Dose-dependent impacts of copper and ocean acidification on Mytilus californianus

Dose-dependent impacts of copper and ocean acidification on Mytilus californianus


Hi everyone thanks for coming first a
couple housekeeping things if you haven’t signed in already please do so
afterwards um there’s a little sheet to sign it over there for those people
tuning in via our webinar you can type your questions into the chat box or
afterwards there’s an option where you could be unmuted by Judith um easier
ones probably just type your question and also just to note that you’re
currently in listen-only mode so I’ll introduce our first speaker
Megan Hall she comes from the University of Southern California Sea
Grant she’s Knauss fellow in the National Ocean Service Policy and
Constituent Affairs Division. She’s originally from Fort Lauderdale, Florida
where she graduated from Duke then to graduated from Duke University in 2009
with a BS in biology with a concentration in marine biology
now she’s currently a PhD candidate at the University of Southern California in
the marine biology and biological oceanography program. Fun fact about
Megan is that she has a black belt in karate. You joke about not being on Megan’s bad side and Megan’s title is
Dose-dependent impacts of copper and ocean acidification on Mytilus
californianus, close enough, and development and gene expression. [Applause] So yeah that is actually just muscle, you can think of it as muscle. Thank you all for coming today I’m gonna talk to you
about one chapter of my dissertation research studying the
interactive effects of copper and ocean acidification on mussel larval
development. Coastal ecosystems face many challenges right now both chemical,
physical, and more these are just a few of them depicted here where I did my
PhD research and in many urban coastal regions around the world copper
pollution has been an issue for a long time. Copper pollution comes from a
variety of sources one of the prominent ones is antifouling paint on the hulls
of boats it also comes from atmospheric
deposition and runoff from land base sources. Meanwhile there are novel
stressors that are emerging rapidly in coastal oceans such as ocean acidification
so in this set of figures from the California Current you can see that pH
of surface waters has been declining rapidly and is predicted to continue to
do so and aragonite or calcium carbonate saturation constants are also decreasing
and so with all these things happening extant stressors and novel ones emerging
it leads us to ask how are these things going to interact in coastal waters and
how are they going to impact key organisms and ecosystems? So when we
think about the impacts of stressors on key ecosystems one of the groups of
organisms that has long been used in this kind of research are calcifying
marine organisms that’s everything from mussels oysters coral reefs all of these
organisms are both foundational to providing habitat they can be ecosystem
engineers and create habitat for Suites of other organisms they also are
important for people in coastal communities and that they confer
protection against flooding and storms and they’re also economically important
often as the target organisms of many fisheries and aquaculture operations
however calcifying marine organisms also happen to be some of the most
susceptible to some of these changes that are facing our marine environments
and so in this rough overview briefly of how ocean acidification occurs a co2
enters coastal waters a series of dissociation reactions progresses in
which hydrogen ions are released into the surrounding water and that’s what
actually drives down the pH of the water the release of those excess hydrogen
ions but the carbonate buffering system has carbonate come in and react with
hydrogen to drive the reaction back the other direction and for more bicarbonate
kind of buffers that pH change so it makes it not as drastic but at the same
time if you look at this reaction at the bottom it drives the reaction of calcium
carbonate formation or other words the molecule that’s
necessary to form the shells of these organisms in the opposite direction and
so essentially it makes it harder for them to maintain their shells and form
their shells you add metal stress to that equation it could create even more
challenges and so copper or other metals in general have been predicted to have a
range of negative effect in addition with ocean acidification organisms one
of which is that changes in carbonate chemistry are predicted to potentially
alter metal bioavailability and thus make the metals more toxic to marine
organisms alternatively just the added stress of dealing with both of these
stressors could pose an energetic challenge to the organisms could target
similar pathways and make it harder for them to cope with these kind of stresses
if you look at larval calcifying organisms in larval bivalves in
particular which is the really early life history stages these problems only
can become worse and so essentially in this first 48 hours of development for
most bivalve larvae that is when the initial shell is formed the calcium
carbonate shell that will they’ll grow and expand for the rest of their lives
and ocean acidification in studies that have happened already has been shown to
delay development of that she’ll create abnormalities in the development of that
shell and in some cases even prevent them from being able to create a shell
at all and so failure of larvae to develop normally could leave the pot
lead to big ecological challenges a declining population and that could also
impact economic factors as well on top of that mollusk larvae are particularly
sensitive to copper as well and in one of my other chapters of research i also
found that copper targets shell formation genes as well as ocean
acidification and so this you can imagine how these problems could be
compounding and create more issues however although it seems like based on
these things it’s all doom and gloom there is some evidence these studies
aren’t but their occasional that ocean
acidification may reduce metal toxicity to some extent in some systems and so
this kind of suite of evidence makes it difficult to predict how metals and
ocean acidification are going to interact to impact the health of
important organisms and that leads me to my two research questions and I’m going
to dress today first of all how will Co exposure to copper and simulated ocean
acidification impact these sensitive early larvae and second of all what are
the molecular and physiological mechanisms of the impacts that we see so
to address these questions I use standard embryo larval toxicity assays
and walk you guys through what exactly that entails in general you’ll take your
parent parents into the lab so this is a female mussel mussel spawn them in the
lab and generate embryos you will then expose the embryos to whatever your
conditions of concern are right so whether it’s copper ocean acidification
you’ll expose them to that usually at a range of different concentrations or
doses in order to detect their effects across that whole range you’ll then
allow the organisms to develop under those conditions for a given amount of
time it can be from often 24 to 96 hours in this specific case we looked at it
for 48 hours and at the end of that time you can measure your endpoints of
interest so my two endpoints and the two
endpoints that are using many of these typical embryo larval development assets
with mussels were a survival and normal development so survival is pretty
self-explanatory in each concentration how many of the organisms lived how many
died normal development maybe a little bit less clear but essentially at that
48-hour period a normal healthy muscle larvae should look like this it should
be what is called a D Vinge larva or a D villager and if they don’t
look like that though it means something’s gone awry so in the figure
in the upper right hand corner here you can see a normal deviant larvae compared
to an abnormal water also at 48 hours and these animals
usually don’t survive long either and are more or less than ecological
equivalent to mortality and so essentially you can look at these end
points across your range of doses and often you see a sort of sigmoid or
response pattern like these curves here dentistry and where those curves fall
along your x-axis your toxin concentration shows you how sensitive
the organisms are to that toxin so for my specific experiments in this case I
looked at seven different copper concentrations with two different
partial pressures of co2 both modern-day co2 which is about 400 parts per million
and one of the end of the century projections which is about 800 parts per
million each of those those condition combinations had three replicate
containers and I repeated this whole thing four times with four different
sets of muscle parents and so if we go back to this first question how will Co
exposure to copper and ocean acidification affect the early larvae
and we look at those two endpoints of survival in normal development this is
what you see so I’ll walk you guys through these grafts on the Left these
for our survival plots and the four different 44 crosses the right these are
normal development plots in the four different crosses the blue line in all
of these cases is the modern-day co2 condition the black dotted line is the
future projected co2 condition the x-axis is copper concentration and the
y-axis is the proportion of your given response variable whether it’s survival
or normal development and so if we look at survival you can see in almost every
case survival is actually more sensitive to cobbler in the lower modern-day co2
treatment than it is at the future co2 treatment black stars above the bars
indicate the if there’s a significant difference in that shift shift which it
is in the first experiment but in general you see that kind of shift
and in the survival making the organisms better off in the higher co2 condition
if you look at normal developes see a similar trend but it’s a little bit
different so when the lower copper doses here you typically saw the animals
developed more normally at the modern-day co2 conditions kind of look
what you might expect but again when you got to mid range calm produces you see
again that that black line is further to the right whether the organisms are
apparently less sensitive to copper in future co2 shows and so this kind of
highlights again the fact that it’s difficult to predict how metals in our
way are going to interact right this kind of points again to those small set
of studies that did show that Oh a can actually reduce toxicity and to kind of
get at the heart of that question we want to ask what are the molecular and
physiological mechanisms of these observed impacts and specifically what
might be driving that reduced copper toxicity so just a few notes on the
benefits of using molecular detection methods so first of all molecular
protection can help you work toward a more mechanistic understanding of what
is happening physiologically it can reveal specific biochemical pathways
that are targeted and it also opens up many other downstream avenues of
research which I’ll discuss a little bit more detailed for the end of the talk on
top of that molecular methods can provide more sensitive indication of
toxicity in that organisms may be responding physiologically at levels
where you can’t actually see growth morphological changes and molecular
methods can pick up on those changes for this talk when I talk about molecular
detection I was specifically referring to gene expression and so to make sure
we’re all on the same page with gene expression we will visit the central
dogma of biology okay so basically all organisms have a genetic code DNA right
and in order to create bodies and function that DNA has to turn into
protein somehow and the way that it does that is and so this is a dynamic process it
changes of time it changes with different environmental conditions and
it can it can be very informative for those reasons it can be very sensitive
to what is happening around the organism and so using RNA sequencing you can take
a snapshot of the RNA expression at any given time or under any set of
environmental conditions in an organism and so you may see different genes being
expressed at different levels and ultimately those that highlight what
biochemical pathways are being targeted what the organism is doing at that most
fundamental level of biological regulation you can then kind of take
those snapshots and compare them across environmental conditions or different
organs and health seats so for example if you look at a normal larva let’s say
you’re looking at just one gene the blue gene in this case if you’re looking at
the blue gene and a normal larva under normal ocean conditions you may see a
pretty high level of expression however if you look at the expression of that
same gene in an abnormal larva under maybe a toxic condition you may see that
that gene isn’t very highly expressed and in that way you can look at the
profile of a genes expression across a range of concentrations of whatever your
environmental condition is a toxin and you can do the same thing and compare
RNA levels of every expressed gene in across the entire genome which is on the
order of tens of thousands of genes and that’s about how many I was working with
in this study and of course all those are going to probably exhibit quite
different patterns so you may have some that exhibit this kind of pattern like I
showed you before that is called down regulation but a gene starts out at a
high level of expression in the control and then decreases with an increase in
concentration of toxin you may also have up regulation you may see up regulation
and down regulation in a linear pattern rather than sigmoid ‘el or you may see
non monotonic responses where genes are actually increasing and then decreasing
vice-versa and so using that kind of profiling we can address how does gene
expression across the transcriptome respond to copper on ocean acidification
and use that to answer this question of what is or might be driving reduced
copper toxicity and so we use these patterns in this study to address to
draw potential theories of what might be happening the first of those is that
copper toxicity could be reduced by a chemical mechanism meaning that before
you even before these things are even entering the organism the interaction of
the chemistry could actually be preventing copper from getting into the
body and the cells as much and this might be the case because of cation
competition with hydrogen ions that are now in excess right if this is the case
you would probably expect to see same the similar genes responding right and
probably responding in the same direction but because less copper is
ultimately entering the body you wouldn’t expect to see that response
until slightly higher nominal copper concentration so for example here the
blue curves the response at modern-day co2 conditions the red curve might be a
response at future co2 conditions and you can potentially expect to see the
expression of genes shift a little bit to the right being less sensitive
alternatively if this is a physiological mechanism that’s conferring the kind of
protection against copper and reduce toxicity you might actually expect to be
seeing a different suite of genes involved or see the genes that are
already there acting in different directions so in this case again blue
modern-day co2 red future co2 you’d see a gene that was decreasing now being
upregulated or you might see a gene that was down regulated in response to copper
at modern co2 suddenly not being responsive at all or you might see a
gene that wasn’t responsive at all at modern co2 suddenly becoming copper
responsive and so all of these might be mechanisms that the organism utilizes to
defend itself against the increase presents are both of these stressors on
his body so getting to the data these are heat maps of the genes that are dose
responsive to copper in both of the co2 treatments right so they they have that
kind of both of them responding pattern a little bit about the anatomy of the
heat map so red or darker colors means higher
gene expression yellow or lighter colors moves lower gene expression the samples
that are surrounded by blue boxes here are all samples from ambient modern-day
co2 conditions and the columns increase from low or control copper to high
copper in these cases and these boxes here are surrounding samples from future
co2 conditions same patterns of copper increasing from control too high and
these on the bottom are the same samples and genes as you have up here but
they’re hierarchically clustered meanings of the samples at all put
together based on how similarly the expression profiles are across the whole
set of genes and so what does this data tell us essentially it kind of reflects
that say morphological pattern and shows that the response at 800 parts per
million is slightly shifted to higher co2 or higher copper concentrations at
the higher co2 bureaus so to walk you guys through where you see that exactly
this is an example of expression at the modern-day co2 conditioning at one of
mid-range copper doses if you look at the expression at that same copper dose
and future cm – you can see it’s a bit dark light it doesn’t really quite look
the same as this one however if you look at that expression profile at the higher
co2 dues at a slightly higher copper concentration you can see that now they
look much more similar maybe even pretty identical if you look at the
hierarchical clustering you see that these two the slightly higher copper
concentration of the future co2 condition
and slightly lower copper concentration modern co2 conditioning cluster together
and are very similar in fact and you see the same pattern in upregulated genes so
all of this is kind of indicative of this sort of gene expression shift same
genes same direction but shifting to higher compositions at the same time we
did also see some novel molecular response to copper at the higher co2
concentration so in these Venn diagrams all of the pink the fully pink area
represents genes that were only responsive to copper at modern-day co2
or in other words have sort of this pattern right on the other hand these
little yellow bits here represent genes that are only responsive to copper at
future co2 conditions or in other words this kind of pattern so these are what
we might call novel markers of the combined effects of co2 and gene
expression to take that a step further and actually ask what are these genes
what are these kind of novel pathways that might be activated we can do a
pathway enrichment analysis and look at the pathways that are specifically
modulated that only low co2 or I mean only high co2 sorry so here you can see
in this Venn diagram on the right there are 45 pathways that were only down
regulated at the future co2 condition and 99 pathways that were only up
regulated at the 800 parts per million co2 condition so these are all things
that are kind of showing that there’s a really unique physiological response
occurring here and so if we actually look at what the functions are that are
involved in these among the down regulated genes there are down regulated
pathways there were a lot of terms related to neurotransmission
neurotransmitters especially especially the glutamate glutamine glutamate cycle
which all involves ultimately the formation of gaba and so all of these
are really important neurotransmitter molecules that can
act organism movement behavior overall well-being at the same time we saw some
down regulation of cation and anion symporter activity and I think this
could probably be involved in modulation of uptake or export of copper from cells
and the up regulated pathways you saw fatty acid synthesis which interestingly
has also been found in the literature before to be upregulated or increased in
production in high co2 conditions and also saw DNA repair being upregulated
only in the future co2 condition and you know I think that could definitely be
explanatory as to the well-being of the animals because copper is known to
damage DNA via oxidative stress so getting back to this question of what is
driving reduced copper talk sisse T so based on the heat maps the first set of
genes that we looked at it is likely that there are chemical impacts that are
preventing as much copper from entering the body right and this expression
signature generally supports that however it does seem like there’s
probably some physiological role as well and potential explanations as to what
the organism could be doing by modulating those specific pathways I
talked about are the first of all more comprehensive down regulation of
transmembrane symporters and neurological signaling molecules could
be used to reduce movement and thus reduce the uptake of copper or even just
the simpler is alone being down my Glu could be resulting in reduced subjective
copper enhanced DNA repair as I kind of already mentioned could also be allowing
for more successful cell replication and proper developmental pathways to occur
so no broad conclusions the interactions of stressors and future coast waters as
we saw from all of this may have very unexpected effects on animal health you
could actually see it’s actually benefits of interactions of these kind
of things that you initially thought would surely create
harm under co2 conditions many copper responsive genes are similar meaning the
same genes are expressed but shift in exhibit shifts and sensitivity and some
unique genes and molecular pathways are targeted by the stressor interaction so
what are the implications of this and potential future avenues of research and
so the implications for understanding molecular drivers of toxicity can be
pretty important first of all these things can be used for novel biomarker
discovery so if you remember again in these Venn diagrams all the genes here
that were uniquely responsive at future co2 conditions those could all be used
in toxicity assays and tests down the road to detect those specific
interactions of chemicals in the water also this could be used for downstream
genetic analysis and selective breeding so essentially areas of the genome that
are responsive and potentially conferring protective mechanisms against
the interaction of these stressors could be analyzed for genetic diversity and if
you find genotypes that are associated with the organism having even more
activity in that specific region and potentially conferring even greater
benefit on ultimate survival and development those kind of specific
genotypes could be used and selected for in selective breeding programs and
finally enhanced predictive models which is what we’re altima trying to get out
here how can you take two stressors you haven’t done a specific experiment with
before for a specific organism and say what the effects there might be and
understanding the underlying molecular method or mechanisms and what exactly is
happening for that organism physiologically is going to be an
essential component of trying to make those connections however we definitely
still need to do a lot more testing on these kind of approaches
first of all take it to the field I think this kind of approaches are great
to get an initial assessment of what’s going on in a controlled environment but
ultimately you need to see that it also plays out in the field right you need to
know are these changes that you see happening
in the lab also happening where you see those same centers of conditions and
additionally we should continue to test these interactions and other systems so
basically right now a lot of the research in this kind of on these kinds
of stressors are primarily focused on bivalves calcifying organisms a little
bit on other species as well but really we need to be looking at a suite of
different organisms in order to ultimately predict how ecosystems are
going to change and we also need to be looking at the effects of other
interactions that they might be facing and ultimately that would hopefully be
able to lead us to develop better predictive mechanisms and so that I want
to thank all of my research support both financially and morally and all of those
ways and I’ll take any questions is relatively conserved yeah I mean in
bivalve molluscs there’s a lot of similarity I mean there are definitely
differences especially when you start looking at the genomes but they’re
compared to a lot of other organisms they’re much more similar yeah all of the experiments that have
kind of showed the beneficial potential beneficial effects have been in the lab
or at least all the ones that I see so yeah I haven’t yet seen anything in the
field that confirms the reduction but there also haven’t been that many field
studies in general and so if you look at the relative proportion of field studies
it’s it’s definitely I think not surprising but you wouldn’t have
necessarily seen that effect emerge yet and it’s especially difficult to study
these early like his three stages in the field in
any kind of controlled way because they’re small and mobile and you can’t oh I see what you’re saying yes no but
they’re also aren’t many areas that have these levels of co2 yet this is pretty
much what’s predicted to occur within the next like 30 to 70 years depending
on which climate projection you’re looking at but yeah would be good to see
yeah that’s a good question I didn’t say that
but so these are copper concentrations that are very typical of ambient water
in slightly contaminated harbors so highly contaminated harbors may have up
to like 30 to 60 parts per billion copper micrograms per liter
these ones are what you might see in kind of like most southern california
harbors on almost any given day potentially a little bit on the low side
of that range but those are definitely concentrations that are environmentally
relevant right right that’s a good question um the copper itself doesn’t
from my understanding have any specific location within the water column I mean
if it’s like if the copper is coming from runoff to them or something fresh
water going to the salt water that will probably say toward the surface because
of just the difference in density but once it’s actually in the water is
probably more or less just gonna get mixed in evenly in the sediment there
are definitely higher levels so in fauna and sediment are often at higher risk
for these kind of things organisms that live near the sediment that and muscle
RV I don’t believe exhibit that much
vertical cycling they’re not really that active of swimmers and movers relative
to other Marine RV so can you can you ask each question one by one I’ll answer
each one as I go along okay so first I’ll say to test the
significant response to copper all the genes I showed here I use a program
called sigmoidal dose response search which is has was used initially to
identify genes that respond to different pharmaceuticals or drugs or metals
potentially in a sort of sigmoidal pattern to look at the actual enrichment
of pathways I used the cytoscape plug-in bingo and I looked at the sets of genes
and I had identified via STRs and looked for significant enrichment there I I did
also in another set of genes not represented in this study used both Dec
2 and edge R to look at differential expression and that all is a whole other
story but I kind of use that data to address a different question yeah was
there I think there was more of a question tell them they can email me and
I will tell them any more detail they want to know. Thank You Megan!

Leave a Reply

Your email address will not be published. Required fields are marked *