[Coral-List] Separation Zooxanthellae-Coral (Carla)

Luc R A Rougee rougee at hawaii.edu
Thu Jun 4 15:07:26 EDT 2009


Dear Coral List Serve, 

 I am trying to respond to the Separation Zooxanthellae-Coral post made by Carla. This is my first attempt at responding, so I am not sure of the procedure. If I need to go through a different method, please let me know. Thank you. 

Dear Carla,

 I have been using a simple method to extract functional protein from just the coral animal tissue. I have applied it to different species and it has worked well for me. The starting point for the method was adapted from the publication by NJ Gassman and CJ Kennedy; Marine Bulletin of Marine Science 50(2):320-330. Here is the method I have developed:

 -  <!--  /* Style Definitions */  p.MsoNormal, li.MsoNormal, div.MsoNormal 	{mso-style-parent:""; 	margin:0in; 	margin-bottom:.0001pt; 	mso-pagination:widow-orphan; 	font-size:12.0pt; 	font-family:"Times New Roman"; 	mso-fareast-font-family:"Times New Roman";} @page Section1 	{size:8.5in 11.0in; 	margin:1.0in 1.25in 1.0in 1.25in; 	mso-header-margin:.5in; 	mso-footer-margin:.5in; 	mso-paper-source:0;} div.Section1 	{page:Section1;} -->    -  <!--  /* Style Definitions */  p.MsoNormal, li.MsoNormal, div.MsoNormal 	{mso-style-parent:""; 	margin:0in; 	margin-bottom:.0001pt; 	mso-pagination:widow-orphan; 	font-size:12.0pt; 	font-family:"Times New Roman"; 	mso-fareast-font-family:"Times New Roman";} @page Section1 	{size:8.5in 11.0in; 	margin:1.0in 1.25in 1.0in 1.25in; 	mso-header-margin:.5in; 	mso-footer-margin:.5in; 	mso-paper-source:0;} div.Section1 	{page:Section1;} -->   
Coral samples were removed and immediately placed into 50 mL Falcon tubes and submerged in liquid nitrogen to prevent further stress. Branches were subsequently removed from the liquid nitrogen, crushed using autoclaved pliers and placed into a 100 mL Erlenmeyer flask, on ice, with 15 mL of cold filtered sea water (FSW). Flasks were covered with Parafilm and shaken for ten minutes, by hand, in a circular motion;  -  <!--  /* Style Definitions */  p.MsoNormal, li.MsoNormal, div.MsoNormal 	{mso-style-parent:""; 	margin:0in; 	margin-bottom:.0001pt; 	mso-pagination:widow-orphan; 	font-size:12.0pt; 	font-family:"Times New Roman"; 	mso-fareast-font-family:"Times New Roman";} @page Section1 	{size:8.5in 11.0in; 	margin:1.0in 1.25in 1.0in 1.25in; 	mso-header-margin:.5in; 	mso-footer-margin:.5in; 	mso-paper-source:0;} div.Section1 	{page:Section1;} --> allowing the coral skeleton to tear and remove the tissue from the fragments. The resulting liquid was transferred to a fresh tube and homogenized on ice using an Ultra-Turrax homogenizer for 30 sec. The homogenate was centrifuged at 2,000 rpm for four mins at 4 °C in an Eppendorf Centrifuge 5810R (15 amp version). The resulting pellet of coral skeleton and zooxanthellae was discarded. The supernatant, containing the coral tissue, was transferred to a new tube and centrifuged at 4,000 rpm for ten mins at 4 °C in the same Eppendorf centrifuge. The supernatant was decanted, the remaining pellet of animal tissue is what is remains. 
  

  
 If you have any question please feel free to contact me directly. Best of luck!

Best Regards,
Luc Rougee
University of Hawaii at Manoa
Kewalo Marine Laboratories
Pacific Biomedical Research Center
41 Ahui St. 
Honolulu, HI 96813
Lab:808-539-7321
Mobile:808-271-2254



----- Original Message -----
From: coral-list-request at coral.aoml.noaa.gov
Date: Thursday, June 4, 2009 6:03
Subject: Coral-List Digest, Vol 10, Issue 4
To: coral-list at coral.aoml.noaa.gov

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> Today's Topics:
> 
>    1. Separation Zooxanthellae-Coral (Carla)
>    2. disease of Diadema mexicanum (Eugene Shinn)
>    3. Diurnal adaptations of coral polyps? (Erin Sams)
> 
> 
> -----------------------------------------------------------------
> -----
> 
> Message: 1
> Date: Wed, 3 Jun 2009 11:26:12 -0300
> From: "Carla" <carlazilber at yahoo.com.br>
> Subject: [Coral-List] Separation Zooxanthellae-Coral
> To: <coral-list at coral.aoml.noaa.gov>
> Message-ID: <C1803FA9CCF14772B8382ED8D9E5C4E2 at Mozi>
> Content-Type: text/plain;	charset="iso-8859-1"
> 
> Dear all, 
> 
>  
> 
> I need to separate the zooxanthellae from the coral tissue in 
> order to
> extract DNA only from the animal. Does anyone know the best way 
> to do that?
> I also would like to know what would be the best way to fix the 
> coral before
> the separation, since I?ve heard that ethanol might disrupt the
> zooxanthellae cell wall?
> 
>  
> 
> Thanks in advance!
> 
> Carla
> 
>  
> 
> **********************************************
> 
> Carla Zilberberg, Ph.D.
> 
> Departamento de Zoologia
> 
> Universidade Federal do Rio de Janeiro
> 
> Ilha do Fund?o, Rio de Janeiro, RJ 
> 
> cep: 21941-590, Brasil
> 
> Tel: 21- 2562-6389/ 21-2562-6361
> 
> **********************************************
> 
>  
> 
> 
> 
> ------------------------------
> 
> Message: 2
> Date: Wed, 3 Jun 2009 12:47:21 -0400
> From: Eugene Shinn <eshinn at marine.usf.edu>
> Subject: [Coral-List] disease of Diadema mexicanum
> To: coral-list at coral.aoml.noaa.gov
> Message-ID: <a06230910c64c57fb0344@[131.247.137.127]>
> Content-Type: text/plain; charset="us-ascii" ; format="flowed"
> 
> Dear listers, 1983, the beginning of the Caribbean-wide Diadema 
> sp. 
> die-off was also the peak of the Acropora sp. Caribbean-wide die 
> off 
> as well as the beginning of the sea fan disease. And, well you 
> know 
> the rest of the story. Lets hope this new event can be watched 
> even 
> more closely. Gene
> -- 
> 
> 
> No Rocks, No Water, No Ecosystem (EAS)
> ------------------------------------ ----------------------------
> -------
> E. A. Shinn, Courtesy Professor
> University of South Florida
> Marine Science Center (room 204)
> 140 Seventh Avenue South
> St. Petersburg, FL 33701
> <eshinn at marine.usf.edu>
> Tel 727 553-1158---------------------------------- 
> -----------------------------------
> 
> 
> ------------------------------
> 
> Message: 3
> Date: Wed, 3 Jun 2009 13:39:47 -0400
> From: Erin Sams <samsee at alumni.hiram.edu>
> Subject: [Coral-List] Diurnal adaptations of coral polyps?
> To: <coral-list at coral.aoml.noaa.gov>
> Message-ID: <BAY119-W7E2208C25DAE62185501FEF4A0 at phx.gbl>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> 
> Hello all,
> 
>  
> 
> Last February (2008), I noticed while diving off Koh Tao, 
> Thailand and the Maldives that many corals - stony as well as 
> Gorgonian - had their tentacles extended during the day, 
> something I've never seen while diving in the Keys or Bahamas. 
> Is this normal for some species? For this region? I have always 
> understood corals to be nocturnal feeders, keeping their 
> tentacles retracted to allow the zooxanthellae to 
> photosynthesize during the day and extending them to feed at 
> night. 
> 
>  
> 
> I am taking a coral reef ecology course through my Masters 
> program in August and would like to address this as my research 
> topic, but I have been unsuccessful in finding explanations of 
> this behavior. Insight or direction to sources of information 
> would be greatly appreciated.
> 
>  
> 
> Best,
> 
>  
> 
> Erin Sams
> 
> samsee at alumni.hiram.edu
> 
> _________________________________________________________________
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> End of Coral-List Digest, Vol 10, Issue 4
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