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Shelly A. Trigg: Hi everyone, my name is Shelley trig and I'm a postdoc at in the School of aquatic and fisheries Sciences at the University of Washington.

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Shelly A. Trigg: And today I'm going to talk about a series of experiments that look at the tolerance of Pacific gooey duck to low pH.

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Shelly A. Trigg: And by, first I want to acknowledge the CO authors on this work, which include collaborators from University of Rhode Island. My advisor Stephen Roberts some others from University of Washington and Washington see grant and also the Jamestown school and point Whitney hatchery

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Shelly A. Trigg: And this work is funded by the US Department of Agriculture foundation for Food and Agriculture Research

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Shelly A. Trigg: So if you're not familiar with the Pacific gooey duck. This is a large burrowing clam that is found in the northeastern part of the Pacific Ocean.

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Shelly A. Trigg: from Alaska down through California and they are among the most valuable farm shellfish on a per acre basis, bringing in over $20 million in annual sales to Washington State alone.

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Shelly A. Trigg: Here is a picture of gooey duck seed, which are about three millimeters wide and these get planted out in these big flat shore areas. This is Puget Sound. And these are pieces of PVC pipe. So one seed goes in each of these tubes and then they are grown until they are full size.

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Shelly A. Trigg: And they come out of that they eventually you know borough below those tubes.

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Shelly A. Trigg: Buddha are also really important ecologically as they serve as bio filters and pre source for a variety of animals and they're also really important culturally, a lot of tribes have sustain themselves on Google Doc as food source. And as a revenue source.

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Shelly A. Trigg: And the question of Cal pH effects GUI de comes from how they might fair under oceans as certifications that are predicted for the future. And then also

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Shelly A. Trigg: From it, wondering if stress conditioning could perhaps be beneficial in agriculture. For instance, if adults are exposed to an environmental stress or controlled conditioning.

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Shelly A. Trigg: Say like low pH, could they perhaps pass on resilience to their offspring, or if juveniles are exposed to locate condition. Could they then

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Shelly A. Trigg: retain a resilience trade as as adults.

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Shelly A. Trigg: So to investigate those questions. We set up these different experiments to look at rootstock performance and reproductive development juvenile development and also carryover effects or in other words traits that are passed down from parents to offspring.

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Shelly A. Trigg: In this first experiment I looked at the impact of static low pH on rootstock and animals were conditioned and either ambient pH or low pH for 93 days.

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Shelly A. Trigg: And I found that the low pH negatively impacted survival.

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Shelly A. Trigg: And it delayed female reproductive development. So here we're looking at images of the gonads of the

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Shelly A. Trigg: This is just representative of the low pH group and the ambient group females. And you can see in the low pH group. There's a lot more pink, which is the connective tissue.

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Shelly A. Trigg: This white area is the follicles and then these purple dots are the eggs and you can see there are fewer eggs. They're a little bit smaller. There's much fewer follicles a lot more connective tissue compared to the ambient and now this is looking at

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Shelly A. Trigg: Quantitative data that was obtained from these images using program using image, J.

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Shelly A. Trigg: And you can see that this is that 72 days. So about here 72 days after the treatment started, there were smaller eggs and the low pH group and this

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Shelly A. Trigg: Changed over time they did mature the egg size did get bigger. However,

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Shelly A. Trigg: In the Ambien group we at the later time points at the end of the experiment we saw more of these animals had actually spawned and start resolving the eggs.

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Shelly A. Trigg: Where the low pH group was a little bit behind that. And interestingly, we did not see the same trend in males. We saw similar proportions of amateurs firm and matures firm.

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Shelly A. Trigg: At the 72 days after treatment and at the end of the experiments we did see that both of these groups continued to develop similarly

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Shelly A. Trigg: Now I'm going to begin talking about a different experiment that was done to look at

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Shelly A. Trigg: The effect of static pH on juvenile development and this is an experiment that was run by Holly Putnam, who's now at University of Rhode Island, but at the time was a postdoc and Stephen Roberts lab.

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Shelly A. Trigg: And she took three month old juveniles and expose them to either an ambient condition a moderate low pH or a very low pH for 23 days and after the 23 days the animals.

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Shelly A. Trigg: were reared in a common garden at an ambient condition and then they were split out again after 112 days in this common garden.

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Shelly A. Trigg: To either an Ambien condition or a low pH condition and this is to look at if there was any impact from this initial exposure on how they respond to a secondary exposure.

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Shelly A. Trigg: And she found that after the initial exposure.

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Shelly A. Trigg: Animals exposed to the very low pH tended to be smaller in size and the other true groups.

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Shelly A. Trigg: Now after 112 days she saw that the animals that were exposed to moderate or low pH initially were now much larger in size. Then they were. If they had only experienced ambient conditions.

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Shelly A. Trigg: And really interestingly

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Shelly A. Trigg: After the second pH exposure. She saw that ambient animals that had never seen the low pH before exhibited this growth big growth difference

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Shelly A. Trigg: Where animals that had only been exposed or that had been exposed to the moderate or the low pH did not experienced a growth difference they

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Shelly A. Trigg: did not experience any delay in growth. So, to try to investigate the molecular mechanisms underlying those growth phenotypes. We sequenced the gooey duck genome using 10 X genomics and phase genomics and we found that there were 18 scaffolds and 942 mega bases.

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Shelly A. Trigg: And about 35,000 genes.

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Shelly A. Trigg: And this is the general methylation landscape of the GU duck genome. And you can see these red bars are jeans and you can see that the blue, the methylation is concentrated within the jeans.

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Shelly A. Trigg: I performed a differentially method deleted region analysis.

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Shelly A. Trigg: Looking across these different time points. And here we're looking at heat maps where each row shows differentially method, a different

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Shelly A. Trigg: Region and their colored by their methylation, so more red means more math related and blue means less methylation.

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Shelly A. Trigg: And on the left side of these plots is the ambient group on the right side is the very low pH and in the middle is the moderate low pH. So you can see I just wanted to show that there are

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Shelly A. Trigg: Differences in the methylation across these different groups. This is after the common garden and this is 10 days after the secondary exposure.

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Shelly A. Trigg: And then I wanted to find out where these differentially method regions are located. If they tend to be located any specific genomic features and I found that, yes.

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Shelly A. Trigg: After 10 days they tended to be located in putative three prime UT are regions and

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Shelly A. Trigg: After the secondary exposure again putative three prime UT are regions and also in trance.

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Shelly A. Trigg: So just going to go over the summary of those results. The initial low pH exposure leads to increase growth after the common garden and protects against a slow growth delay exhibited by naive clams under re exposure.

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Shelly A. Trigg: And DNA methylation occurs primarily in jeans and low pH and do DNA methylation changes tend to be an interest and regions just downstream genes. And these are related to biological processes.

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Shelly A. Trigg: Of the situs skeleton activity which is related to the increase growth after the common garden and nucleic acid binding and mitochondria associated processes related to protection against slow growth exhibited by naive clamps under re exposure.

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Shelly A. Trigg: So now I'm going to talk about a different experiment. This is the impact of variable low pH on rootstock. So these are very similar conditions to the first experiment I showed you, only now it's looking at a variable treatment.

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Shelly A. Trigg: And you can see that the variable low pH exposure did not affect brutes doc survival.

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Shelly A. Trigg: And to follow up sort of on the performance of these animals. We looked, I looked at their the effect of the variable low pH treatment for the parents on their larval offspring.

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Shelly A. Trigg: So this is work that was done by Sam girl who is a grad student at University of Rhode Island.

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Shelly A. Trigg: And we're looking at size of the larval offspring over time. And in general, you can see there's very little impact from the parental conditioning on the larval growth. So the blue indicates animals that had ambient conditions animals were the yellow indicates animals that had

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Shelly A. Trigg: Variable low pH conditioned animals. And there are a couple time points where there's some size difference. But in general, they're pretty similar in their development.

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Shelly A. Trigg: And interestingly, Sam also measured metabolic rate over time. And he found that in general animals that had parents that were exposed to variable low pH tended to have a lower metabolic rate over time.

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Shelly A. Trigg: Consistently over time and

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Shelly A. Trigg: So like, even though the animals were all similar in size. They had this lower metabolic rate.

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Shelly A. Trigg: I followed this experiment up looking at the animals as juveniles. So those same animals were reared until juvenile stage. And then I expose them to the similar conditions that their parents were exposed to.

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Shelly A. Trigg: And I found at the end of the experiment, there was a difference in survival but it

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Shelly A. Trigg: Was regardless of the parental conditioning. So the parental conditioning did not help the animals survive better if they were exposed

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Shelly A. Trigg: Too low pH, which is the pink bars. This is the parental exposure here. This is the juvenile exposures. The color if they were exposed to the variable a PhD just kind of had low survival in general.

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Shelly A. Trigg: And there is a marginal effect, but of

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Shelly A. Trigg: The parental exposure, but it was not significant.

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Shelly A. Trigg: So then I also looked at the end point size distribution and I found an interactive effect of parental conditioning and juvenile exposure and that combination of low pH condition parents and low pH exposed juveniles actually were larger in size.

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Shelly A. Trigg: And also measured metabolic rate over time. And in general, I saw that

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Shelly A. Trigg: Parental variable low pH exposure led to lower metabolic rate in the juveniles.

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Shelly A. Trigg: So now I'm just going to summarize all these experiments.

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Shelly A. Trigg: I found that longer duration of the pH stress and the constant low pH is more detrimental to survival than the variable pH at a shorter time

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Shelly A. Trigg: And also that female reproductive developments is more sensitive to the low pH then males.

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Shelly A. Trigg: In regards to juvenile development. We found evidence for an environmental memory and that the initial low pH exposure led to larger animals, four months after they saw the low pH and this was associated with methylation changes and jeans related to cite a skeleton activity.

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Shelly A. Trigg: And the initial low pH exposure also prevented a growth delay that was seen in naive juveniles and this was associated with methylation changes and jeans related to nucleic acid binding and mitochondrial activity.

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Shelly A. Trigg: In terms of carryover effects.

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Shelly A. Trigg: I found that parental variable low pH conditioning leads to reduced energy demand an offspring that achieves the same or larger size under low pH conditions. So overall, it seems that

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Shelly A. Trigg: Variable low pH exposure could be beneficial to animals in the long run.

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Shelly A. Trigg: There needs to be some more. Follow, follow up experiments in terms of optimizing these conditions, we definitely went with a lower

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Shelly A. Trigg: A pretty extreme low pH and there. It's possible that finding something that's a little more moderate may still lead to these beneficial effects.

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Shelly A. Trigg: And overall, there

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Shelly A. Trigg: Seems to be different. Yeah, there are different things happening at different stages in the animals lives.

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Shelly A. Trigg: So thank you for tuning in. I hope you enjoyed the talk and please feel free to follow up.

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Shelly A. Trigg: With any questions. Here's my my email is a string at University of washington.edu. Thank you so much.