Larval Bivalve Responses to Ocean Acidification | George Waldbusser

[00:00:00]excited to have Dr George wal Buster assistant professor at the College of Earth ocean and atmospheric sciences of Oregon State University with us before moving to OSU in 2009 George was resident at chesap biological lab in southern Maryland and obtained his PhD in 2008 examining the effects of Bic inana on poor water advection and biogeochemistry in locations from the Pacific Northwest to the Southeastern us he's conducted he conducted his first oyster research examining recruitment Dynamics in the hudston Hudson raran Estuary in New York City in the mid1 1990s George's long interest in human environment interactions and the interface between ecology and biogeochemistry led him to begin working on ocean acidification impacts on Marine bals in 2007 with Dr Mark green George serves on the editorial board for the Journal of Shel off research and as an associate editor for limnology and oceanography methods outside of his academic career George grows organic apples and has been experimenting with different hard cider recipes for the past five years George I'm going to hand the control over to you for the um presentation great thanks Paula and I just as we get started here um want to thank thank uh National Science Foundation for funding this work and the no OA program for hosting uh the seminar webinar Series so before I get started I just I need to acknowledge a a huge number of people who've been involved with this work that I'm going to present here um particularly Burk Hales Chris langon who have been sort of critical in both the sort of uh chemistry and and the Lal rearing aspects of this work and then a suite of postto and under and graduate graduate students to have been really crucial in um actually carrying out the work so um so what I'm going to talk about today is really how uh the work we've been doing to understand laral B valve responses to ocean acidification and we're focusing on larvi because we know that they are the probably the most sensitive stage for many species and really a population bottleneck and we'll talk a bit more about that so and then just down in the center here I just I just this is one of my favorite scanning electron micrographs that we've Ed so far this is actually Olympia oyster larve that is developing a shell and it just looks similarly like Pac-Man so I couldn't help myself to uh make a Pac-Man reference here so we have the Olympia oyster here developing a shell and a fully shelled uh Pacific oyster here these are not to scale I should say so I just want to give a quick outline of the talk and so first to start with some some background and context uh about ocean acidification variability and environmental change so really thinking about Coastal and estrian habitats then moving into why saturates the state uh could and should matter to BU valve larve and then uh talking about the experiments we've been doing where we've decoupled carbonate system parameters to understand what in fact the B valve larve are sensitive to and then uh add on to that a little bit and talk a little bit about Nativity of middle larve and so we've looked at Native and non-native lby to the Pacific Northwest here and examine their responses and then sort of wrap it up with thinking about OA as a multiple stressor in itself and then uh and then sort of bring it home with the mighty Olympia oyster so uh which I'm pretty excited about some new data we've collected there okay so uh we Jan Kai gave a great webinar uh a couple weeks ago and so I'm not going to revisit that really too much but I think just a couple important points is that really acidification the coastal zone as we can think about that is sort of the weather that the organisms are exposed to the same way we think about climate change um and global warming and and the effects of climate change on localized weather we know that there are a number of processes that are going to affect carbonate chemistry in the coastal zone and these processes are are generally additive and so even though chemistry can be or CO2 levels can be higher we know in the global ocean areas where they have already high CO2 their increase in CO2 is increasing um at the same rate as places that have lower CO2 and so that Baseline shift is it appears to be ubiquitous across different variable environments so just because a coastal zones are more more variable it doesn't mean that the Baseline shift doesn't matter and I think a great example of this is some work that Brian helmouth has done looking at the temperature exposure of muscles in the inner tidal Zone and so what I'm showing you here is is um sort of the average temperature and these are for data loggers that were put out inside mimic muscle shells across nearly 10 years in the interner tial in Northern California and what I want to highlight is two years in particular here you can see the running average uh means for 2004 and 2007 you know roughly appear to be the same in what they're experiencing uh so the mean conditions are pretty similar but what's becomes really important is if we look at uh extreme events and so these are uh now on the same uh x-axis with year on here and the number of days above 30° C which is a temperature starts to create real thermal stress for these organisms and well you'll notice in 2007 there were nearly 27 days above 30 degreesc where there was some thermal stress versus 2004 where it was only about nine or 10 days and these numbers associated with the dots are just a frequency of return events and so I think this really the work uh in the thermal temperature um groups that have been looking at physiological responses some of them have really sort of I think provide us a a path to to be thinking about this a little bit more coherently and that while climate change is a global phenomenon to an organism all relative environmental changes are very local as the organism moves through space and time so this brings us to some uh conceptualization that I've done with a paper I wrote with Joe Salsbury and thinking about uh what contributes to that weather patterns that organisms experience and the perturbations of those weather patterns so to speak and so this is essentially just a cartoon hypothesis if you will of um the different wavelength of processes that affect carbonate chemistry in any given area and so starting from kind of uh tides at about the 12h hour time frame up to tens of thousands of years it's geologic hundreds of thousands of years in geologic time and I think the key thing to think about is that on the same process time scales there's biological organizational levels from Shell formation to laral periods and orders of days to weeks out to sort of evolution adaptation occurring at these sort of longer time scales and so what we know is that the frequency and magnitude of the contribution of these changes are smaller we get out to these longer time scales in terms of what the organism experiences but the the permanence of the perturbation also increases as you head out to those longer time scales so we know when there's been big geologic change in the CO2 of the of the planet there's been big changes in the composition of species that are present so what I really want to focus on now is is thinking about the vi valve life history and bottlenecks in that that are really we know related to sensitive areas in the life history for uh ocean acidification or other environmental stressors and I just want to highlight three and then we're going to focus on really one and so the first is really this development of the dehin shell which is really what we've been focusing a lot of our work on previously I've worked a lot of the work I did with Mark green I was looking at this period um from the swimming to the settlement stage um and again another bottleneck has to do with big developmental changes and energetic lows and then finally um habitat which has been the focus of a lot of restoration work and sort of Persistence of shells and we have some new work that's been funded to start looking at that as well but what what we're going to talk mostly about today is this bottleneck occurring uh in that development of that Deen shell because we know that this is a very sensitive stage for most invertebrates in B valves in particular and it is a bottleneck for populations so uh really the question we're trying to get at is what component of the carbon in chemistry Matters Most by about lity and and why does it matter and so one of the big differences between today and the past in terms of CO2 changes is the rate at which it's occurring uh this is some really nice modeling work that's been done by barbell Hanish and others um and this what this is showing is essentially different simulations of a doubling of CO2 in the atmosphere going from about 10 years to uh 10,000 years and what you'll see is that the faster that increase in CO2 the the faster or the more the the mean saturation state of the ocean drops in the surface water and so if we increase CO2 much more slowly which has generally happened in the geologic time the the saturation State more or less stabilizes we do get a decrease in PH which isn't as bad um but that's really the big difference about what's happening today in what's our previous geologic periods um it matters also because in coastal zones and estuaries freshwater inputs will change alkalinity and we also know that many estuaries have different alkalinity end members of rivers coming in so that becomes important as well to understand what components of the carbonate chemistry lower alkalinity Waters will basically have respond more um readily and and severely to increases in CO2 and then finally the variables in the carbonate system don't respond linearly to increasing CO2 and so this was some work from our recent paper nature climate change just illustrating what happens to pH in saturation State as CO2 in in pco2 increases and what you'll notice is that there's a more rapid decrease in saturation State initially relative to pH um and we just annotated on here some aspects from the literature where we start to see chronic effects on balve larv then where we start to see more acute effects at different saturation States and this delta3 pH unit is a value that's been banded around in some of the meta analysis that have been conducted looking at responses and so what really is that and what's important is that all of the previous almost all of the previous work that's been done on this has really bubbled CO2 and all the parameters change in some way and it's there's very little ability to differentiate what really matters to the b l so this should start to lead us to understanding what traits ultimately result in resiliency or susceptibility of B valve lar acidification and we'll come back to revisit this but there's a number of things that have been talked about in the literature so far one of the big ones is the ability to compensate internal acid base chemistry um and this is a primary mechanism that's been shown very clearly for adult organisms um energy Avail availability and so having access to more energy is certainly really important um that's going to help to some point and there's physiological limitations to that there's a great example that gets thrown around about um clam or muscles that live at hydrothermal vents in these highly corrosive environments and it turns out they're chemosynthetic and so they essentially have an unlimited uh supply of energy from the sulfide leaking out of those hydrothermal vents and so that's probably one of the mechanisms that allows them to live in a really highly corrosive environment gel minerology has been talked about quite a bit and that's really the thermodynamics and I think that um in most most cases we've seen very little evidence of that in experimental data is that being a determinant of susceptibility to ocean acidification and then a lot of the work our group's been doing recently is understand the role of kinetics or how rapidly uh shell formation is occurring and particularly focusing in on this early larel stage because it is very rapid shell forming period so really I just want to one here and say let's not forget natural selection works on traits and thus it should be a key uh to understanding what OA ultimately holds for marine species and so we need to be thinking about traits um and what's really going to matter to the organism so here's a suite of possible traits we talked about or I mentioned and we're going to just really kind of get into the kinetic side a bit more so first I want to start with why saturation State should or could matter to B valve larv so this is just the standard idealized model of organismal physiology under OA it's a really nice work that's been done by Hans pner and many of the other physiologists working on these sorts of question and the sort of important features of this kind of model is that the calcification surfaces are within the body so they're protected from the outside environment everything is linked through metabolism and really it's the regulation of the internal acid base chemistry that is key um so according to this model only the external factors that affect the internal acid based chemistry can affect the organism all responses are generally thought to be related to how quickly bicarbonate accumulation occurs and so how quickly can they compensate that internal the organisms can compensate that internal acid based chemistry um and all the work that's been done so far shows that this generally occurs over the course of a few days and kind of the fastest responses that have been noted so at least takes them a few days to compensate for this change and we know from Plenty of measurements in coastal environments that within a day the changes can be quite significant because of iral variability production and respiration cycle so this clearly is an important mechanism but it may not be capturing everything I just wanted to highlight this is a a new study just came out last week uh out of Donald Monahan's Group by panl this is in proceedings of national eies and really the the key thing that they found here and these are SE urant larvey was that uh these are looking at essentially the energy budgets or the ATP allocation to different components of the energy budget the black areas are in these pie charts are are uh to protein synthesis and the gray is to proton pumping so sodium potassium link atpa activity and the white is sort of everything else and basically what the argument is and it's really nice work shows that under acidification stress in these red circles here there's a really significant increase uh in the energy allocated for protein synthesis um and so clearly proton pumping and and um acid base regulation is being upregulated but there's a significant portion of that energy that's being reallocated just simply synthesizing and regenerating protein in some way so it's clear here's one example it's clear it's not just intercellular pH regulation and I'm not saying that's not important but we're we have an evolving model of understanding this and I think in order to understand these responses we have to be open-minded that there are more than one potential Avenue for acidification effects on these organisms so the work we've been specifically doing is looking at early shell formation so we call this a pro Aon one shell and uh some people refer to that as dehin shell and just to summarize and I'll show you some data here in a minute is the reason this formation uh this this stage is so sensitive um to saturation state in particular is the calcification surfaces are exposed and so I'll show you those isotope data in a second here we know there's limited energy until they develop that shell and they can attach the vum and start swimming and feeding and the calcification during this period is extremely rapid and so this is some isotope work stabil isotope work that's been done by a former graduate student of mine Elizabeth Brunner um and what you're looking at here is sort of uh We've sampled these are Pacific oyster lar we sampled over uh development from fertilization couple days or day after fertilization out to roughly settlement uh or competent to settle and S of the key thing in the top graph here is to note that in the Shell there's a much higher heavier Del 13 signal which indicates greater incorporation of diic from seawater if this was 100% DIC that they're making putting into the shell uh in aragonite it should be about I think about 2.7 and so what we can do is because we know what the metabolic signal should be from the food we can calculate the proportion of metabolic carbon in the shell and so what we notice is that two days there's about 7% metabolic carbon in the shell and by the time that they're ready to settle that's up just over 14% metabolic carbon in the shell and so we interpret that in part as being this these early surfaces are more dependent on DIC uh these early shell development is more dependent on DIC and it's more exposed to external seawater and as those they grow and develop new organs and uh the ability to close off those calcification surfaces more readily then you get more of the metabolic carbon in the Shell it's also important to note that at this stage at this early stage when they're building the shell their Mass specific metabolic rate is is is almost an order of magnitude higher than what happens later so they're respiring in an a phenomenal rate but most of that is not being captured in the Shell because we believe these these we think this data shows us that those surfaces are more exposed the bottom graph I just just to point this out all this really shows is that from the early period on they're more dependent on the maternal carbon derived from the egg and as they start feeding that proportion goes down and there's still measurable amounts of maternal carbon in those organisms out to uh just about a week and a half okay and just finally to show this rapid calcification on the right here this is about a six-h hour time frame they go from an unshell lar you can see the back of the hinge here and this is the initial per ostrome that's formed the outer organic coating of the shell and within about six hours that has been fully formed and calcified so what we're arguing is that saturation States sensitivity is really about the kinetics it's because this this rate of shell formation is very fast um so this is some work again by uh my my former grab student elizabth Runner and this is just showing our estimated calcification rates which are very conservative actually relative to the more recent sem work we've been doing but if we take the averages over days we can see there's about a really big drop in calcification rate from that early calcification uh of that first shell onward um and what that implies is that if we take that calcification rate and this is the the abiotic equation for calcium carbonate precipitation we take those calcification rates and solve for the rate constant needed under a decreasing saturation State what we see is that that rate constant increases exponentially and it increases much more rapidly at higher rates and so what we're clearly saying is that this isn't the equation that defines biocalcification but it's a constraint on the system that biology clearly overcomes but it's a physiochemical constraint on the on the biology that they have to contend with in some way so I guess the analogy I've thought of recently is that birds fly in spite of gravity but that doesn't mean it doesn't exist for them right so the physical forces are still there the physical chemical forces are still there biolog is very good at overcoming them but it's still there's a cost for doing this and that's why it becomes much we believe much more energetically expensive to calcify um during this early period when those calcification rates are high these rates are hundreds to thousand times greater than than abiotic precipitation rates it's clearly an acceleration that goes on during this period and throughout the life later life stages of V Valves and all other calcifiers so the egg reserves then provide that initial energy for the shell formation and so really the egg size say the Lal lunch bag I'm just going to move through this to keep things going but we see egg lipids are high they drop during that initial period they start feeding and then accumulate the again preparing for settlement so it's calcification is clearly biological mediated and requires energy there's proton pumping to maintain these uh uh the saturation state in these these calcification surfaces whether that's pumping protons out of hemocytes or out of an internal surface area or space where they're calcifying from and then there's also the organic Matrix which is the sort of scaffolding or the framework for the shell that they must produce which is actually the bigger the two costs which is exciting in the context of the findings of panad L showing that there's a big increase in organic in protein synthesis under OA stress so I wanted to show you a little evidence we have um for Less effort in the initial shell we tend to think about it in my group as the construction trailer when they make that initial shell and once they get that set then they can start building a better shell um and so here's a cross-section of a Lar shell um for my group and you'll see this initial shell layer is up here in the top we've done some transmission electron microscopy on this and as the beam resides across this entire section um what you'll see is that this this calcium carbonate is softer in this initial shell so this is the same exposure to electrons here but we see in that initial layer that's deposited that there's this beam damage is much greater and we see that consistently across the board we've also done some uh dis usion experiments on laral shell so these are not live larv we're putting into acid where we've cleaned the larv out of here and we're just looking at the sort of pitting and where the shell dissolves and basically what we see is that the mineral acid uh preferentially pits and dissolves that initial shell area the prison one and appears to be more dissolvable than other larel shell micro structures so we're going to really focus on the formation of this prison one shell which I've highlighted here in this electron m MRA from the Pacific oyster l so this is a shell that gets put down in that first really rapid period for the Pacific oysters and then show you some data from experiments we've been doing that to illustrate the saturation State uh importance of this at this stage so what I'm going to start showing you now is experimental data we've collected with our group the work we've done at the half field Marine Science Center um where we've decoupled the carbonate parameters understand larel V valve responses particularly at this sort of first stage going from an unshelled developing embryo to a shelled uh swimming straet hinge vager so this is just a couple examples of some of the experiments um I'm going to show you data from middle Gallop pralis is the Mediterranean muscle which is a incredibly important uh aquaculture species that's grown here and elsewhere and then crosio gigus again the Pacific oyster and is also non-native to the US Northwest W Coast um but also sort of the key oyster for the oyster industry on the west coast here so what we've basically done is manipulate the DIC and alkalinity to decouple the carbonate system parameters and so what I have plotted here on in the y- axis is the pco2 and on the x-axis is saturation state with respect to aragonite the gray circles and squares are uh different experiments and what these show are essentially our treatment values so we've done this four levels of CO2 four levels of saturation state with respect to aragonite in a factorial design and what you'll notice is that on these isop plats or pH and so what you can see is we can generate we can't decouple completely the three without a third um manipulating Factor there which becomes exorbitantly um big experiments to run these are already large uh very complicated experiments to put together but what you'll notice is that we can generate conditions where we have low CO2 and low saturation State high CO2 High saturation State and all sorts of combinations in between and then we also have conditions where both the CO2 and saturation State change but we have constant um pH on these isop plats in many cases so it allows us to essentially evaluate the role of these three different variables on the effects of the uh shell develop and some other physiological responses so just to give you quick background on the methods these are all closed bottle incubations for everything other than what I'll the last set of experiments I'll show you they're two-day uh incubations uh for the muscles and for the Pacific oysters we do about five days for the Olympia oysters that take longer to develop in that same period the uh we have 16 chemistry treatments we have three replicate bod bottles per treatment we do three counts per each bottle and then what we're doing primarily is measuring shell length uh we do triplicate counts for development and so we're scoring whether the shell is developed normally or not and we're measuring shell length of all the normal lar that we collect in those TOs and so we end up scoring about 100 larv per sub replicate which means about 300 larv per uh bod bottle um and so it's a and we have about 60 bod bottles including controls and so we have a a really very large sample size to sort of put together some of this data and I think the important thing I really want to point out here is that we're only measuring acute effects and so we're only measuring how this affects the ability for the larv to develop and grow in that first kind of Developmental perod so I'm going to show you series of plots that look very similar to this uh on this set here again we're going to focus on the Mediterranean muscle and the Pacific oyster I have on the y- axis for all these plots the proportion normal um and on the x-axis is on the top set is pco2 the middle scale is pH and the bottom one is saturation State these data are published in our nature climate change paper which came out in print in the March Edition and it's basically proportion normal so it's the number of uh larve that build a normal shell of two days relative to the total number of larve that we count in that sample and so what we find is that um at extremely high CO2 levels we see normal shell development uh on par with what we see at lower Lev levels up to over 3,000 micro atmospheres almost 3,500 micro atmospheres in some cases we get relatively good shell development uh pH I should point out that these shading are the essentially the the saturation State treatment so darker saturation darker symbols indicate lower saturation States and so you can see where this pattern where we're going with this but for both the muscles and the and the Pacific oysters we see in general there's very good development even down to pH is 7.4 saturation State still high the muscles appear to be maybe a little bit more sensitive to pH but again a lot of this has to do with the co-variance with the saturation State and here the the the picture becomes very clear in the response to uh saturation State we see very clear threshold response it's repeatable so each of these we've run these experiments twice for both species and uh and we see very very similar responses so so really for this early stage this early shell development saturation state is a primary variable of importance for these larv and there's really little to no impact that we can determine from pH until you get to these really low PH values and typically only when conditions are undersaturated with respect to the mineral so I'm going to show you data on shell length and so these are the basically measuring how big the shells of the normal LV are so we're excluding anything that's abnormal because we're trying to not bias the results and look at sort of okay okay if they can develop normally does it does it then not matter at all and so um just to pull these up we did these only on one of the two sets of experiments and what you'll notice again is we see at really high CO2 uh really no effect on shell length of the normal RV same thing with ph but it becomes very clear in this saturation State uh space that that's what's actually driving the response of shell length and so the decrease and saturation states make smaller normal RV and what this really indicates is there's a smaller scope for growth meaning the shell is more expensive to make or they're using the energy somewhere else uh during this initial shell building period you might look at this and notice these little Oddities so so for the high saturation state it actually looks like pH is having a negative effect or the CO2 seems to maybe have a slight positive effect those are probably real and that's probably related to Total DIC concentrations and so that's something that's been shown to be important in um Coral calcification and so increasing the amount of DIC seems to help uh them corals grow more quickly and it might be true here as well but again that is a really small effect relative to the saturation State effect that we document okay so we're going to move on and compare um muscles from uh Portland or from Oregon so muscles here with really nice wealth formed groomed beards and our uh Gallo provalis muscles which were actually collected or gathered from uh carlbad Aqua Farms down in carlbad in Southern California so they have very different exposure histories you know the chemistry down uh in this region of the West Coast is much more stable it's not subjected to the strong upwelling pressures we see in in the North Coast and so if we anticipate that exposure to uh these different environments might pre-adapt these organisms to some kind of sensitivity or make them more uh stronger than we would expect the californianus muscles that are found in Oregon to be more more resilient to um ocean C ification and in fact it's not what we see at all so these are the data from the previous experiment I showed you for proportion normal and also shell length for the Gallow provencial in white and gray and the californianus in black and you'll see there's really no discernable difference between their response is in proportion normal development and we we see essentially this very similar response between them with perhaps slightly higher growth rates at higher calc at higher saturation States for the Gallo Cialis so we're taking this indicat is that hanging out in OA Hotpot such as the Oregon coast where really strong upwelling doesn't seem to help California anus at least in terms of this early developmental stage okay so couple more slides here uh OA is a multiple stressor so there's been a lot of interest in thinking about multiple stressors uh in the environment and so what we've done is take those same experiments where we've looked at Shell development shell length and then also measured respiration rates and feeding a feeding metric for californ or the native middle here on the Oregon coast so similar plots again this is respiration rate on the y axis now pco2 pH and saturation State we had to do this A reduced set of treatments because it just became um logistically impossible to do this across the entire Suite so what we focused on was trying to get a few values in the mid PH range that have different saturation States um and then a high pH and a low ph and what we basically see is that pH is driving the show and this is not this is basically what we'd expect and so we can say and this is clear from other work as well that pH is actually affecting the respiration rate it's not saturation State and it's not CO2 so even in high CO2 rates if pH is low as it here there's really no discernable effect of CO2 and if saturation State even well undersaturated we see no effect on respiration rate it's only when we have that really low PH don't have any measurements between 78 and 7.4 so we don't know if this increases linearly we don't know if it steps makes a step function here but we do know that the CO2 or the pH is affecting respiration rate and then this is a feeding metric we'll call proportion feeding so this is at 44 hours post fertilization on larv that have been exposed to the same condition treatments and we're measuring with some techniques that Matthew Gray and Chine working out with using beads to look at the proportion feeding and so the strongest effect here is really due to CO2 we see potentially some slight effects of pH at the really high CO2 uh but generally speaking it's really looks appears to be um a CO2 effect on the number of organism number of lar that are feeding at this early stage so uh I just said all that so um then I'm just going to wrap it up here with the mighty Olympia oyster and so these are basically the same experiments uh as above and so I'm going to show you shell development and shell growth but we've extracted the brooding lar to measure their responses under the same developmental phase as the other organisms and so the difference is Olympia oysters brewed the larv for one to two weeks before their released and um so they were maintained within the maternal Brew chamber and so there's been very little work done on that really early stage and so some really great work that Matt Gray has done uh down at Hatfield worked out the techniques to be able to extract day old fertilized Olympia oyster embryos and develop them grow them normally and we find basically no effect of their uh on development and growth if they're outside the Brew chamber then when they're they're inside under kind of normal conditions and so and I'll show you some some controls for that as well in these data so basically what we see for the Olympia oysters is really almost no response to any of the variables um again these are the same graph CO2 pH and saturation State proportion normal here on this on this group on the left and shell on the right we see what might be uh look a little bit like a negative response as we get to these really high saturation States and we haven't really figured out what that is but we also ran control so we we have controls for all of our experiments where we keep them under kind of good water and we see that uh even with the bruted larv uh that we extract later at the end of the experiment that um there's really almost no difference between them so so this doesn't really mean that lur are impervious to O just this early developmental stage um and again I'd point you to the really nice work that's been done by an heter Jerel when she was down at you gavis on carryover effects in Lura from exposure and so this really got us thinking about what is it about Lura that gives them some trade or some resiliency to OA so I'm going to show you this and I I've showed you the sems previously but these are light micro microscope images and cross polarized light mic microscope images at 18 hours 24 hours so this is a different cohort than I showed you previously but it shows about the same 6-h hour window from where we first start to see some initial calcification which shows up as this glowing color all the way out to a fully calcified shell so this happens in about six hours for Lura this takes about 24 hours and so these are again light micrographs cross polarized light showing where the calcium carbonate is forming we know it forms starts at the Hing of the shell and extends outward and they get to be about fully calcified in 24 hours there these the concurrent sems and for this group in the same time frame you can see that sort of puffing up where they're starting to fill that shell out and then within about uh 24 hours from when that calcification is initiated it actually completes and they form that shell so um we've actually measured the calcification rates on the larv so we've counted out the larv and measured very carefully the the total calcium content and so these are the Pacific oysters uh during this early shell development period and the Olympia oysters here um and what we basically see is that the ltic calcification rate is about seven times slower during the same developmental stage and so although the timing is different this is a this is when that same pd1 shell uh formation period begins I'm not sure these are a couple measurements we had here there's a lot of error in these measurements and I'm not sure what how to interpret them quite yet but I've left them out of this regression analysis just for the sake of this presentation here so so what we've learned so far is that saturation state does matter to BU valve lve and if embryos can't develop then po then their population population impacts clearly if if the larv can't get past that early stage nothing else really matters later on um OA appears to have potentially to have multi-stressor type effects across B valve life history um but again that saturation state matters most first for that developmental period but I think this is really a key issue when we think about Cal uh coastal zone acidification because all these estuaries are going to respond differently with these different drivers and so although I I'm not advocating against the other multi-stressor thinking about oxygen and temperature alongside ins solinity um I think what we're sort of pointing towards is that we really just starting to truly understand the responses of some of these organisms in these environments uh Nativity to an O A Hotpot in itself does not convey resiliency and we saw that with the middle IDs um however slow shell development during pd1 appears to convey some resiliency and supports our idea of a kinetic hypothesis for this early Lal sensitivity um and again traits are what natural selection acts on and so the rate of shell formation during pd1 might be a critical one at this stage is what our argument is so what we're doing now to follow up some of this work is we're uh We've Now set up and beginning trials on a flowthrough system system that we can explore these same types of questions where we decouple the carbonate chemistry and we can look at how pH saturation State and CO2 affect the organism over the entire entire larval life history and track them out all the way uh until settlement so I've run a little bit long and so that's about all I got and thanks for listening and I guess I am now ready to take questions via the chat so okay thanks thank you very much George that was a a wonderful presentation uh so yeah we're going to um open the floor now for any questions from for for George and if you could type your questions or comments um into the questions box on the come the go-to webinar control panel on the right of your screen and we already have um at least one question uh from Libby JWT and she uh wants to know she she just makes a statement she says maybe U Middle is California and it's just hasn't adjusted to the new ocean yet or adapted to the new ocean if you reduce the habitat such that uh newly set spat are in perhaps acidic mud instead of up in the water column I presume that would also cost the fat energy for Shell formation how oh how are you able to parse out differences in feeding growth Etc I'm sorry that was another question okay well let me let me start with the first one from liy I guess we've always had an upwelling system here so they've always been exposed to elevated CO2 to some level and The Gallows are originally from the Mediterranean which has very stable CO2 and much higher alkalinity the Mediterranean muscles from these experiments from Carl's bat have been there for 20 years and they've been confirmed with genetic testing that there's no hybridization so they've been exposed to very different conditions clearly the conditions are getting worse um but if they've already been exposed and are exposed to upwelling conditions well before we're putting CO2 into the atmosphere so I guess we would anticipate that in that environment that they would have adapted in some way to that higher higher CO2 that's there um and I think so they in there was a clam question after that uh yeah um Evan asked how are you able to parse uh out differences in feeding growth Etc due to pH versus PC2 uh that's the whole th those are that's the whole point of the experiments and so let me try to go back here if you're whoever asked that's on the um that's Libby um but but basically we we essentially we do this by we we're manipulating the total Bic and alkalinity in experiments and so by changing the total amount of those and the ratio of those we can create these different conditions and so we have conditions where we have you can look across say on this diagonal here of 7.7 on the left graph that I'm pointing to now we have a constant pH but we have a CO2 that increases from 500 to well over 3,000 um and we can do that on any one of these isop plats for pH here and so it's the experimental manipulations and it's the full factorial design with four levels of each of the CO2 and aragonite saturation state that allows us to do that okay thank you here's another question um from Libby have you tried experiments Crossing uh OA and hypoxia yet no nope okay yeah we haven't and just because that would put another we like I said we have usually somewhere between 60 and 70 bod bottles and to do these counts each experiment we end up counting somewhere around 6,000 to 10,000 lar per experiment and so if we add another factor in there um but but certainly you'd expect some other effects there and so we you know we could in theory you could do this and put uh hypoxia on top of saturation State and um pH and CO2 and see what those effects are but it would it would it just starts to increase exponentially the number of containers in experimental size that we're doing and we already have a fairly large team but it's a great question because it we really need to look at whether or not the hypoxia effects are different when you tease apart these different carbonate system parameters okay uh Jim POI asked uh you've shown data on shell link normaly but do you have any data on laral Survival across the various carbonate system parameters um we take the development as a survival usually once they can't once they are malformed they usually don't survive that's it's a toxical assay that's been used for decades um and that's part of the point of the new experiments it's a great question it's part of the point of our our experiments we spinning up now for the longer term stud I to see how many actually make it and are able to metamorphose and settle and so I guess I'd anticipate that even those smaller ones even if they're normal that they're they'll make it they might very well could make it to settlement but often what the hatcheries have seen is they they will often see and we know from natural populations that there's often a lot of mortality that occurs during metamorphosis because it's a highly energetic period And if their energy stores are already hit because they been subjected to some of the stress um then we would anticipate that and I I believe some of the work by Chris gobler has shown that as well that there's you can see fairly good survival through the larel period um that metamorphosis tends to knock them out and I guess it's what I'm really curious about is whether or not that effect on the saturation state of the early development how much that becomes important later on so we would anticipate a sort of sublethal effect of pH during that Lal period and maybe the saturation state becomes a little less important and what we're hoping to do with these longer term experiments is really tease those apart okay uh Robert Brock uh wants to know what actions if any uh can be taken as a natural resource manager to reduce impacts of ocean acidification on shell bearing organisms um well I guess the first one is we reduce carbon emissions but we we I think we all know that that you know is is a is a challenging Prospect in itself there's a lot of great work going on looking at um mitigating effects and so the work that's been done in hatcheries at least with buffering but clearly we can't buffer the entire ocean or it'd be really difficult to do that um there's I think I think that there's some work that's just gotten funded up in Washington state looking at growing macro algae in association with some of the culture uh work oyster culture in the environment I mean I guess overall I would say that and we're doing some work with shells now to try to understand the role of sort of Shelly habitats those are often removed and are are highly diminish from what they once were and they provide a potential buffering Source in in uh Shelly environments but I guess ultimately you know we've done work thinking about policy issues and dealing with sort of the other drivers of U Coastal carbonate chemistry and so reducing nutrification but really getting out restoring the resiliency in coastal ecosystems and taking a whole wide holistic habitat of restoration and restoring habitats I think is really a crucial one thank you uh Matt says thanks George for your talk and Dick fely says excellent talk uh what do you think might be the impacts of on natural populations of oyers and muscles over the next 50 years um are these different species of lar and the water column oh that's another question sorry yeah um I it's a great question and I think we're just starting to get some ideas about how to get at that but um you know there's interesting differences in the in the in the reproductive biology between the the primarily aquaculture species the Pacific oyster and the native oyster here um and so you know I think it's going to take time to discern those differences um and we haven't really there hasn't been a lot of focus on the post LEL stages there's been some work clearly that's been done um and we're spinning up some work now as well to try to look at those exposure histories so I you know I would never say that all the oysters are going to die um clearly there's been recruitment issues in Willa Bay and I've been involved with some work that Burke HS has in review now um and this is just one of many factors in in all these Coastal environments that are going to impact the organisms and so I would say the the the responses that I'm showing here we've removed all the other potential effects that are going to occur in those environments including hypoxia different thermal regimes changes in food availability so these are kind of the best case scenarios I think of what organisms will how they will respond to OA um you know the other thing to point out too is that in all of our experiments even at some very low conditions we do get some LV that develop normally now those are generally much smaller than the other ones but there's potential there for breeding as well and some nice work um that Chris Lon do is doing as well as Dennis hedgcock in looking at sort of genetics and breeding uh is one way but again a lot of this is manipulated and we're talking about a very small number of species that have a high economic value and there's lots of species out there that we just simply don't either know what their responses are or there isn't the interest in them and so I think it's fair warning that you know we're going to see some impacts and there are likely some things that might do better than others but there's you know there's going to definitely be some losses of species if we don't address the emission issues um and Beth Turner uh wants to know uh are these different species in the um the water column let's see I'm sorry just yeah are these different species in the water column at the same time um might there be impacts on differential survival or growth that would lead uh to one species having an advantage over another yeah that's that's a great question I mean you know that there's a real for the oyster species that we've looked at so far that this pacifics and the Olympia oysters they have very different breeding strategies and they very different cues for what triggers reproduction we know much less about the Austria Olympia oysters Costa need a certain temperature to spawn and again that this work that's in review that BRK has led looking at will bay chemistry and temperature data you know seems to indicate that they're shrinking Windows of time when the good chemistry and the good thermal regime overlap now the difference with Austria is that they they tend to reproduce much earlier they don't need the warm water and they they tend to have Peaks um that sort of bracket to some degree the upwelling window they they will reproduce throughout the summer period of upwelling but if they're reproducing before upwelling really kicks in and they get the larvey through those early stages then they're have a they clearly have an advantage um um you know in terms of getting past those susceptible life history stages but again the challenge is you don't really have good time series data for a lot of these places to try to put together um the reproductive periods the LEL periods with um with when the chemistry and temperature really look good for these things so it's it's again it's a great question I think it's the next step for a lot of this work is to really understand that relationship between the variability the seasonal variability in the chemistry and the the reproductive timing for these different organisms because if they do time it differently then then they might find a way to get past those bad periods thank you Shan Newton uh asked do you think outplanting a limpiate oers is a useful adaptation strategy or is that premature yeah I mean there's there's great efforts going on in Oregon here already there's in earts Bay there's a native oyster restoration site and there Yin Bay as well some work with uh Oregon oyster and the Nature conserva has been involved with both of those efforts they've been putting out um Olympia oysters and I there's also been some work in kzb planning Olympia oysters and I think all many of those cases they're really for restoration and I think again many of the estuaries in Oregon and in Washington were once full of these Olympia oysters and so I I see no reason to not make efforts to restore that and once those populations are in place it gives us a better opportunity for for those organisms to rebound potentially um I you know I don't think we should give up hope and say it's all going to be bad so we should stop trying I think that's the absolutely wrong uh approach to take so I would strongly advocate for that and there is a small Market even for Olympia oysters um both Taylor Shellfish and Oregon oyster produce Olympia oysters for market so and and they're I I know Oregon oyster is heavily involved in some of those restoration efforts as well so okay great and uh this is the last question uh given the and it's uh asked by Matthew libal um given the importance of the maternal carve into the muscles or the maternal brooding for the oysters the health of the parent seems to have a big influence uh will sublethal stresses to the parents necessarily affect the larby or are there other traits working to help sick parents have healthy larby yeah it's a that's another great question and as we we do more work we can start to put some of the story together better there's been some work out of the Australian group led by Laura Parker where they've looked at um maternal effects and so what they found with Sydney rock oysters was some some uh I would say sort of weak effects anyway of exposure during the maternal phase seeming to convey a little bit of resiliency for the larv we've done some work here with pafic oysters which which haven't hasn't fallen in line with that so even short exposure windows for the um even short exposure windows for the um parents for Pacific oysters seem to result in poor development and growth for the larve and so it's certainly another aspect of this and those exposures that we did with those experiments were only two weeks long and so I think it's another component of this and the hatcheries have known this for a long time so they look at things like egg size and quality um but it's certainly another component of of this this problem but in general those adults are going to be more resilient I would say and I don't know that we have a good test yet of how important that is relative to the exposure and I think as until we start getting more integrative across the life history and doing longer experiments where we measure all these things uh it's hard to really put those things together and assign um importance or value to how or how much each of those are relevant to the organism that's a great question again there's lots of questions to be asked so yeah there are and in fact there are few that we're not going to be able to get to today but um what we will do George is we will send those to you and um then get you to answer those and then we will post those uh online with the uh presentation in the PDF so again thank you very much for a fantastic presentation thank you yes and uh so uh again if we didn't get to your questions we will um get those to George and have those um posted and uh if you are interested in joining the soan uh list serve you can find that um here on on this slide and um the webinar and the PDF will be posted um at the first link that you see there if you have any questions about soan um you can uh contact the link there uh in the middle and you can keep up with the latest happenings of soan um by joining the list serve which is the last link that you see here uh we'd like to thank you for attending the soan state of the science webinar today it was most informative uh we welcome any feedback or further questions or suggestions that you have for topics for this webinar series you could submit input by replying to the follow-up email that you'll be sending in a couple of days or by emailing us at one of these links that you see here uh the recording of This webinar and a PDF again will be available on the soan website that you can find um listed uh on the Press on listed here our next uh webinar will be uh two weeks from today on Tuesday April the 21st Chris chambers of Noah's Northeast Fisheries Science Center will discuss effects of elevated CO2 on early life stages of marine fishes and potential consequences of ocean acidification that will be again at 12: noon uh eastern time we hope that you'll join us thanks again George for this very informative presentation and to all of you uh for your thoughtful questions and for taking the time to join us today we hope to see you at Stan's next state of the science conversation this is the end of the webinar session