4 23 2015 Heat/Mechanical Shock 24 Hour Time Point

Today I completed the 24 hour time point collection and the collection of the controls for the experiment. Tonight I ran out of liquid nitrogen before doing the control groups. I put them on ice and placed them in the -80 ASAP.

Heat Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml screw cap tube dropped into liquid nitrogen -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Mechanical Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml tube dropped into liquid nitrogen - then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Controls

For controls sample 8 from each group, this should be done on day 2 as less things are going on. Protocol is the same. These oyster should have remained in holding tank-throughout and should simply be removed and sampled. There is no treatment. Using sterile techniqes.

• ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
• 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
• mantle sample (50-100 mg) in 1.5 ml tube dropped into liquid nitrogen then to -80°C
• remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
• shells placed in labeled plastic bags so correspond to sample including oyster id

4 22 2015 Heat/Mechanical Shock Experiment

Today I ran the Heat and Mechanical Shock experiment on my oysters. While most groups got liquid nitrogen treatment, the mechanical H and S groups did not due to lack of Liquid N2. For those samples I placed them on ice and moved them to the -80 as soon as possible.  The procedure went as follows.

Pre-Experiment

Oysters

• 8°C
• fed daily with 5-10 ml per tank commercial shellfish diet (Isochrysis 1800 mix Marine Algae)
• Partial water changes once per week (remove 5 gallons, replace with fresh 5 gallons)
  • Seawater from Seattle Aquarium
• Aeration with aquarium aerator/stone
• circulation with underwater pump
• Tanks 10 gallon plastic storage tubs (costco storage tubs)

Homogenization tubes for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Collection 1.5 ml tubes (screw cap) for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

Collection 1.5 ml tubes (screw cap) for mantle prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

15 ml conical tubes for whole oyster prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Warm fresh seawater in 500 ml beaker to 38°C by immersing it in a water bath at 38°C.

Experiment

Heat Stress

• 22 oysters each pop in a mesh bag were lowered (stagger each pop by 1 hour) into 500 ml of prewarmed 38°C sea water (1 hr timer started)
  
  • N pop first, H pop next, S pop last
• after 1 hour exposure heat stress ends, animals returned to 8°C water.
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id

Mechanical Stress

• 22 oysters from 1 pop placed in bucket in centrifuge and spun for 5 minutes at 1000 rpm.  Samples staggered by 1 hour. Completed after all heat shock treatments and samples were taken.
  • N pop first, H pop next, S pop last
• after 5 minutes of mechanical stress, animals returned to 8°C water
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id

4 1 2015 EZNA Dabob pt 2 and Oyster pt 3

Today I continued the seed DNA isolation. I completed the 32 Dabob Seeds and the 32 Oyster Bay Seeds as appropriate. You can read about the previous Dabob isolation here. You can read about the other Oyster bay Isolations here and here.

Before using the EZNA Kit I dissected out whole body tissue from 14 seed oysters into the homogenization tube. The oysters are labeled SN 31-32 and HL 21-32. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

1. Added 350 ul ML1 Buffer
2. Added 25 ul Proteinase K solution
3. Used pestle in homogenization tube to grind tissue in solution
4. Vortexed
5. Incubated at 60 C for 30 minutes
6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
7. Vortexed
8. Centrifuged 10,000 g for 2 minutes
9. Transferred the upper aqueous phase to new tube (~300 ul)
10. Added 300 ul MBL Buffer
11. Added 10 ul RNase A
12. Vortexed for 15 seconds
13. Incubated at 70C (started at 67.5 C) for 10 minutes
14. Cooled to room temperature sitting for 5 minutes
15. Added 300 ul 100% EtOH
16. Vortexed for 15 seconds
17. Put spin column in collection tube
18. Added 750 ul sample solution to column
19. Centrifuged at 10,000 g for 1 minute at 4C
20. Discarded flowthrough
21. Repeated 18-20 with remaining sample
22. Discarded collection tube and replaced with a new one. 
23. Added 500 ul HBC solution. 
24. Centrifuged at 10,000 g for 30 seconds at 4C
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute at 4C
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g at 4C
31. Discarded collection tube and put column into microcentrifuge tube for sample collection
32. Added 100 ul preheated 70C Elution Buffer
33. Incubated for 2 minutes
34. Centrifuged at 10,000 g for 1 minute at 4C
35. Repeated 32-34. 
36. Stored DNA at -20 C

DNA is in a box labelled:

Seed Oly DNA HL 

3/23/15 Jake Heare

Seed Oly DNA SN 

3/31/15 JH 2 of 2

3 31 2015 EZNA with Oyster Bay oysters Pt. 2

Today started using the new EZNA 200 reaction kit we received yesterday. Before beginning I added Isopropanol to the HBC buffer and 100% EtOH to one bottle of DNA Wash buffer. Then I continued the procedure as normal. These samples are related to the samples I extracted yesterday.

Before using the EZNA Kit I dissected out whole body tissue from 24 seed oysters into the homogenization tube. The oysters are labeled SN 7-30. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

1. Added 350 ul ML1 Buffer
2. Added 25 ul Proteinase K solution
3. Used pestle in homogenization tube to grind tissue in solution
4. Vortexed
5. Incubated at 60 C for 30 minutes
6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
7. Vortexed
8. Centrifuged 10,000 g for 2 minutes
9. Transferred the upper aqueous phase to new tube (~300 ul)
10. Added 300 ul MBL Buffer
11. Added 10 ul RNase A
12. Vortexed for 15 seconds
13. Incubated at 70C (started at 67.5 C) for 10 minutes
14. Cooled to room temperature sitting for 5 minutes
15. Added 300 ul 100% EtOH
16. Vortexed for 15 seconds
17. Put spin column in collection tube
18. Added 750 ul sample solution to column
19. Centrifuged at 10,000 g for 1 minute at 4C
20. Discarded flowthrough
21. Repeated 18-20 with remaining sample
22. Discarded collection tube and replaced with a new one. 
23. Added 500 ul HBC solution. 
24. Centrifuged at 10,000 g for 30 seconds at 4C
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute at 4C
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g at 4C
31. Discarded collection tube and put column into microcentrifuge tube for sample collection
32. Added 100 ul preheated 70C Elution Buffer
33. Incubated for 2 minutes
34. Centrifuged at 10,000 g for 1 minute at 4C
35. Repeated 32-34. 
36. Stored DNA at -20 C

DNA is in a box labelled:

Seed Oly DNA SN 

3/31/15 JH 2 of 2

3 30 2015 EZNA with Oyster Bay

Last week I started isolating DNA from seed oyster from Dabob and Fidalgo. Yesterday the new kit didn't come in until later so I started with the remaining six reactions from the previous kit. I got busy and forgot to post about it so here it is. The samples are labelled SN 1-6.

Before using the EZNA Kit I dissected out whole body tissue from 6 seed oysters into the homogenization tube. The oysters are labeled SN 1-6. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

1. Added 350 ul ML1 Buffer
2. Added 25 ul Proteinase K solution
3. Used pestle in homogenization tube to grind tissue in solution
4. Vortexed
5. Incubated at 60 C for 30 minutes
6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
7. Vortexed
8. Centrifuged 10,000 g for 2 minutes
9. Transferred the upper aqueous phase to new tube (~300 ul)
10. Added 300 ul MBL Buffer
11. Added 10 ul RNase A
12. Vortexed for 15 seconds
13. Incubated at 70C (started at 67.5 C) for 10 minutes
14. Cooled to room temperature sitting for 5 minutes
15. Added 300 ul 100% EtOH
16. Vortexed for 15 seconds
17. Put spin column in collection tube
18. Added 750 ul sample solution to column
19. Centrifuged at 10,000 g for 1 minute 
20. Discarded flowthrough
21. Repeated 18-20 with remaining sample
22. Discarded collection tube and replaced with a new one. 
23. Added 500 ul HBC solution. 
24. Centrifuged at 10,000 g for 30 seconds 
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute 
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g 
31. Discarded collection tube and put column into microcentrifuge tube for sample collection
32. Added 100 ul preheated 70C Elution Buffer
33. Incubated for 2 minutes
34. Centrifuged at 10,000 g for 1 minute 
1. Internal lid in centrifuge failed which resulted in tube caps being sheared off. 
2. Pipetted sample in collection tube to new tube
3. Moved the spin columns to the new tubes
35. Repeated as normal 32-34. 
1. Under calculated amount of Elution buffer needed to be preheated. 
2. SN 4, 5, and 6 had cold Elution buffer added instead
36. Stored DNA at -20 C

2 13 2015 oyster bay sample move

Oyster Bay Wa
1030 am to 1 pm
Mid 50s foggy.

Moved the samples today from the calm fresh dock to the taylor mussel raft. 

Checked the samples quickly for mortality. Low to no mortality in each container. All animal look good and healthy. Fidalgo animals look much larger than the other two pops. They are also super dense. They feel like they weigh 50-75 grams. Compared to oyster and dabob which feel between 30-50 grams. 

Due to time constraints for the taylor boat, I was not able to image any populations for size. I pulled the logger data on both tags.  Samples were hung on the taylor rafts with bright orange tags with contact info.

Finally the new dock at crab fresh will be completed by the end of march. I plan to move the samples back from the taylor raft in mid april.

10 30 2014 Dual Y Axis Graph (ggplot2) % Brooding vs Temperature Oyster Bay

Here I'm showing how to produce a dual Y axis graph with ggplot2 package in R. If you've tried to do this before you know it downright impossible. I found a great work around in the rpubs from a user named Hadley. Below is my code to produce a dual axis bar and line graph. I'm doing this to show the percent of animals brooding at each time point along with the associated temperature at that time point. 

Broodperctemp.R

Jake H

Thu Oct 30 13:23:08 2014

require(plyr)

## Loading required package: plyr

require(ggplot2)

## Loading required package: ggplot2

require(scales)

## Loading required package: scales

require(grid)

## Loading required package: grid

require(gtable)

## Loading required package: gtable

grid.newpage()
p1<-ggplot(data=oysbay, aes(x=Date, weight=Percent, colour=Pop, fill=Pop))+
  geom_bar(binwidth=10, position=position_dodge())+
  theme(axis.text.x=element_text(angle=90, size=10, vjust=0.5))+
  scale_colour_manual(values=c("blue","purple","orange"),labels=c("Dabob","Fidalgo","Oyster Bay"))+
  scale_fill_manual(values=c("blue","purple","orange"),labels=c("Dabob","Fidalgo","Oyster Bay"))+
  labs(x="Sample Date", y="Percent Brooding",title="Percent Brooding vs. Temperature")+
  theme_bw()+
  theme(legend.justification=c(0,1),
        legend.position=c(0,1),
        plot.title=element_text(size=30,vjust=1),
        axis.text.x=element_text(size=20),
        axis.text.y=element_text(size=20),
        axis.title.x=element_text(size=20),
        axis.title.y=element_text(size=20))
oysy1edit<-read.csv("TempData/oysY1fixed.csv")
oysy1edit$Date<-as.Date(oysy1edit$Date, "%m/%d/%Y")
oysmintemp<-ddply(oysy1edit,.(Date),summarise,min_temp=min(Temp,na.rm=T))
oysmintemprep<-subset(oysmintemp, Date>="2014-05-01"& Date<="2014-08-07")
p2<-ggplot()+geom_line(data=oysmintemprep,
            aes(x=Date, y=min_temp), color="red")+
  geom_hline(yintercept=12.5, color="forestgreen")+
  theme_bw() %+replace% 
  theme(panel.background = element_rect(fill = NA),
        panel.grid.major.x=element_blank(),
        panel.grid.minor.x=element_blank(),
        panel.grid.major.y=element_blank(),
        panel.grid.minor.y=element_blank(),
        axis.text.y=element_text(size=20,color="red"),
        axis.title.y=element_text(size=20))

g1<-ggplot_gtable(ggplot_build(p1))
g2<-ggplot_gtable(ggplot_build(p2))

pp<-c(subset(g1$layout,name=="panel",se=t:r))
g<-gtable_add_grob(g1, g2$grobs[[which(g2$layout$name=="panel")]],pp$t,pp$l,pp$b,pp$l)

ia<-which(g2$layout$name=="axis-l")
ga <- g2$grobs[[ia]]
ax <- ga$children[[2]]
ax$widths <- rev(ax$widths)
ax$grobs <- rev(ax$grobs)
ax$grobs[[1]]$x <- ax$grobs[[1]]$x - unit(1, "npc") + unit(0.15, "cm")
g <- gtable_add_cols(g, g2$widths[g2$layout[ia, ]$l], length(g$widths) - 1)
g <- gtable_add_grob(g, ax, pp$t, length(g$widths) - 1, pp$b)

grid.draw(g)

It's really important to note that I have subset the temperature data for only the daily minimums and  only from the date of the first sampling to the date of the last sampling. If you don't do this, the temp graph will be fit onto the graph regardless of whether it correlates to the dates on the x axis. My first version was really screwy because of this. Also note that for some reason when using this method you cannot create a legend for the line graph nor can you generate a title for the right hand y axis. It's not the best but it will make a good looking graph for you if you know these limitations.