During the experiment only one primer was added to a sample at a time. Therefore, it
should be clear when the samples match one of the seven species of sea turtles. It is expected
that a majority of these samples will match the marker of the species that the vendors claimed the
meat and eggs to be from.
We expect to find that the collected samples are identified to species using DNA
barcodes. Each species of sea turtle should be represented in the samples. Depending on the
location of collection certain species should be found in a higher concentration.
Discussion
This study will be undertaken in order to determine the level of threat that sea turtles face
from a market for their eggs and meat. By determining if the DNA is bound to a primer barcode
it will be apparent which species the sample came from. This will allow researchers to determine
which species were trafficked and eaten in certain areas of the world. This data can be used in
order to create a map of where each species is harvested most. Hopefully, this information can be
used in order to aid the conservation efforts currently underway. The locations of trafficking can
be monitored and the harvesting of the sea turtle species can be regulated. Therefore, sea turtle
populations could be given a chance to increase.
The data populated from this study may be used to inspire further studies not only
involving sea turtles but also other endangered species. DNA markers are currently being used
by many researchers in studies similar to this one. These methods could be applied to several
other taxonomic groups that are threatened due to human interactions and harvesting. Zoonotic
diseases may also be affected by meat markets and the consumption of exotic animals (Chomel,
Belotto, Meslin, 2007). A future study could use the methods developed in this study to aid in
identifying the species of infected meat in meat markets. The information collected here will aid
in the conservation of sea turtles and hopefully contribute to the conservation of other species as
well.
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