5 24 2014 fidalgo repro

Anacortes, WA

Temps mid 50 to 60
Light rain

Participants L. Christine savolainen and Jake heare

Checked the third stack for reproductive activities using Anesthesia SOP. Also checked the first stack to see if there were any mortality events. The first stack was completely fine with minimal mortalities.

The third stack had no signs of brooding.

Numbers for pops as follows

2H9-12
Brood. 0
Gaping. 24
Dead 0

2N13-16
Brood. 0
Gaping 53
Dead 0

2S1-4
Brood. 0
Gaping. 77
Dead. 0

#priorities

5 23 2014 oyster bay emergency sampling

Oyster bay, WA

Mid 60s 70s

Participants Steven Roberts, Brent vadopalas, and Jake heare

We came back to oyster bay today to check if mortality occurred across the board.

Upon inspection of the three remaining stacks. There was no indication that the mortality event was spread to the other trays. It seems that a mix of extended exposure, high temperatures, and the treatment caused a mass mortality event. Dabob was the first group to be treated and thus did not experience the same duration of high heat and low oxygen that the north and south sound trays did. We decided to same from the affected trays anyway.  We collected 8 from the north sound tray, 4 from the south sound tray, and 10 from the dabob tray. We also individually bagged the shells from the dead oyster in hopes of using them for future data efforts.

On a side note, there appeared to be 2 dabob oysters that had recently spawned as evidenced by the post gonadal canals. Since they showed no signs of brooding it is assumed they spawned as male and may spawn again in the near future as female.

We also did a semi brood check on the other full set of trays and found no brooders.

Shells were placed on dry ice, tissue samples stored in rna later. Will be transferred to cold storage in the near future.

Will continue repro check at fidalgo tomorrow.

Enjoy this selfie of Brent and I in the car as I write reports.

5 22 2014 oyster bay repro

Oyster bay, WA

High mid 70s.

Participants Katie Jackson and Jake heare.

Tested for reproductive active today. Very bad news.

There has been a huge mortality event at oyster bay. Numbers for brooding, gaping, dead, and alive but not open are as follows.

1n13-16
Brood. 0
Gaping. 3
Dead 93
Alive 5

1s5-8
Brood 0
Gaping 2
Dead 68
Alive 4

1h5-8
Brood 0
Gaping 47
Dead  51
Alive. 9

Clearly dabob has survived whatever mortality event much better than north and south sounds populations. We will be sampling this site tomorrow to ensure fresh samples.

5 21 2014 Manchester repro

Did a brood check today at manchester. Found no brooders. 

Numbers as follows. 

4S5-8

Brood. 0

Gaping. 76

Dead.  3

4H13-16

Brood. 0

Gaping. 16

Dead.  2

4N13-16

Brood. 0

Gaping.  40

Dead.  3

Enjoy this picture of an eagle.

Capsized boat at Fidalgo Marina

Apparently a boat capsized roughly 500 feet from the samples at Fidalgo Bay. Depending on what leaked into the water, the size of the wake and other disturbances caused by rescue efforts. This might cause some issues with the animals. You can watch a time lapse video of the recovery effort here.

5-14 to 15-16 2014 Reproduction Work Up

5-14-2014

Manchester WA

Participants: Brent Vadopalas and Jake Heare

We retrieved the next set of trays in the dosing sequence and allowed each tray to dessicate for 45 minutes. Trays were then treated in a 10 gallon bath of 50% sea water to 50% freshwater mixed with 7 lbs of epsom salt. The tray was treated for 45 minutes, at which point it was removed and gaping animals were examined for signs of brooding. After examination the trays were then placed into a recovery tub with 100% sea water until the last tray examined had be in the recovery tub for 45 minutes. After each treatment the treatment water was replaced with fresh treatment to reduce temperature flux.  Then trays were rebuilt into a stack and hung off the dock. 

We counted the number of gaping animals and brooders for each tray. 

They are as follows:

4H1-4

brooders 0

Gaping 25

Closed 72
% Open  25.8%

4S9-12

Brooders 0

Gaping  43

Closed  55
% Open  43.9%

4N9-12

Brooders 0

Gaping 31

Closed  28
% Open 52.5%

5-15-2014

Oyster Bay WA

Participants: Katie Jackson and Jake Heare

We followed a procedure similar to that at Manchester. The difference being that the treatment water was not replaced after each treatment. We also counted the dead in each tray.

1H1-4

Brooders 0

Gaping    49

Dead      7

Closed   33
% Open  59.8%

1N5-8

Brooders 0

Gaping    46

Dead      7

Closed  14
% Open  76.7%

1S13-16

Brooders 0

Gaping     59

Dead        8

Closed    26
% Open  69.4%

5-16-2014

Fidalgo WA

Participants: Steven Roberts and Jake Heare

Same procedure as at Oyster Bay. 

2N1-4

Brood 0

Open 53

Dead  0

Closed  46
% Open  53.5%

2S13-16

Brood 0

Gaping  55

Dead   0

Closed 39
% Open  58.5%

2H5-8

Brood 0

Gaping 48

Dead  0
Closed  52
% Open   48%

DNA Extraction Attempt 2 5-12-14

Attempting to extract DNA from 5-10 mm whole oysters minus the shell. This is needed so that sequencing can occur in the future with completed extractions. Unlike the last attempt I will be switching out 95% EtOH for Isopropanol to get better extraction results.

Protocol is as follows:

Incubation for 24 hours at room temp:

1	ml	DNAzol
2.35	ul	Proteinase K (100ug/ml)
1	whole	oyster 5-10 mm
X5		Reactions

1. Pipette 1 ul DNAzol and 2.35 ul Proteinase K in tube containing oyster
2. Homogenize each oyster with disposable pestle.
3. Centrifuge briefly
4. Incubate overnight at ROOM TEMP.

Isolation:

500	ul	Isopropanol
1	ml	75% EtOH
1	ml	75% EtOH
200	ul	Sterile H2O

1. Centrifuge @ 10,000 g for 10 minutes.
2. Collect and Transfer Supernant; Discard pellet
3. Add 500 ul Isopropanol
4. Mix through inversion
5. Incubate for 1 minute at ROOM TEMP.
6. Spin down DNA precipitate
7. Remove supernant
8. Wash with 1 ml 75% EtOH
9. Spin down
10. Remove supernant
11. Wash with 1 ml 75% EtOH
12. Spin down until solid pellet
13. Elute with 200 ul Sterile H2O
14. Quant.

Spawning Oly Predictions

After looking through the new temp data I pulled off the temp loggers yesterday I notice a few trends that have lead to some predictions as to which site will spawn first.

Oyster Bay

Temps are trending upward to the 12.5 C mark that the animals require to begin spawning. It seems that they will have hit that temperature this week and will possibly begin spawning next week. The forecast for next week is also encouraging with the daily ambient air temps to be 75 or higher. This will bring these animals to fruitition. The only problem being that it will be during a spring tide which is not good for spawning. 

If they don't spawn next week it will occur during the following neap tide during the second to last week in May.

Manchester

While these animals have experienced a warming trend, it seems as in recent weeks there has been a stalling of the warm temps. Water temperatures need to hit the 12.5 C mark and maintain them for several days to iniate spawning. It looks like it might be a slow start at Manchester or even a possible abortive spawn if the temps don't stay high enough long enough. I expect these guys to spawn in the last week of May, first week in June. 

Fidalgo

Temps here are colder than elsewhere but are experiencing consistent increases throughout time. It will probably be the last week of May when they spawn. 

5-8-2014 Anesthetization Issues

Dr. Roberts recently asked me why I was getting such a low response rate from the treatments. I was seeing between 5 and 50% of the animals become anesthetized. Monitoring the situation I have a few ideas on why. Which I stated in a response to him in a message which is copied below.

I think it was a combination of the warm ambient air temps that caused an increase in treatment temperature which led to animals not opening in the treatment.

I also checked my treatment calculations and the epsom salt concentration was about half. I calculated it fo 5 gallons instead of the full ten. I'll just double the amount of epsom salt to make it the proper concentration. Hopefully it will increase response from the animals.

Brent has also suggested doing sub samples instead of dosing the whole tray to limit exposure by all the animals and reduce the amount of epsom salt we are using. I think its better to dose the whole tray because I can't guarantee that the percentage of animals that open will stay the same. On top of this variable percentage, there will only be 10-20% of the animals brooding at any given time. If we don't treat enough animals we will be more likely to miss spawning effort. 

Brent also had some useful suggestions about previous work with the treatments as well as some recalculations of the treatment solution to increase treatment concentration.

Katie consistently saw about 30% response (without dessicating them first. I don't have her data on dessication, but response rate was higher).  If we stick with the 5% threshold to see brooders and a 30% response,  you'd need to treat 67 oysters to = 1.  If the response with dessication is closer to 60%, you can decrease the subsample by half.  
It sounds like the oysters may have been too warm to respond--important to bring a thermometer, and probably some ice packs to control treatment temp.  
85g/L = ~7 lbs of MgSO4 for 10 gallons.

It can be argued that we only treat half the tray at any given time to limit downstream effects from the treatment which may affect spawning including abortive spawns or gonadal regression. The problem with this is that too see any signs of spawning we would be looking for 1-2 animals in any group to be brooding. I don't think this is a reliable way to determine if the populations are spawning. We would be better served in treating the whole tray to avoid mixups and hopefully have a higher number of brooding animals to strongly suggest that they animals have spawned.

Fidalgo Bay 5-2-14

5-2-14

Anacortes WA
Fidalgo Bay
Start Time: 9:30 AM
Finish Time: 5:00 PM
Temps: Mid 60s to Mid 70s

Participants: Jake Heare

I arrived earlier in the morning. I pulled up the hanging devices and broke them down into individual trays. I collected dead, counted live, and photographed trays for size/growth for later analysis. Upon completing these metrics the trays were reorganized into stacks containing one tray from each population at a random level in stack (top, middle, bottom). 3 stacks were then rebuilt and hung off the docks.


The 4th stack was treated with an epsom salt mixture to anesthetize the animals for brooding larvae inspection. One tray was dosed at a time in a 10 gallon mixture of 50/50 sea water/freshwater with 6-8 cups of epsom salt in them (roughly 75-80 g/L). Gaping oysters in each tray were individually inspected for brooding larvae. Once inspection was completed, trays were then transferred to a tub filled with 10-15 gallons of fresh seawater to recover from treatment for 30 minutes to 1 hour depending on recovery rate. In the first tray we had 25 animals open up, in the second we had 40 animals open, in the third there were 30 animals. None of these animals showed any signs of brooding larvae. 

Temperatures logged at Fidalgo Bay from Mar 1 to May 2, 2014. Temps logged every 15 minutes. 

Fidalgo	2N1-4	2N5-8	2N9-12	2N13-16	Total
Live	99	100	96	110	405
	2H1-4	2H5-8	2H9-12	2H13-16	Total
	91	100	83	94	368
	2S1-4	2S5-8	2S9-12	2S13-16	Total
	92	104	93	94	383

Calm Day at Fidalgo

Sea Otter hangin out by the dock

Sea Otter Hunting for Prey

Sea Otter after capturing large red rock crab

Sea Otter Eating Large Eel.

Oyster Bay 5-1-14

5-1-14

Shelton WA
Oyster Bay/Totten Inlet
Start Time: 9:30 AM
Finish Time: 5:30 PM
Temps: Mid 60s to Mid 80s

Participants: Jake Heare

I arrived earlier in the morning. I pulled up the hanging devices and broke them down into individual trays. I collected dead, counted live, and photographed trays for size/growth for later analysis. Upon completing these metrics the trays were reorganized into stacks containing one tray from each population at a random level in stack (top, middle, bottom). Since one stack is missing we were unable to completely reorganize the trays as such. One hanging tray has 2 Hood Canal Pops and 1 North Sound pop tray. 2 stacks were then rebuilt and hung off the docks.


The 3rd stack was treated with an epsom salt mixture to anesthetize the animals for brooding larvae inspection. One tray was dosed at a time in a 10 gallon mixture of 50/50 sea water/freshwater with 6-8 cups of epsom salt in them (roughly 75-80 g/L). Gaping oysters in each tray were individually inspected for brooding larvae. Once inspection was completed, trays were then transferred to a tub filled with 10-15 gallons of fresh seawater to recover from treatment for 30 minutes to 1 hour depending on recovery rate. In the first tray we had 25 animals open, in the second we had 3, in the third we had 4. None of these animals showed signs of brooding larvae. 

Temperatures logged at Oyster bay from Feb 28th to May 1st, 2014. Temps logged every 15 minutes. 

Oyster Bay	1N1-4	1N5-8	1N9-12	1N13-16	Total
Live	84	67		100	251
	1H1-4	1H5-8	1H9-12	1H13-16	Total
	89	101	96	14	300
	1S1-4	1S5-8	1S9-12	1S13-16	Total
		74		93	167

Treatment set up at Oyster Bay

Artsy photo of science

Gaping Oyster

Jack trying to help out

Jack decided to drink some epsom salt water behind my back. He made this face when I caught him. 

Manchester 4-30-14

4-30-14

Manchester WA
Clam Bay
Start Time: 10:00 AM
Finish Time: 6:00 PM
Temps: Mid 60s to Mid 80s

Participants: Sammi Brombacher and Jake Heare

We arrived earlier in the morning. We pulled up the hanging devices and broke them down into individual trays. We collected dead, counted live, and photographed trays for size/growth for later analysis. Upon completing these metrics the trays were reorganized into stacks containing one tray from each population at a random level in stack (top, middle, bottom). 3 stacks were then rebuilt and hung off the docks.


The 4th stack was treated with an epsom salt mixture to anesthetize the animals for brooding larvae inspection. One tray was dosed at a time in a 10 gallon mixture of 50/50 sea water/freshwater with 6-8 cups of epsom salt in them (roughly 75-80 g/L). Gaping oysters in each tray were individually inspected for brooding larvae. Once inspection was completed, trays were then transferred to a tub filled with 10-15 gallons of fresh seawater to recover from treatment for 30 minutes to 1 hour depending on recovery rate. In the first tray we had 30 animals open, in the second tray we had 20, in the 3rd tray we had 45. None of these animals had any signs of brooding larvae. 

Temps logged for Manchester from Feb 28th to April 30th, 2014. Temps logged every 15 minutes. 

Manchester	4N1-4	4N5-8	4N9-12	4N13-16	Total
Live	90	97	59	91	337
	4H1-4	4H5-8	4H9-12	4H13-16	Total
	97	85	89	84	355
	4S1-4	4S5-8	4S9-12	4S13-16	Total
	92	91	98	94	375

Tray being treated with epsom salt

Oysters being treated with Epsom Salt

1 tray being treated, 1 tray being examined, 1 tray recovering

Gaping oyster

Sammi processing Trays.

Ferry on the Sound.

For More info see the oly wiki

Dabob Final Sampling 4-18-14

4-18-2014

Dabob WA

Tarboo Creek

Start Time: 11:30 AM

Finish Time: 2:00 PM

Temp: Low to Mid 60's

Participants: Sammi Brombacher and Jake Heare

We reached the site at roughly 10 AM on Friday morning. There was a slight breeze, misty rain, and overall warm temperatures. We walked out to the site at 11:30 when the tide was low enough to reach the trays. We collected all the trays and placed them on a plastic sled and then dragged them back to the truck. This took approximately 2 hours and some swear words. The trays were then driven back to Seattle and cold stored at 12C. Trays stayed here for 14 hours before being processed the following day by Jake Heare.

Logged Temps for Dabob from Jan 30th to April 17th 2014. Temps logged every 15 minutes. 

Dabob	3H1-4	3H5-8	3H9-12	3H13-16	Total
Live	2	16	32	19	69
Dead	1	1	1	0	3
	3N1-4	3N5-8	3N9-12	3N13-16	Total
Live	0	16	0	17	33
Dead	0	7	2	11	20
	3S1-4	3S5-8	3S9-12	3S13-16	Total
Live	14	19	11	15	59
Dead	12	6	8	6	32

For more info see the Oly Wiki

Inaugural Post

This is the first post in what hopes to be a long running record of the scientific work performed by Jake Heare. This should include wet lab, computer science, and field work posts with informational videos and links to supportive information.