2/7/2024

Spent 2 hours inputting data for Grace Crandall.

Mercuric Chloride Disposal

1/8/24: Began emptying mercuric chloride into carboys and Sam helped me get started.

1/12/24: Made more strides with disposal but still was not finished.

1/23/24: Completed putting the waste into the carboys and but glass bottles in provided carboard waste boxes or in back in the boxes they were being stores in.

Chris Mantegna Data Input Complete

I have finished inputting all of Chri’s data for her mussel project.

Chris Mantegna Data Updates

10/5/23: Began Inputting Scans of Physical Data into the Google Sheet

10/18/23: Finished Inputting Measurement Data for all Individuals

After this was completed, I have been able to start inputting the Weight, Length, etc. data for all measured individuals.

11/2/23: Finished inputting all of Chirs’s data into the sheet and uploaded it to github. The data is now in csv file form and can be manipulated on github.

11/7/23: Started inputting additional data Chris provided me to the thickness section of the google sheet.

11/8/23: For a little bit this morning I transcribed more data into the thickness section.

11/28/23: I finished inputting all thickness data (up until individual 312). Next, I have to finish inputting the final morphometrics sheets up until individual 312 (currently I am on individual 220).

11/30/23: All of the mussel data input for Chris is complete. All morphometrics and thickness data has been input up to individual 312.

Link to mussel data:

https://docs.google.com/spreadsheets/d/1lIc47D1CZP6pt14n_RKokHp_sfRg1B3QpD-X9_kJtYY/edit#gid=885630881

Grace Crandall Data Updates

10/13/23: Picture Uploads

Using the binder of data Grace took over the summer, I took a picture of each individual sheet and uploaded them to the repository.

10/14/23: Begin putting data into excel

I began inputting the data from the experiments into one large datasheet on excel.

10/20/23: Transfer data to sheets and continue to input data

I had to switch over all the data I had put into excel to google sheets. After this was done, I continued to input the following data sheets into google sheets. I am still inputting data from the FHW experiment.

10/24/23: Finished inputting data up until sheet 6-54-22-ercf.

10/25/23: Finished inputting data up until sheet 7-74-22-ercf.

10/26/23: Finished inputting data up until sheet 8-52-22-ercf.

10/31/23: Finished inputting data up until sheet 10-9-22-ercf.

11/17/23: Finished inputting data up until sheet 11-59-hkcf.

Link to new sheets document for data input:

https://docs.google.com/spreadsheets/d/1GI1T-1ZhzvtqRf8o0Kq5C66q0jj7SuTnvZdMtOCF0ho/edit#gid=0

Project-Cod-Temperature Graphs

Week of August 7th-11th

This week is the week where we wrapped up our research and finished up the poster. We finalized the analysis of the final qPCR tests and formulated a complete and robust data set. Then, once this was complete, we began to fully piece together the poster. We put an abstract on the poster as well as a methods graphic and conclusions/takeaways section. To begin with I had a lot more text on the poster which made this a little too jumbled such as a a long introduction section. Fortunately, this was removed in favor of fount size and visuals. I also was able to present to my lab during our weekly lab meeting this week in order to begin to prepare for my presentation that will come on the 16th of August. All in all, this was a productive week that involved a lot of poster discussion and ended with a finalized product I feel comfortable with presenting/

July 31st – August 4th Summary

This is the week where I started to code and make all of the graphs with the data we gained from our PCR tests. I coded and made all of the graphs on r studio. The graphs I made were box plots that displayed the expressions values of all four treatments as well as the color coded the data points so one could tell between tumbled and non-tumbled. To add visual intrigue, I also color coded the graphs. I also performed so two-factor Anova tests to see whether or not our data was significant. It unfortunately was not despite the major differences we observed, and we assume this is the case due to the low number of replicates. Once the graph making was complete, I moved on to the poster at the end of the week.

Summary + Key Points of 3 Oyster Stress Articles

Article 1: Thompson Oyster CO2 paper

Key Findings: This paper aimed to find out if changes in ph which are currently taking place thanks to ocean acidification will affect oysters’ ability to handle general secondary stress such as heat or mechanical stress. This paper found that pH did not have a significant effect on oysters’ response to mechanical stress. However, this paper did find that the expression of HSP70 increases dramatically when oysters are exposed to acidic conditions. This paper explained how shock proteins fold and re-fold damaged proteins. Despite being labeled as a heat shock protein and generally being known to increase in expression when an organism is exposed to heat stress, HSP70 in this instance and others tends to be upregulated in a variety of stressful conditions and thus acts as a Biomarker for harmful environments. This paper also believes that the mechanical stress was simply not extreme enough in order to illicit a response since it did not move the needle in any way when it comes to the expression of the three genes tested. Another possibility could be that the mechanical stress became too in some instances and decreased general expression, but this does not necessarily line up with the controls.

Summary: This paper began with giving a brief overview of what ocean acidification is, why it i such a serious threat and the immediate implications of what a general decrease in ph means for our oceans. Then the paper goes on to explain what they are aiming to discover with their research. This study is trying to look at how adding an additional stressor to oysters exposed to an acidic environment will affect the expression of certain stress and disease genes. Since ocean acidification is already a significant stressor, this study wants to look at how oysters already under the stress of OA respond to an additional stressor that in theory they would also have to allocate resources to. The way in which this paper planned on carrying this test out is by exposing oysters to four different treatments. The first treatment is neutral normalized ph conditions and no mechanical stress, the second is the same conditions but with stress, the third treatment is no stress, but the oysters are exposed to acidic ph conditions due to elevated CO2 levels and the 4th is by default the same conditions but with stress. The three genes used in this study is HSP70, Metallothionein IV and Glutathione peroxidase. To measure expression the paper ran qPCR tests once the treatments were complete. The major finding of this paper was the uptick in the expression of HSP70 when exposed to acidic conditions. This paper recommends that future research to explore how stressors outside of mechanical stress as well as other conditions affect oyster immune response, reproduction and lifespan. It is also important to note that a similar study should be run again but instead with more extreme mechanical stress.

Article 2: Differential response to stress in Ostrea lurida as measured by gene expression

Key Findings: Due to high expression and the having the highest reproductive activity of the three areas, the Oyster Bay population is considered to be the best at handling stressors. There are a multitude of reasons for this that the paper mulls over. The main ones are that the Oyster Bay has high phenotypic plasticity meaning that the population is more apt to dealing with varying conditions. Another explanation could be that this population allocates less energy to reproduction and growth while the oyster bay oysters instead allocate more energy to maintenance.

Summary: Since the oyster population native to Puget Sound is decreasing, hatcheries are attempting to supplement this loss with their own oysters. However, it has been found that oyster populations located in different regions of Puget sound respond uniquely to stress, thus meaning that adding hatchery oysters may disrupt the unique strategies these mini populations have developed for their habitats. This means it is key to understand the phenotypic plasticity of each region. In order to find the answers to their questions, this paper took a sample of populations located in these three different locations: Oyster Bay, Fidalgo Bay and Dabob Bay. Then they put all these oysters into an isolated environment. They exposed these oysters to three different treatments: control, mechanical stress and heat stress. After this they ran qPCR tests on samples taken from the oysters. Some of the genes they targeted with these tests was Heat Shock Protein 70 or HSP70 which tends to be expressed during on organisms’ immune response, especially when the organism is exposed to heat. Another gene that the qPCR tests run this paper targeted was Actin which is a gene that regulates growth. Once these tests were complete the paper graphed and analyzed their results. The main takeaway the scientists had after analyzing their data was that the Oyster Bay oysters had the best and most efficient response to the stressors it was exposed to.

Article 3: Immune response and mechanical stress susceptibility in diseased
oysters, Crassostrea virginica

Key Findings: This study looked at how the P. Marinus infection affects how oysters are able to varying conditions and stress. It appears that that infected oysters have trouble performing apoptosis and therefore have increased numbers of apoptosis while non-infected oysters are able to more frequently engage in hemocyte phagocytosis. Uninfected oysters are also able to better handle mechanical stress and are able to better express stress genes in the presence of it.

Summary: This point of this study is to see how oysters handle mechanical stress when infected with the P. Marinus disease. The study used oysters from the east coast of the U.S called Crassostrea virginica. This disease causes hemocytes to infiltrate the connective tissue and the atrophy of the digestive tract. It also reduces shell growth. The tests that this study planned on performing included two groups: an infected and uninfected group. They would then have two subgroups within each of these two groups: one that is exposed to mechanical stress and a control group that is not. Once the initial tests are finished, the scientists will run PCR tests on 4 specific genes. These genes are CIAPIN, LP, HMGP, and CATL. Once these PCR tests were completed, they graphed the expression of these genes across all groups in order to fully analyze them. The main takeaway this paper had from its study is that uninfected oyster expressed the stress genes needed to handle mechanical stress better than the infected oysters.

Information on all Genes Targeted throughout the Project

HSP70:

HSP70 is a gene that generally codes for heat shock proteins. The main functions of the proteins HSP70 codes for are protecting cells from the effects of physiological stress and protein folding. Specifically, HSP70 proteins protects cells from heat or oxidative stress. HSP70 also frequently dispose of damage and defective cells. These stressors often lead to the unfolding of proteins and HSP70 is able to prevent that to some extent. For example, HSP70 has the ability to inhibit apoptosis. To summarize, HSP70 is stress gene that helps cells curb the negative effects of stressors such as heat and maintain their structure as well as their function.

HSP90:

HSP90 essentially has the same function as HSP70. They both are activated in response to stress and are key for protein remodeling as well as the prevention of degradation/unfolding. A few functions unique to HSP90 is the ability to DnaK system to further facilitate the process of remodeling. Also, the way HSP90 and HSP70 are named is based on how many kilodaltons the different proteins weigh.

GADPH:

GADPH is a gene that codes from an enzyme which catalyzes the 6th step of glycolysis. Despite mainly being associated with its function within the metabolic process, GADPH also frequently initiates transcription and induces apoptosis in cells. In or study GADPH is supposed to be a normalizing gene since its activity is typically constant across all individuals in spite of conditions.

Actin:

Actin is a gene that codes for proteins which form to make microfilaments in the cytoskeleton and thin filaments in muscle fibers. It is a structural protein for the cell. There are typically two types of filaments actin proteins are able to create: microfilaments and thin filaments. Since actins activity is also typically constant across organisms such as teh oyster, this gene was also meant to be a normalizing gene.

VIPERIN:

VIPERIN is a gene that code for proteins which inhibit viruses that attack DNA and RNA. It is stimulated by interferon which is a protein released in the presence of viruses. Specifically, VIPERIN is able to inhibit HCMV viral infection and down-regulate viral structural proteins.

18s:

18s codes for structural rRNA and makes up the small component of eukaryotic cytoplasmic ribosomes. This gene is frequently used as teh subject of PCR and can be found universally across organisms.

ATPsynthase:

ATPsynthase like its name suggests catalyzes the formation of ATP which is an energy storage molecule and is responsible providing the organism with a vast majority of its energy throughout the day. ATPsynthase uses ADP and inorganic phosphate to produce ATP. When an organism requires more energy, they tend to increase ATPsynthase gene activity. This would likely mean in our case that the organism was in a stressful environment which would lead to them to require more energy for maintenance.

Citratesynthase:

Citrate synthase is a gene that codes for an enzyme which acts as a pacemaker for the first step of the citric acid cycle. Citrate synthase takes acetyl-CoA and four-carbon oxaloacetate to create six-carbon citrate. The enzyme plays a key role in the citric acid cycle step of respiration. Occasionally this enzyme is used as an indicator of whether or not the mitochondrion within various cells is intact. Also, another aspect of this enzyme to keep in mind is that it can be inhibited by high ATP:ADP and NADH:NAD ratios, along with acetyl-CoA.

Defensin:

Defensin is a gene that codes for host defense peptides that attack bacteria, fungi and viral infections. They are part of the immune response and are produced by the innate immune system and epithelial cells. Defensin combats microbes in a variety of ways, such as the disruption of the microbial cell membrane.

IL-17_internal:

This gene codes for cystine knot cytokines which are produced by helper T cells and their production is typically stimulated by inflammation. IL-17 also tends to induce the productions of chemokines which recruit immune cells such as neutrophils. Generally, IL-17 is responsible for inducing and mediating proinflammatory responses and a higher level of expression of IL-17 would possible mean that a specific organism has been subject to more disease which may be the side effect of stress exposure.