Took two weeks to get an appointment with Dr. lewis but was able to and it was extremely helpful. She explained that I was looking at the biolumenescence reaction wrong or not fullly. I was looking to effect the main reaction but had the genetic information for luceferase. luceferase is likely produced consistently and stored. by manipulating the oxygen levels it would effect the initial reaction but would likely have no effect on the animals producing luceferase. So when it went to check if levels of luceferase had been manipulating it by changing the amount of oxygen it may effect the initial reaction and change the amount of light produced but since oxygen is not related to the production of luceferase when i checked genetic information it is likely their would be not change.
oxygen depletion and ocean acidification manipulation have many other effects on the animals and it would be hard to tell if that is actually the cause of the change in the luminescence or it is just causing extra stress and not directly effecting the animal. Dr. Al-noori brought up in class that and easy way to effect the reaction would be to effect the inter-cellular calcium storage where the calcium used to initiate the release of luceferase from storage would be. using a calcium kelator, or inter-cellular calcium channel blocker may be more efficient since it would not allow the release of the luceferase. Notes from the meeting are above.
One thing to look at may be to look into checking if luceferase is indeed constantly produced or if it is only produced when needed. it is likely since the animal uses this as a defensive function that it would make sure that the levels were never depleted by constantly producing luceferase. Doing a MRNA-CDNA-PCR on incoming specimens and then another after light production may answer this question and clear up any questions. Dr Lewis mentioned that in order the truly understand the entire genes responsible for luminescence I would need to look at the entire genome by using a microaray. i will need to find out more information it may be beneficial to locate the genome since I do not believe it has been done as of yet.
The enclosure is complete setup and put together as well as the artemia enclosure. I placed 7 2 gallon buckets in the bottom with sifted find sand donated by the fisheries department. A timer with light has been setup but will need to be worked with to make sure it is on the correct light cycle.
Greg will be acquiring up the animals this coming Saturday 8/9/2014 from Bainbridge island Crystal Springs pier were I will pick them up after the dive and transfer them to uw
Have not been able to complete history portion of the proposal since I have had to many questions on the process of the experiment and do not feel confident enough to finish it but now that I was able to talk to Dr. Lewis, and Al-noori i think I have a clearer idea on what to do.
For the manipulation experiment I am going to put the animals in both and oxygen depletion, calcium manipulation and ocean acidification environment and then measure the illuminations with a camera to see if their is any change. This will also give me the ability to check the genetics to see if the production of luceferase is being manipulated. or if I am able to get the entire genome I may be able to look at more of the reaction and see what all genes are responsible for the process. I will talk to Dr. Roberts to look into it.
I also saw some information that states that the sea pens may illuminate at night so I would like to find a way to recorded the live stream and be able to look over it to see if this is true not only will it be wonderful to see but could give me the ability to test their luminescence without having to stress the animal out by touching it but will not know till they arrive and are comfortable in the setup.
i need to really comb through the reaction and pull as much information I am going to look into a permanent dry erase board that I can have access to to write out the entire reaction and be able to study and develop. It seems to help me understand better when I can step back and look and the entire thing. The quarter is coming to and end so I will need to continue the research next quarter which will give me the chance to do all of my experiments and know the pens are being taken care of. Building access was figured out so I can come in and take care of the pens even on weekends.
7/17
Today I finished moving tank and put it into place. Almost completely finished with plumbing just a few more connections to finish up tomorrow. Tank was filled with tap with bleach to sanitize. will drain and refill with saltwater when all plumbing is in place and leak free. Currently no leaks.


Revision to Methods
Instead of just having a sand bottom to the tanks and Trash cans Jon recommend that I fill 2 gallon buckets that are 9" deep with sand and place them throughout the tank so that when the specimens need to bee transported I can bring up the bucket with sand hopefully not disturbing the specimen to much and transfer to a trash can. The specimen will be in the same substrate that it is used to hopefully making it easier to take samples.
Will also make it easier to collect substrate.
Primer information for coelenterazine
was amplified by PCR with primers containing the sequence encoding NdeI site, a bacterial periplasm localization signal (pelB) and XhoI site at the 5ʹ end and PstI site at the 3ʹ end.
Anaesthetizing
<span style="font-family: Arial,Helvetica,sans-serif;">Cnidaria folks and vwlau
For sea anemones, I pour an isotonic, fresh-water solution of 7 1/2%
Epsom Salts = magnesium sulfate (from the drugstore - that’s the hydrated
form, with 7 chemically associated water molecules, not the chemical reagent
stuff without the associated water) into the container with the relaxed
anemones to make a final solution that is about 1/2 seawater and 1/2 mgSo4
solution.
The tricks I use are to prevent reaction/contraction by the animals
is to cool the solution to the same temperature and aerate it thoroughly,
and to condition the animal to water motion (by putting it near the water
inflow area), first.
Good luck!
Liz Francis</span>
<span style="font-family: Arial,Helvetica,sans-serif;">http://maillists.uci.edu/pipermail/cnidaria/2004-March/000767.html</span>
<span style="font-family: Arial,Helvetica,sans-serif;">http://www.google.com.ni/patents/US6436682</span>
A meeting with Jake cleared up a lot of the confusion in the procedures of my research.
Found a book with realted information to the system used for biolumenescence in the orange sea pen
Photoproteins in Bioanalysis edited by Sylvia Daunert, Sapna K. Deo looking into picking it up from UW libraries.
Methods- working on expanding need to speak with Dr. Roberts to discuss his intended kits so I can download info on use of each.
Animal collection and care
Tissue Sampling (Magnesium Sulfate Anesthesia?)
DNA Isolation (Qiagen DNEasy Kit, DNAzol method, etc)
Pick primer for PCR
PCR
RNA Isolation
cDNA
PCR/qPCR
Manipulation experiment
Luminescence Quantification
RNA/PCR/qPCR
Gene Sequencing?
Attended Lab meeting online had a few technical difficulties but I believe I have them all worked out if I can not make the meeting in the future I can Attend online and be able to interact.
Spoke with Jon about setting up the enclosure this week. We are still waiting to hear back from Greg about being able to use the Aquarium room to house the enclosure. Greg replied and said it would be fine so Jon and I will get everything up and running thursday at 1. He plans to move the Tank early tomorrow and we can hopefully have it up and running by thursday.
Started working on the proposal but I will need to speak with Dr. Roberts about some parts of it . Sent an email to schedule a meeting with him as well as Jake. (update-going to put together questions for Dr. Roberts and include them in an e-mail after meeting with Jake)
Created google Doc "https://docs.google.com/document/d/15zQqsOAc0LP0_hbktm-GUAUWwspLUh6cN15MgJzoEng/edit?usp=sharing" to make proposal more accessible
Created possible questions for research
1. What is the genetic sequence and proteins responsible for luminescence in P. gurneyi.
2. Does changes in P. gurneyi environment effect its ability to produce light when touched. (not sure on setup yet, but ocean acidification may be best fit since it has a direct effect on levels of Calcium often used in luminescent reactions but still need to verify this.)
Heard back from Tim Carpenter the curator of fish and invertebrates at the Seattle Aquarium.
- He recommeded 1.25-1.5 gpm for the flow rate and designing a chaotic flow rate instead of direct flow.
- Keeping the Pens between 48 to 53 degrees to slow down algal and any other potential problem growths.
- They feed them Artemia nauplii daily he said anything that would be used to promote coral or planktivorous anemones.
- Dim lighting
- 8" for sand bed is what his are kept in
He also confirmed alki pipeline and reef are good locations to find them. Every once in a while they can be found at golden gardens.
( North of the Alki Fishing Reef if you have a boat or can scooter from Laurelhurst beach, 40-50 ft depth.
At the end of the Alki Pipeline (at 63rd Ave. south of Alki point) – long swim out, about 40 feet depth.
Faye Bainbridge Park, Bainbridge Island – 30-40 feet, easy shore access.
Twin Spits near Port Gamble – easy shore dive. )
Asked if he had any knowledge of the resilience to research since I will need to take samples for the genetic, an protein area of my research.
Meeting with Jon to start putting together a plan to setup enclosures. Found one ~200 gallon tank that should fit the needs of the project. He recommended that we setup in aquarium room but need to speak with Greg to verify. Tank is current located in storage in salmon hatchery and will need to be transferred over. Jon is not available to do so until next week so we are planning to move everything then. Will need to locate equipment to setup overflow and add circulation to the tank.
Jon also recommended finding a different food supply since culturing my own is not plausible for the size of experiment. I noticed in my reading (posts 7/7) that the Pens may not actually feed on phytoplankton but mainly detritus so their are many options to look into.
Learned I cannot collect specimens by myself because of permit needed. Uw has a permit but only certain individuals are covered under that permit. Will speak with Greg to see if he is willing to collect specimens for me. May need to look into getting a collection permit for myself for current and future research.
Meeting with Dr. Roberts
Reviewed lab calendar, lab wiki, archiving data, google+, lab meetings, blog posting and signed contract.
Joined Northwest dive club.
Unable to dive with Sonia but got in contact with some of her connections and found optimal dive sites with confirmed sitings of specimens (4 mile barges, fox island west wall, Sunny-side south of pipeline, Les Davis, Whidbey, Bainbridge island as well as alki reef.) Alki reef is supposed to have tons of them but I am not sure about acquiring specimens since Alki is a no take zone. Reviewed and signed lab contract.
requested access to lab wiki, and signed in a created document folder on school hard drive for storing files.
Need to make appointment with Sam White when he returns to go through lab safety and lab tour.
Scheduled Meeting with Jon to go over and plan setup for aquarium, as well as feeding resources for specimens.
Proposal due next Friday, Dr. Roberts will be in Friday Harbor so it needs to put as priority for next week.
Personal notes:
Rottifers may not be a valid food supply, Jon said he had problems with them in the past looking into an alternative may be nessacary refer to 7/7 blog post.
Species selected (Ptilosarcus gurneyi)
Aquarium Needs: large sand/Mud bed (species full grown average 2 feet, collect specimens 1' or smaller)
Species needs constant water flow (filter feeder, planktonic food supply needed)
Sump system for water circulation possible breeding ground for Plankton (copepods? Tigriopus californicus)
- Sump
- Aquarium
- pump
- overflow box
- intake circulation powerheads/circulation pump
- growth lights for sump and aquarium (add sand to sump as well as local marco algae for copepods)
- mechanical filtration?
Decide to house in Roberts controlled Temp room or possibly talk with Greg about housing it in the Aquarium room so I could connect in to his system which likely already has a large supply of planktonic food available circulating in the system.
Species collection Locations Bainbridge island, Whidbey island, Alki (spoke with LFS in the area no one knew of any good locataions but Greg has said that Bainbridge was a good location to find plus read (http://www.wallawalla.edu/academics/departments/biology/rosario/inverts/Cnidaria/Class-Anthozoa/Subclass_Alcyonaria/Order_Pennatulacea/Ptilosarcus_gurneyi.html ) an article stating that they are often found "behind whidbey island"
Got in contact with Jon Wittouck <wittouck@uw.edu> to put together
everything needed for aquarium setup
Update: Jon replied and told me we should not have any
trouble getting everything set up raised a good question
to check "biomass of plankton filtered out of water per day" to establish feeding needs
Species creates luminescent mucous?
Species needs large sandbed or lower stalk can becomen inflamed.
T.carpenter@seattleaquarium.org-e-mail sent requesting information on Sea pen needs and possible diving sites.
LFS
BARRIER REEF renton(copepods)
1717 NE 44th St, Renton, WA 98056 (425) 277-7670
Saltwater city, bellevue(copepods)
14150 NE 20th St F3, Bellevue, WA 98007 (425) 644-7050
The fish store, lake city
12320 Lake City Way NE, Seattle, WA 98125 (206) 522-5259
Denny's pretty world, Kirkland
12534 120th Ave NE, Kirkland, WA 98034 (425) 821-3800
Sierra pets, renton(copepods)
601 S Grady Way, Renton, WA 98057 (425) 226-3215
http://www.advancedaquarist.com/2003/10/inverts
Basic Aquarium handling and care
Diurnal pattern of expansion
Culture of Rotifiers recommended or artifical plankton SDMP (ESV's spray-dried marine phytoplankont), APR (Artificial Plankton - Rotifer), or "Size I" (50-100µm), Cyclop EEze if grouded up to small bits. Feeding on a regular basis
Check other types of plankton as better food supply
Online research shows mechanical filtration better for cold water systems since the cold temperature slows metabolic rates making it difficult to establish bacteria to properly cycle tank, and local live rock not porous enough to act as a good biological filter. (Check if tropical live rock/dead rock could be used as a replacement).live rock is a good habitat for copepods as well as macro algae (join aquarium blog to ask about cycling coldwater aquariums and types of filtration)
Passive Suspension Feeding in a Sea Pen: Effects of
Ambient Flow on Volume Flow Rate and
Filtering Efficiency
BARBARA A. BEST'
Department of Zoology, Duke University, Durham, North Carolina 27706
Reference:Biol.Bull.175:332-342. (December, 1988)
http://www.biolbull.org/content/175/3/332.full.pdf
Flow rate:
Small optimum 5-6 cm/s data shows optimum efficiency at 1.5 cm/s
Mediuim/large 8-9 cm/s
data shows optimum efficiency at 1.5 cm/s for both all sizes change from 1.5 to 6.0 decrease efficiency from 42.8-29.9%
optimum food particle size 10-25 microns
Height stats: sea pen with a rachis height of 7 cm, medium sea pen 15 cm high, and large sea pen 25 cm high
http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=52424
KingdomAnimalia – Animal, animaux, animals
SubkingdomRadiata
PhylumCnidaria Hatschek, 1888 – cnidarians, coelenterates, cnidaires, coelentérés, água viva, anêmona, caravela, cnidario, coral, hidra
SubphylumAnthozoa
ClassAnthozoa Ehrenberg, 1834 – corals, flower animals, sea anemones, anémones de mer, coraux, água viva, anêmona, antozoário, caravela, corais, gorgônia
SubclassOctocorallia Haeckel, 1866
OrderPennatulacea Verrill, 1865 – sea pens, sea panzies, sea pens
SuborderSubselliflorae Kükenthal, 1915
FamilyPennatulidae Ehrenberg, 1828
GenusPtilosarcus Verrill, 1865
SpeciesPtilosarcus gurneyi (Gray, 1860)
http://data.gbif.org/species/2258713/?extent=-129%2B41%2B-109%2B51&zoom=5&minMapLong=-129&minMapLat=41&maxMapLong=-109&maxMapLat=51&c[0].s=20&c[0].p=0&c[0].o=2258713
http://www.asnailsodyssey.com/LEARNABOUT/PEN/penFeed.php
Consolidation of research information
the food particles are unhatched brine-shrimp Artemia cysts