Hi everyone, good
afternoon, to reintroduce this is the
Brown Bag Committees, Brown Bag hour
and I want to thank our first
speaker today Carrie for an excellent
and informative presentation. Next up we have Dr.
Skylar Bayer who's gonna be talking about
an environmental DNA or eDNA approach for
investigating the reproductive biology
of sea scallops. Skylar is a 2018 Knauss
Fellow in the Senate Environment and Public
Works Committee Minority office. Skylar
received her bachelor's in marine
biology from Brown University, her
master's in biological oceanography
from MIT-WHOI joint program and her PhD in
marine biology from the School of Marine
Sciences at the University of Maine. Her
research focuses on reproduction ecology
and population dynamics of marine
invertebrates. Excited to have you! [Applause] Can you guys hear me okay? I don't know about all of you out
there in space um so just to sort of
give you a summary up front if you were
at the AFS meeting this is the same talk
um I'm this talk is designed for anyone
who doesn't know anything about eDNA and
there's also a bit in the beginning
about sort of the motivation for how I
ended up here which has a lot to do with
traditional ways of measuring
fertilization success in the field so I
have a bit of that and a little bit of
explanation of terms in the middle so if
you just want results you can tune out
for a little bit towards the end until
the end um if you are on Twitter my
Twitter is up here in a cool cartoon
that you can pay someone 20 bucks to
make those for you it's great I put
on all my talks. So to begin
with motivation at NOAA we probably all
know that seafood needs to be able to
reproduce to create more seafood you
have to allow animals to reproduce in
order to have more of it to fish or eat
or just admire and
that means understanding how they
produce where they produce spawning
fertilization rates how many of them
develop into larvae where the larvae are
the entire larval black box okay um and
so marine protected areas and fishery
closure designs really should be
considering reproductive requirements
and sometimes with best available data
we can do a really good job and
sometimes we're kind of just guessing so
there's still there's still a lot of
room to sort of figure out because
there's a lot of biology we don't know
about how animals reproduce to sort of
improve those management needs um
and the sort of third point on this
slide that I really want to make its
method of reproduction that is
commonplace in the ocean across all
phyla is broadcast bonding and that's
the release of egg and sperm into the
water column where they meet somehow in
a very turbulent world sounds like the
dating scene but it's actually quite
literal and this ranges from fin fish
here's a picture of red snapper spawning
and you can see this beautiful cloud of
eggs and presumably sperm what these fin
fish and actually a lot of fun fish are
able to do is they come into
aggregations are able to swim really
well and in a lot of cases there are
fish species whose sperm can only live
maybe a few seconds because if they're
any farther away then that would never
fertilize an egg anyway so they're
adapted to being able to swarm these
aggregations then many marine
invertebrates like this to spine sea
star release eggs and sperm from the sea
floor and sort of hope they're close
enough to neighbors and often these
species that might be kind of close to
each other but far enough away have
sperm and egg adapted to sort of longer
time periods of survival so that they
might be able to meet however they're
sort of a lot of question marks on all
of those characteristics of these
gametes so the study system I spent
seven years studying PhD and beyond is
the sea scallop or platypus
magellanic estancia Suman harvesting yet
I'm very heavily exploited the federal
fishery brings about 500 million dollars
a year doing pretty well I think it's
like second or third right now in terms
of fishery species someone here now
probably knows better than I do
the Maine fishery it brings in about 10
million dollars a year which is pretty
good Maine I think is the only state
that has a sea scallop fishery at the
moment and it's benefited greatly from
fishery closures on Georges Bank on some
of that might have to do with currents
that affect larval circulation but
there's still a lot unknown actually
about how reproduction plays into that
equation they're gone a Christic meaning
they have male and females occasionally
find from aphrodite's but it's like one
in a thousand or three thousand and on
the right-hand side of this slide I have
a female and a male the female gonads
are orange and the male is sort of a
white or cream color and during peak
spawning season before they spawn these
gonads can make up like 32 sometimes 40
percent of their body weight
wet body weight so spawning scallops
sort of an interesting process I spent
seven summers figuring out how to do
this and usually after sort of the peak
temperature highest temperature in Maine
you give McCulloch chop for females it's
usually has to do with a lunar cycle
although after the last few years I'm
not sure what part of the lunar cycle
now this is a picture of the collecting
eggs that have been spawned by but the
female scallops males actually spawn
kind of randomly in comparison and so
coordinating experiments on
fertilization can actually be quite
difficult but to figure out when sea
scallops are spawning in the field will
collect eggs in lab you steal them and
then we put them in these tiny little
Night x mesh chambers that are about 45
microns the eggs are about 70 microns so
in theory you can put all these eggs in
them put them out in the field next to
some hopefully male scallops
gets in maybe fertilizes the eggs and
the fertilized eggs stay in the chambers
and we hang these next two populations
you there naturally spawning or under
docks where we've isolated them a nets
and so then we collect them after 24
hours and we count all these eggs then
we score them by stage then we come up
with a number and my point here is that
this takes a lot of time a lot of work
and people don't appreciate how much how
much it takes just to get some numbers
that you hope are like kind of accurate
um and so one of the things that we did
to look at naturally spawning
populations is we would actually put
these little chambers on blocks and then
we set up these populations that we had
used scuba divers to lay them out and
sort of hope that they kind of stay
there they don't move too much and the
map on the right side of this slide
shows the DMC or darling Marine Center
River based and then the LD site
indicates a low density site that we
made of scallops and H D indicates high
density site they're about tenfold
difference in population density and so
whenever we could get the scallops to
spawn in lab we would deploy the Nets or
deploy the baskets and then we would get
a time series and the take-home here
where you see the site's again on the
left and these time series on the right
is that you are seeing these sort of
peak spawning events we think because
these fertilized eggs are being
fertilized by sperm in the water and
actually what's most interesting about
this experiment is that the high density
and low density site showed peak
fertilization at the same time in the
summer and actually the low density had
about the same fertilization as the high
density so mmm the problem with this
method is that spawning season waits for
no one
and it's really hard to get control of
the situation
it's Harrison Ford is reminding us in
this gif but um you can't you have to
wait for the scallops you can't sample
every day because they don't spawn every
day in lab so we started thinking about
is what about sampling water for a DNA
maybe we
can just capture the spawning events
that are happening in the natural
population without having to get lab
scallops to spawn so what we did is we
identified and isolated a genetic marker
for sea scallops which we hadn't really
found in the literature before and then
we developed a quantitative real-time
PCR method to estimate sperm
concentration which presumably is an
indicator of a spawning event in both
the laboratory and then the field and
I'm gonna walk through some of the terms
here now so if you're you know all this
you can just ignore it um so PCR
polymerase chain reaction its molecular
technique used to amplify DNA small
quantities which it's easier to measure
you can see how much is there
quantitative PCR is when you measure
this PCR amplification over time I TS
which is the region that we isolated the
inner genic transcribed spacer region
which is located between the small and
large ribosomal RNA gene so basically
from what I understand almost every
organism has these genes so it's a very
reliable region it's easy to
differentiate between species and
actually there's a paper but I think
laying at all in 2006 that looked at I
TS regions for a lot of commercially
exploited by valve species and found
that it was a really good marker for
distinguishing between species when
sampling by valve tissue and then
finally the last thing is the weaves
attack man method there's another
super-green method but that we did not
use that if you know what those are and
so basically use a fluorescent probe to
quantify DNA and so if you have some
questions about that later I might be
able to answer them some who might not
might not be able to so this is the
steps we took we developed this program
primer after we isolated the the DNA
region IPS region then we did a clonal
dilution series so we knew what genes we
had and then we created a series to look
at C Q which I'll explain in a minute
to create a nice relationship then we
did a sperm dilution series where we
looked at cell count versus gene copy we
did test this out with other species I
don't have time to get into it but
basically we know that this works for
just sea scallops and it's not going to
attach to any other species and then we
did a field test so walking through the
extraction process we have a scallop
over here on my right shoulder of that
cartoon you saw earlier we extract the
DNA from the tissue we isolate and clone
this I TS region we actually found eight
different variants of this clone so this
is important because when you design
probing primers you want to make sure if
it's for all the variants and you're not
just sort of targeting one variant um
then we sequenced all of these and then
we came up with our probe and primers
with a program called allele ID so
simple enough um next we looked at the
CQ value which some people here might
know what that means but basically you
have a fluorescent threshold that you're
looking for your sample to cross um and
it's over a number of PCR cycles of
warming and cooling and the number that
we cared about is called the CQ value
which is when your sample crosses the
fluorescent threshold at what PCR cycle
and that's what CQ is and if it crosses
earlier at a lower CQ value that means
you have more DNA of your target region
so um we tested out this method on our
clones we found it very very nice linear
relationship so we know that this works
really well if anyone cares there's a
little table in the bottom of the probes
and primers but I'm submitting this in a
couple days so hopefully it'll be in the
literature soon next we got the male's
to spawn which is hard and we did we did
five sets of the sperm dilution series
where we collected concentrated gametes
and did dilutions twice
and then we did cell counts so the
dilution series you have the five series
so those are the five different times
that we did this and we diluted by
tenfold each time and we did the cell
count that we extracted across the
dilution series and the gene copy number
and this is actually a very good
relationship who I work with whose peak
count when he's an expert in these
matters so that this was like a very
clean line so this is great because what
this means is when we take our field
samples we can get the C Q value then
the gene copy number and then sort of
guesstimate how many sperm cells were
getting so for the field methods we
collected scallops by scuba then we held
some of them in a lantern net so we knew
they weren't going anywhere and then we
did sampling during what we thought was
a spawning season and we sampled only on
low and high tides because we wanted the
least amount of water movement as
possible and then we filtered these
samples with a 20 micron filter and
that's because we were specifically
looking for sperm anything above 20
microns could be eggs could be pseudo
feces it could be all sorts of things so
size fractionation is actually really
important for us to target after those
sperm then we also did this thing where
we followed the gonad index going at
indices our traditional method of
detecting spawning by basically killing
animals dissecting them and seeing what
percentage of their body weight changes
so we did the traditional like murder
method at another site to see if it
correspondent with some of the the peaks
we were expecting to get from our
samples so this is so this is the big
slide if you're paying attention
somewhere else um we we tried to sample
actually the most when we thought they
were gonna spawn France number 12 which
actually wasn't when they spawned they
seem to be spawning towards the end of
August and end of September and we had
really these sort of two very clear
peaks which we were actually not
expecting and when you look at the gonad
indices um and I've highlighted in
yellow
these regions were you see a decline in
gonad index which indicates that there's
some spawning and these these Peaks are
within those declines so that implies
that these were actually from spawning
events so in summary we isolated these
new regions which is very exciting to us
anyway um we developed this really
species specific probe and in primer set
and then we were actually able to detect
spawning in near-real-time
which is huge because for invertebrates
like I've been able to find anywhere
else doing this yet um for future work
there's this is actually part of an EPS
core proposal that hopefully will get
funded and we're hoping that maybe we
can use this even a method attract both
spawning events and also larval supply
which people have done work on but we
could do more of it and we can expand
beyond scallops so that's it thank you
for your attention and hope I didn't
bore you too much so firming eggs are
viable how that relates to DNA possible
that you're picking up DNA but it is
possible
oh sorry the question was how long eggs
and sperm were viable and if they aren't
viable anymore is it possible to still
pick up the DNA right okay so sperm can
live think a couple hours eggs can are
still viable for about up to 24 hours we
think that sort of like really the end
um of course you can pick up DNA and
assume I think what's more interesting
in in this case and our results is the
the peaks like the difference because
there is sort of a background level that
we're picking up those lower dots and
that might be you know anything but the
fact that there's so many would indicate
based on the size fractionation that it
could be but but if it were a bunch of
sperm maybe you caught a spawning event
that's only a couple hours old which is
better than gonad indices which usually
is just month or maybe week I mean the
problem of going at indices is it's
lethal so you're killing off your
population and if with like sea scallops
you don't have that many to kill it can
actually impact your study quite a bit I
don't know that answers your question
that was the extraction part we took
like mantle tissue from the cartoon much
earlier and then that's how we got our
our DNA clones and then designer probes
and primer off of the adult you know I
think there are a lot of oh the question
right um I'm sorry I'm gonna have to say
it again I think so does the can the DNA
be detected and different be able to
take DNA from her goals yeah so the
question is if you take DNA from adults
in different areas can you detect
broadcast spawning
like where the origin of them are so the
so then your question is more about like
within a population like population
genetics level this is a species level
so I'm sure that you could get to that
point if you're curious about hey this
this genetically distinct population of
adults versus another right within a
species that would require sort of
another level of genetics this is only
species specific so
that's as much as this will tell you but
for population genetics I'm sure someone
could work on it yeah okay so first one
is could this or would this lead to
on-site sensors that might trigger
commercial activities if they had a
series of movies that would notify
interested parties of a spawning event
and with that influence decision makers
about changing operations do I need to
repeat your question okay
um it could I mean if you have a
spawning event and then you know the if
you're concerned about whether or not
you're harvesting that error and if you
want to know if it's like say a source
of larvae but you're getting a lot of
spawning activity maybe it'd be a good
idea to close it or study it better if
you're interested in how much larval
output in the origin so you can inform
it into models to figure out maybe what
kind of larval sets or whatever
you're going to get that year so it
really depends I think like what you
want to do but you could definitely do
something like that which i think is one
of the hopes of proposals like that for
project kind of Maine and Bigelow. How
far away from the spawning event do you
think you will be able to detect the
scallops?
um that's a great question we played it
super safe with our little field study
because this is the first time we did it
so it was sort of a methodological
development and we hung them in nets and
we took samples right next to the net at
lower high tide so slack tide so it's a
great question
we don't know yet. Could there have been other scallop cells in the water column that you also detected?  yes and so that's why
when you look at that figure I have
there are baseline values um and what we
really cared about are those peaks so
that we're assuming there's got to be
you know they're stuffing off cells
there's their pseudo feces and regular
feces and all sorts of stuff in the
water so what we were most
thrilled about were those Peaks and the
fact that those Peaks were at size of
less than 20 microns which indicates
problem that it's probably sperm why do
you think the peak is funding was
different between sets of years
well the climate change affecting
temperature salinity exactly yeah that's
a great question I'd like to know the
answer to but probably 2012 which is
what that first set of data I had from
sort of the hard labor method of
fertilization studies 2012 is an
exceptionally warm year and those
scallops spawned and like early or not
early for what we thought they would but
it was like late July and since then we
watched them spawn later and later every
year I am convinced that there's some
sort of lunar cue involved we are also
in a tidal estuary so it may not be like
exactly lunar but it might be more of a
tide flow cue but it's a really good
question and I do not know the answer
yeah and last one this research can it
be applied to other stations yes that's
the hope so I did scallops because it's
when I studied forever and it was really
easy but you know be great to do it for
lots of species and there's actually
there's a there's some literature from
about 10 years ago where a bunch of
people did this with larvae and they did
it for all sorts of species and then it
just seemed to the literature some of it
just seemed to kind of disappear maybe
it was too expensive or not interesting
but it seems like now that this
technology is a lot cheaper and more
accessible that these kinds of more
larger scale versions of these projects
might be much more useful to industry
and management
yeah so sure that's great
I had the clicker here and this might be
actually um so we sample because it was
literally just me and um we had
basically no funds to do this project so
it was when I could while I was working
on another project um so we went I went
the most which was daily and actually
sometimes twice a day
that week in September and then for the
other ones it was like once or twice a
week and that was based on what I've
seen with going at indices declining
when they often decline and during what
part of the lunar phase I've seen them
decline and sort of a side now this is
male focused but most of my work has
been a female focused and they seem to
be which oddly they seem to be picking
up different cues or they're just not
quite synched up so...
Any more questions?
Thank you so much Skylar! [Applause]
