Importance of Natural Resources

Biology, Ecology and Genomic Studies of Florida Bottlenose Dolphins

We’re over at the Population Biology/Behavioral
Ecology Group lead by Dr. Greg O’Corry-Crowe. He couldn’t be here today so my name is Heidi
Pagán and this is Sarah Rodgers and Tatiana Ferrer. We’re all going to be presenting updates
on our projects. A little bit of background on our role in dolphin research: We are lucky
to be part of Harbor Branch who has this long term study on the dolphins and as we’ve already
seen there have been years of data collected already on the unusual mortality event just
recently and then the years of strandings and disease and general dolphin health. So our goal is to come to in and incorporate
a genetics aspect to this research. And this is our website for our group. I’m going to
go ahead and start by handing this over to Sarah. We are going to talk about a three-part
investigation and she’s going to be starting it off. Thank you Heidi. All right. So my name is
Sarah Rodgers again. I’m going to talk to you today about the population structure and
dispersal of the dolphins along the east coast of Florida. So quickly bottlenose dolphins.
They are a very common mammal. They’re found worldwide. They live in these interchanging
communities. They have sex specific associations and they’re apex predators. They’ve also been
characterized as you heard before as a sentinel species which means changes in their environment
may alter their physiology or behavior which serves as an early warning signal to potential
risks of their ecosystem and anything of lower trophic levels. Now bottlenose dolphins along the east coast
of Florida are managed in groups called stocks and these are groups of marine mammals of
the same species who share common spatial arrangement and who interbreed when they’re
mature. There are four main estuarine stocks and two offshore stocks of the east coast
of Florida. Now dolphin studies have reported that there’s an estimated around 1,300 animals
within the Indian River Lagoon but this is unpublished and it’s considered one stock
all the way from New Smyrna Beach and Mosquito Lagoon down to Jupiter Inlet. There’s three to five communities within the
IRL based on whether you look at ranging patterns of dolphins or social affiliations. The IRL
is a separate to an adjacent coastal population of dolphins that has been observed having
limited to no movement between the two, essentially making the IRL dolphins a closed population.
Finally the population structure published to date has used a limited number of samples
or nontraditional methods leading to misinterpretations of results. Now you heard before about all of these problems
for dolphins. Adam mentioned the prevalence of infectious diseases, the pollutants. You
heard about unusual mortality events with Steve, the interactions with crab pots and
boats and human encroachment and competition for their prey. So it’s of extreme importance
that we resolve the patterns of the dispersal, gene flow, population structure and immune
response in IRL dolphins. We first need to know who these animals are and where they
go so that we can then determine who specifically is actually at risk. Only then can we start
to minimize the negative impacts dolphins face within the IRL. Now there’s two main ways to identify structure.
One is observational studies such as photo ID and radio tracking. But as Heidi mentioned
observation studies can only tell us so much. The second way to identify population structure
is using genetics. For more concrete answers genetic analysis using a multitude of markers
is needed to understand what really may be going on within and among populations. For this study we used two types of DNA, mitochondrial
DNA seen in red and nuclear DNA seen here in blue. Mitochondrial DNA or MTDNA is found
in the mitochondria. It’s maternally inherited meaning you only get it from your mother.
It’s relatively high in mutation rate but it only provides information to us on female
patterns of dispersal. Nuclear DNA; specifically I used these regions called short tandem repeats
called microsatellites that you actually heard from Josh as well. These are found in the
nucleus. They’re biparentaly inherited. They’re genetically hyper variable and they offer
insights on breeding patterns and male mediated gene flow . Now this study used over 600 samples that
were collected over ten years from New Smyrna Beach all the way down to Jupiter inside and
outside of the lagoon along the Atlantic. Now skin was taken from dead or live animals
and was immediately preserved or frozen. Once in the lab the skin samples were extracted
using a standard salt method and DNA was quantified and archived. We then amplified mitochondrial DNA using
standard primers and an additional ten microsatellite markers were used to amplify their respective
regions. Sequencing on both strands of the mitochondrial DNA and genotyping of the nuclear
DNA were done in house here at Harbor Branch as was the alignment and analysis of all the
data that was generated. Again from start to finish nothing in our lab gets sent out.
Everything is done in house to insure optimal quality control and proper interpretation
of the results that we find. So what questions can we ask with all of these
samples covering such a large area over all these years? Well we decided to take a four
ecological avenue approach. We first started with an ecotype question which was in the
form of comparing dolphins compared within two ecosystems, the estuarine and the coastal
dolphins. Our second hypothesis was based on subdivision of water bodies or separate
embayments. Mosquito Lagoon separate from the IRL proper versus the Atlantic. Third was a habitat-based approach specifically
using discreet hydrologic and geographic segments as potential boundaries. Again you’ve seen
this earlier on the segment maps. Fourth we implored a more biological based approach
using the results from one of our collaborative studies Adam mentioned using social networking
and association patterns of IRL dolphins. Here we tested those – population structure
behind the six communities. So what did we find? We found that dolphins
sampled from the IRL estuarine system are genetically differentiated from animals sampled
along the Atlantic coast. Our markers showed a difference in the sexes, that males moving
from both populations versus our IRL females which seem to remain homebodies. We also were
able to determine these differences have been in effect for a long period of time. And finally
with our extensively large sample size spanning the entire Indian River Lagoon estuarine system
and all along the Atlantic we were able to detect immigrants in both populations seen
here. In hypothesis two when we looked at water
bodies we found Mosquito Lagoon dolphins were actually more genetically similar to Atlantic
dolphins than to IRL proper dolphins. Even more surprising was that these dolphins weren’t
a separate population. They’re what’s called a mixed stock and that appears to have a minimal
breeding between those two source species. Very interesting ecological situation that
we’re definitely looking at right now. When we tested the hydrologic and geographic
segments that have been used in many previous dolphin papers as potential groupings we found
that there was minimal to no differences between these groups. And therefore our segment map
actually looks more like this and we conclude that IRL dolphins aren’t actually using the
water management districts as boundaries to separate themselves genetically. Finally using
a biological boundary estimator of dolphin communities we found similar to the segment’s
hypothesis that dolphins sampled living within each of these community boundaries do not
appear to be kin based. And again our results show the fluidity of genes within the lagoon. “So all of this work helps us how?”, you may
ask. Well you have to start with the solid concrete foundation to build bigger and better
projects upon. These findings will be the basis of all future dolphin related studies
and we'”re very, very thankful for the plate supporters that have helped us finally nail
down these answers. Further, your donation funds are continuously growing tissue and
DNA archive which we bank samples for future studies with collaborators all around the
world. So now we can identify who actually is an
IRL dolphin regardless where they may be found and how these two populations overlap. So
we can ask major questions like “Do dolphins with IRL genes have different immune potential
than Atlantic dolphins? Could those male dolphins be the disease vectors between the IRL and
the Atlantic dolphin populations? Could dolphins with Atlantic genes be more adept to deal
with pollutants, metals and other toxic contaminants?” “Could IRL dolphins have a stronger immunity
during those mass die offs, those unusual mortality events? Is that why we haven?t seen
the mass mortality within the lagoon that we’ve seen during the coast? And what if we
were able to determine which populations may be more at risk for these types of events
ahead of time?” Now answers to these questions and more may be in the genes involved in the
immune response which lucky for you Tatiana will now tell you about in part two of our
investigation. Thanks Sarah. So first I want to talk to you
guys about why diversity in MHC loci matter. So understanding the genetic aspects of the
immune response provides insight to the determinants of individual fitness and population viability.
Immunocompetence is an individual’s ability to produce a normal immune response post antigenic
exposure. Pathogens are some of the strongest selective forces. Individuals are constantly
exposed to them especially in changing environments. Immunogenic diversity is associated with decreased
fitness and increased risk of extinction. So the major histocompatibility complex of
MHC is the immunologically most important family of genes determining a population’s
ability to detect and combat pathogens. It’s comprised of about four million base pairs
and contains the most variable functional genes described in vertebrates. So the MHC is central to the vertebrate immune
system. It is a system of recognition and presentation that initiates the immuno cascade.
It responds to pathogens by recognizing nonself peptides and it determines the disease susceptibility
or resistance at the genetic level. Here we have a basic schematic depicting a pathogen
being engulfed by a macrophage, broken into pieces and presented by a receptor on the
surface of the cell. A T cell then binds, activates and turns on the cell initiating
the immune cascade. For this project we are focusing on the binding and presenting that
initiates the immune response in the individual, seen here in pink. So talking about our specific locus the DQ
is a heterodimer comprised of an alpha and a beta chain. DQ genes play prominent roles
in adaptive immunity. Many associations have been made between the polymorphism seen in
DQ genes and resistance or susceptibility to a broad variety of infectious or autoimmune
diseases. Information on non-model organisms is lacking. Wild or semi-wild animals like
our bottlenose dolphins are considered to be better models for setting adaptive survival
on their pathogenic conditions. And finally DQ genes will hopefully provide insight on
how different environments can influence MHC evolution in marine mammals. Of the DQ genes DQA is one of the least studied
in cetaceans. We targeted exon 2 for this study because it codes for the peptide binding
region which is responsible for antigen recognition, binding and presentation to the T cell receptor
which initiates the immune response in vertebrates. So what did we find? First we were able to isolate and sequence
the entire DQA exon 2. To date this has never been done for bottlenose dolphins. Then we
determined that it is possible to genotype individuals rapidly with direct sequencing.
We also found diversity at this locus. We cleaned our PCI products to verify that the
alleles were legitimate and then to determine the allelic composition of a heterozygous
individual. We detected patterns of geographic variation in allelic diversity finding very
different allele profiles for IRL and Atlantic dolphins. We have evidence of positive selection indicating
that this variation is happening despite genetic drift and gene flow that Sarah just told you
about. And finally we identified variation in the peptide binding pockets. So paving
the way for future work. Now that we have been able to characterize diversity in exon
2 for bottlenose dolphins and we’ve identified crucial variation in the pockets of the peptide
binding region we need to remember that this is just a part of the DQA gene. So some of the questions we asked ourselves
during this project were: “How does variation in the rest of the gene compare to exon 2?
How can we look at the whole gene given the limitations of Sanger sequencing?” And finally:
“How does variation in another immune response gene compare to DQA?” Well with that we’ll
let Dr. Heidi Pagán tell you about part three. All right. Thank you Tatiana. So like she
said I’m doing this last part. I couldn’t talk about my project first without going
over theirs because they laid the groundwork. Sarah determined the population structure
and then my project is an extension of Tatiana’s project where I’m looking at two different
genes. So here’s the gene that she was talking about, DQA. And just for a brief refresher
this gene has five exons and it has a promoter region at the very beginning. Transcription factors have to come and bind
to that region before the gene can be turned on. When it’s turned on it’s transcribed into
an RNA molecule to translate it into a protein. Then that protein is further processed by
going through folding to become a specific confirmation that allows it to form part of
the DQ receptor molecule. Now it takes two genes to completely form that DQ receptor.
She just told you about the importance of exon 2 in forming the peptide binding region.
So I want us to look at variation of this in both genes because that will tell us about
what types of different pathogens can be bound by the DQ receptor but I also wanted to look
at variations in promoter regions because that’s going to tell us about how this gene
can be turned on or off. So there are four different loci that I was
looking at here, the DQA promoter, DQA exon 2, DQB promoter and DQB exon 2. The next part
of my project was to incorporate next generation sequencing methods. These are high throughput
methods. That means we can cover a lot more samples in a shorter amount of time and for
a lower cost. The trick is that all the samples have to be combined into a single tube before
they’re loaded into the sequencer. So when we’re amplifying our region of interest we
put on unique bar code pairs. That’s a known segment of DNA at the beginning and the end
of each region we are interested in. So each dolphin ends up having four unique bar code
pairs. Did that for 95 dolphins and then combined
all of those into a single tube. That sample was loaded onto a chip in the Ion Torrent
personal genome machine and sequenced. We got more than five million sequence reads
from this and here’s a little snippet of what the data looks like. It’s not just the DNA
sequence. It includes a bunch of symbols below that which are quality values which tells
how confident we are in each position of the sequence. So we did some filtering steps and we ended
up being left with more than two million sequence reads that were the correct length and had
the identifiable bar code at the beginning and end of the sequence. Here’s an example
of the data processing. If these were our sequence reads we’d sort them by the bar code.
So here’s a subset that have the same bar code at the beginning and end of the sequences.
We would then trim off the barcodes and align it to the reference sequence and then we do
our variant analysis. This is where – I’m sorry if you can’t see
very well – but there are green positions that are different from our reference sequence
so that’s the parts that we’re interested in. And here’s an example of this. For DQA
exon 2 for one dolphin. It had over 1,000 sequence reads and the positions that are
a little bit lighter are the ones that are different from the reference sequence. So
this was a nice sample because they fell very cleanly into two distinct groups. That’s because
we have two copies of the gene – two alleles, one from mom, one from dad. But most of our
data was not actually that nice so here’s an example where there’s a lot of noise. There are sequencing error and other amplification
errors that we have to consider. So we had to run a bunch of controls as well and I’m
analyzing the data of the two different programs to see how well they line up and then I’m
ground truthing this because I include some of the same dolphins that Tatiana ran and
I can look at her data and mine and make sure they match up for those individuals and that
way I can confirm that this is a good method for using because she’s doing traditional
and then I’m doing next generation. So I’m still finishing up this analysis. When I’m
done with that this will be the most comprehensive MHC genotyping project for any cetacean. That was a lot to throw at you so I’m going
to do a quick summary of the three projects. The first one presented by Sarah was population
structure and dispersal using non-coding loci. She was able to detect a genetic profile between
estuarine and coastal dolphins and immigrants of each. She was able to resolve population
boundaries and movements of Indian River Lagoon and adjacent Atlantic dolphins essential to
proper management and the basis of all future Indian River Lagoon dolphin research. Tatiana’s project was differentiation using
an immune related locus. These are the first time this type of research has ever been documented
for Tursiops truncatus, provides us with a blueprint for the geographic variation in
crucial binding pockets of the Indian River Lagoon and coastal Atlantic bottlenose dolphins
at an important immune response gene. And then finally my project was a differentiation
using multiple immune loci at high through put frequency and this process can now be
easily applied to additional populations and species. With that I would like to thank everybody
here at Harbor Branch who made this research possible as well as other organizations and
the license plate that allowed us to do this research and thank you all for your time.

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