Note: Please distribute this to as many sources, forums, clubs, magazines, stores, or other places as possible. Please refer interested parties to this article, or contact me by email at eborneman@uh.edu.

Background and Introduction

For many years, elegance corals (Catalaphyllia jardinei) were among the easiest corals to keep in aquaria. Over the past five years, most entering the trade are doomed because of a condition for which there is no known cause or cure. In this condition, the coral adopts a relatively swollen oral disk with a fringe of unextended tentacles. The coral tissue eventually shrinks, and the coral dies despite all manner of experimental intervention.

In some cases, a white opaque mucus-like web may be present. I am not sure if this is an entirely separate condition, somehow related, secondary to the primary condition, or part of the same condition.

There has been much speculation as to why this condition now occurs, and various sources have suggested causes and even cures. But I stress that no research to my knowledge has been done on this condition, and to date none of the potential causes, solutions, or cures seems to have much validity.

These corals are extremely beautiful and desirable. Unfortunately today, their poor survival rate in captivity puts them in a similar class with Goniopora stokesi where survival rates are too low to justify the large-scale collection of them from the wild. In fact, Catalaphyllia appear to be relatively rare species and may be highly overcollected so that populations in some collection areas are threatened or even locally extinct. To continue to collect rare species that have extremely low survival is bad for everyone - it is an economic loss, a resource waste, and a source of great frustration for all those who purchase and attempt to keep them alive.

Not all Catalaphyllia shows signs of this condition. Occasionally, I see them in stores with a normal healthy appearance. During surveys of coral collection areas, I never saw one with this condition in the wild, and of hundreds being held in tanks for export, only a single specimen showed the signs of the pathology. To be sure, Catalaphyllia are being collected from dramatically different types of habitats, and may be collected from very different places from where they were collected years ago when they were easy-to-maintain. I could speculate logically as to many potential reasons for their current conditions and demise, but unfortunately this speculation would be no better than the complete lack of understanding of this condition that currently exists.

Because of the desirability and immense popularity of Catalaphyllia, as well as to learn more about this highly unstudied species, and to help ensure the populations of wild elegance corals and their success in captivity, I propose to conduct a formal study of the condition to attempt to determine its cause and any possible solutions so that, once again, we can enjoy healthy elegance corals in our tanks.

My research field is the investigation of coral diseases with currently unknown etiologies. I would like to volunteer my services to help provide answers to the elegance coral condition. Together with collaborative work from some of my colleagues, I believe we can determine the cause of high mortality resulting from this condition. I will attempt to do this in the most economical and efficacious manner possible, and will provide results to all applicable forums upon its completion.

Methods

I propose to collect funding and material to conduct this work, and to do so in phases so as not to require more funding or material than is necessary.

Catalaphyllia will need to be acquired from various sources, both healthy and affected with the condition. In some cases, special shipping arrangements might be required to avoid delays or exposures that might confound any pre-exisiting conditions from the wild. Corals will be sent either live, or dead and preserved for analyses. Generally, a type of formalin or alcohol fixative will be used, and not everyone will have access to some of the fixative material. I will then examine the corals working from the most obvious to the less obvious. External and gross changes will be documented in live samples with a clear description of the signs, changes, time frames and fate of the coral over which the condition occurs. I will look at the colonization of the surface flora and fauna by using sterile swabs and take samples for live material and freeze them for any future molecular work that might be required. I will prepare histoslides to examine microstructure and look for obvious abnormalities in tissue or zooxanthellae and the presence or absence of intracellular parasites or pathogens. At this point, something may or may not make itself apparent. I will prepare a report, offer my best suggestions for the next phase if the cause is still unknown, and outline the next funding and materials request.

Initial Requirements

Initially, at least, the only thing I would need is corals - as diverse in time and space as possible. For example, ordering 100 corals from the same wholesaler on the same date would be far less valuable than a handful from a number of sources over various time periods to increase the sample diversity. I will need elegance corals from as many sources as possible. The following are sources that should ideally be exploited for sample material.

Direct from the wild
Export facilities
Wholesale facilities
Retail facilities
Home aquaria

Healthy Corals - The Hard Part

Live corals
I would like to obtain 5 -10 healthy elegance corals with at least two of these arranged to be shipped directly from the source to my lab, or to a facility who is willing to ensure that the specimens go directly into a clean covered tank with freshly made seawater using bottled distilled water and clean gloved hands. See protocols below. I will be attempting to contact sources for this material, although if anyone has such contacts and abilities, their efforts would be greatly appreciated.

As for the remainder of the healthy samples, it would require someone with enough experience to determine if a given specimen truly appears to be one unaffected by the condition. This requires either a facility with a regular turnover of Catalaphyllia or volunteers policing local stores to find the rare ones that are healthy. In any case, I think a photo should be sent to me prior to acquisition or shipping for confirmation that it does appear to not be affected. If it turns out to be affected, it's not a problem as it will still contribute to the study. However, I have to acquire live healthy specimens as a control. If a healthy (or suspected to be healthy) coral is located and provided, I need that coral to be carefully collected and shipped to avoid any new sources of contamination. The process will be described below in the "techniques" section.

Tissue samples
I know I have seen at least several photos from aquarists' tanks who have apparently healthy elegance coral in their tanks. I have one, as well. For those who have healthy elegance corals and want to keep them in their tanks (likely everyone!), a tissue sample can be used. The techniques section below outline the procedure.

Diseased Corals

Live corals
Catalaphyllia showing signs of the condition can be prepared and sent using the same protocol for healthy corals. These can be from any source where they are found.

It is very important to ensure that no additional contamination occurs until I receive them. For example, if this condition is caused by a bacterium, and aquarists grab the coral with their hands and I run surface samples and find most or all have the unusual presence of non-marine types of staphylococci on them, I would erroneously look at the staph bacteria as a possible causative agent. It would be wasteful to run culture and reinfection studies looking at fulfilling Koch's postulates when these microbes were all artifacts of human handling. The same is true of air exposure - we really want to limit the number of mold, fungus, bacteria, and other microbial organisms that are common in the air and on surfaces that could confound the tests and make the study longer, more expensive, and more difficult.

Fixed dead corals
If an affected Catalaphyllia is found and cannot be shipped live to me, I can use the whole coral or tissue samples of fixed material. For example, if someone has purchased or seen a Catalaphyllia that they feel has no chance for survival, and might not even last through the shipping process, it can be killed and fixed for study. Similarly, stores and aquarists with access to affected corals that are willing to donate tissue samples or the corals, but only if they are not alive for whatever reason, can do so through fixation. The techniques section below outlines this procedure.

Skeletons, Photography, Direct Counts, and Surface Swabs

If anyone wants to contribute to this study but cannot contribute in any of the ways above, there are still some possibly important bits of data that could be obtained. I can use skeletons of dead Catalaphyllia, photos of affected living Catalaphyllia and photos of the skeletons of recently dead Catalaphyllia, and sterile surface swabs of affected corals from tanks or from facilities. None of these do I feel will provide conclusive evidence of anything, but could potentially be used to support other findings.

Skeletons can be valuable because of the presence or absence of various organisms. The presence or absence of boring algae, fungi, mollusks, crustaceans, and other flora and fauna may be occasionally or consistently present and might deserve further investigation as to whether they play a role in the condition.

Photography is supportive and may be good in terms of documentation. It may also be able to provide evidence of gross changes or factors involved in the condition.

Another bit of information that would be helpful is epizootiological. It would be good to have some idea of the occurrence of this condition. If anyone is visiting a facility such as a fish store, wholesaler, or other source, a simple count of the number of elegance corals present and the number of affected colonies would be very helpful. Please provide the date of the count, your name and contact information, and the name of the facility, along with the count information. You can email me the information at eborneman@uh.edu.

Sterile swabs of coral surfaces could be valuable if the condition involves changes to normal biotic flora, or if there is a parasite or pathogenic microbe that effects the coral by colonizing its surface. If this is the case, sterile swabs could be supportive if detailed tissue work shows this to be the cause.

All protocols for this work are outlined below in the techniques section below.

Techniques

To provide live corals to the study (healthy or diseased)

The water in which the coral resides or freshly-made seawater should be made using bottled distilled water and clean salt scoops from closed containers of salt. The coral or any tools coming in contact with the water or the coral should not be used unless previously treated aseptically using alcohol or a commercial aseptic scrub like Hibiclens.

To make or collect water, use a glass container that has been wiped down thoroughly with rubbing alcohol (preferably 90%) or ethanol. All tools used to add salt and stir the container should also be cleaned. Hands should be gloved with latex exam gloves wiped with alcohol or hands should be washed with an aseptic scrub like Hibiclens or wiped with alcohol. Once washed, avoid touching unwashed skin, hair, clothing, or other surfaces.

Water should then be poured into the shipping bag or container. If a bag (Ziploc or fish bag), the bag should be new and relatively sealed and having not been left open to the air. If a container (Tupperware or similar), the container should have its inside wiped with alcohol prior to adding the water.

Corals should be quickly removed from the tank and placed into the prepared water which is in the shipping container. The tissue itself should be handled as little as possible, grabbing the skeleton instead. For aquarists who need to use a local store for bagging and shipping, the closed container should be taken to the store and the same procedure repeated for transfer to another shipping bag. Ideally, corals will be shipped using a double bag with oxygen, with more air than water in the shipping bag. If compressed air is used, the filling tip should be wiped with alcohol prior to inserting it in the bag for filling.

The container should then be quickly sealed and shipped. All of these procedures should be timed carefully to minimize shipping durations. Generally, this means working in the afternoon for a later afternoon pickup. See shipping protocol below.

Tissue sample collection

If tissue samples or fixed coral specimens are being provided, please follow the same aseptic techniques described above, even if it is a dead coral or skeleton. Tissue samples on Catalaphyllia can be obtained easily by using a scalpel, razor blade, or sharp scissors. Tissue samples usually require some holding or handling of the coral, so please make sure gloves or aseptically cleansed hands are used. Once the tissue sample is obtained it should be immediately placed into the fixative and sealed. The same would be true of a whole colony that is affected. The colony would simply be dropped into the fixative. I will take care of all subsequent steps once I receive it. The amount of tissue required should be at least 1cm x 1cm and incorporate tentacle and oral disk, if possible and in the more affected parts of the coral. Please do not send tissue samples without fixation. They will deteriorate rapidly in seawater and be unusable for study. See fixative instruction for methods.

Photography

I will gratefully accept any photographic documentation of Catalaphyllia with the diseased condition. It might help to just take a digital camera to a few fish stores and snap some photos. I am especially grateful for photographic support of corals that are either sampled or sent to me alive or fixed. In fact, I would be very pleased to receive photos prior to receiving coral material to ensure that it is the right condition. I will also appreciate any additional photos of the tank as a whole, especially in relationship to the elegance coral. If anyone has lost an elegance coral to this condition, but has retained the skeleton, I would like to see photos of it from all angles and extremely close-up, if the camera has this capability. Please do not provide photos that are out-of focus or do not show clearly the structures or animals being photographed. You can email digital or scanned images to the email provided above. I do not have any practical limitation on size of files, and I have very fast internet lines at home and work, so don't hold back on file size or number of files. Please provide the following information with each photo:

Your name
Date taken
Basic photo description
Contact information
If the photo accompanies other material being sent or is a stand-alone photo documentation.

I will probably ask other questions upon receiving them, but this information is fine for the time being.

Shipping

Live corals
Please use an overnight delivery service, and try to arrange for the latest possible pick-up time so that the coral spends as little time in transit as possible. Before you send anything, confirm with me the planned ship dates so I can be sure to be available or have someone available. Do not send any live animals to my lab address as there could be significant delays before they make it to my lab.

Send all live shipments to my home. Please contact me by email to eborneman@uh.edu for my home shipping address. You do not need to require a signature release. All the drivers know me.

After confirmation with me for receiving a shipment, please email me the tracking number to eborneman@uh.edu.

Live corals should be packed in bags or containers and ensured that they do not leak. Double bagging or sealing of containers is highly recommended. Make sure that the boxes are well insulated with peanuts or Styrofoam, and if you are shipping in cold weather, to make sure to use a heat pack. A fish store will likely be able to provide these and the proper number for the climate at the time. Generally, one or two will be fine for small boxes. If the weather is extremely cold (or hot) please wait until more moderate conditions occur.

Fixed tissue samples, skeletons, and other non-living material

Please send these by regular ground service of your choice to the following address and make sure that if liquids are present, especially alcohol or other fixatives, that containers are rigid and well sealed, preferably with screw-on lids rather than snap-on lids. If you are sending glass containers, make sure the box is well packed and padded to avoid breakage.

Eric Borneman
Department of Biology
University of Houston
Science and Research Building II
4800 Calhoun Rd.
Houston, TX 77204
Ph (713) 743-2667

Fixation instructions

I have pasted a bit of text on fixation below from Histotechnique for information purposes. The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues or soon after death to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.

Formalin is used for all routine tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly.

There are two major groups of fixatives for the work required in this project, classified according to mechanism of action:

• Aldehydes

• Alcohols

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin, although for coral tissues, 10% formalin in seawater will work. There are a number of available fixatives that are better, including a modified Helley's solution, paraformaledhyde recipes and a brand called Z-fix, that are especially good for coral tissue. If anyone has access to these chemicals, let me know via email because this would be the best for most of the histology assays I will use. I can provide detailed instructions if this is something you can do.

Glutaraldehyde fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde. I do not expect to have to do EM at this point, although I might in the future. However, I can use tissue from affected live corals and really don't want anyone unskilled to use glutaraldehyde. It is pretty nasty stuff.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness. Because coral tissue is only two cell layers thick, alcohol works. It is difficult to work with the tissue later on, and some assays cannot be performed if alcohol is used as a fixative, but I will be able to get some results if they are used. They are the most easily available for most persons. Ideally, ethanol should be used by purchasing small bottles of pure grain alcohol from a liquor store and diluting it to 70% with freshly made artificial seawater. I would use about 1/4 of a teaspoon of sodium bicarbonate (baking soda) in the mix, as well, to act as a buffer, if the fixative volume is around 250-500ml (8-16 ounces).

The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Because corals have skeletons, this must be included since the fixative will soak into porous skeleton and be unavailable to penetrate coral tissue.

Sources for materials

90% rubbing (isopropyl) alcohol for surface sterilization is available at most major drugstores.

70% rubbing (isopropyl) alcohol for fixation and less effective surface sterilization is available at drugstores, supermarkets, convenience stores, etc.

99-100% ethanol is available as pure grain alchohol from liquor stores. It is often sold under the name Everclear. Ethanol is worth the effort to acquire, though it costs more than rubbing alcohol. It is an extremely good surface sterilizer, evaporates very quickly, is much more ideal for fixation of tissues, and is relatively non-toxic to humans and organisms in the aquarium.

Sterile swabs, syringes and containers are available at some drugstores and laboratory supply houses. Most veterinarians or your physician will likely be willing to part with some, too, if you ask them.

Please contact me by email for further inquiries.

Support for the study

If you cannot contribute coral material but want to support the study by helping to pay for the costs involved in the study of the condition, you may make donations to the Elegance Coral Project Fund. Please send donations in the form of check, money order, or electronic payment to the following address. Funds will be maintained and used as needed by drawing from the account. Any unused funds over 10% of the donation total will be refunded if the research is completed without using all the funding. Refunding 10% of donation totals will be cost and labor prohibitive, and this remaining money will either be saved for any future projects or donated to the investigator for his efforts in the project, to be designated by the request of the donating party.

I am currently working on establishing the fund, and will post the information shortly in The Coral Forum.

Expected costs

The costs of this initial phase of the project will be directly related to the amount of sample material obtained. Materials costs may vary depending on what is already available to the party. My estimates of average costs per sample are:

It is conceivable, though very unlikely, that the per sample cost if a coral is provided for free, all materials and shipping are available or free, and that the cause of this condition is relatively obvious, that the project could be completed for the cost of the aquarium facilities (unless donated) plus $500.00.

I suspect this initial phase of the project, hopefully successful and conclusive, and excluding the costs associated with obtaining the material for a good sample size of 50 corals, will cost around $3500.00. I think this would be a reasonable goal to try and reach in terms of initial funding.

Absolutely Required Information on All Contributions
          (except financial only, which is voluntary)

1. Contact information including name, address, email (phone number optional but highly recommended if I need to talk with you quickly)          

2. Tank Information

   1. Size

   2. Equipment used

   3. Maintenance routine, including water changes and salt brand, and any major changes in brands or routines implemented during the ownership of the affected coral.

   4. Additives, including food, supplements, and medications, and any major changes in brands or routines implemented during the ownership of the affected coral.

   5. Water parameters, including but not limited to pH, alkalinity, ammonia, nitrate, phosphate, calcium, temperature, salinity or specific gravity, and any major changes in brands or routines implemented during the ownership of the affected coral.

   6. Length of time the tank has been set-up.

   7. Any unusual problems or events that may have contributed to this condition.

3. Coral Information

   1. How long the coral has been in the current tank (date of acquisition).

   2. If alive, how long did it take before signs of the condition were noticed?

   3. If dead, how long did the coral live before it died? Please include the length of time it appeared healthy, and the length of time it looked diseased.

   4. Location where it was purchased or obtained.

   5. A written description of the condition and any accompanying photographs.

   6. Any changes in the signs of the condition over the time the coral was in the tank.

   7. Any other background information on the coral that is known (Fiji, Indonesia, in three other tanks before the current one, was dropped on the floor by accident, etc.).

4. If this is not the first Catalaphyllia owned, please provide the information above for others and their fate.

5. The date when the coral was removed and sent in for this study.

Example:

Joe Aquarist
15 Griggs Street
Houston, TX 77252
(713) 555-1111
email: jaqua@earthlink.net

   - 75 gallon tank
   - 2 years 7 months in operation
   - 4 MaxiJet 1200 powerheads, 20 gallon sump, ETS Gemini skimmer, Mag 5 return, 2 x 175 watt 6500K metal halide, 4" CaribSea oolitic sand bed, 65 lbs. live rock, Red Sea ozonizer, 1 liter ESV carbon changed every six months
   - Kent Calcium 100 ml/week
   - Kent Strontium 20 ml/week
   - Brine shrimp - approx. 1 tablespoon per day
   - DT's phytoplankton, 50 ml/week
   - Red Slime Remover used once, 50 mg, 10/2003
   - 10% water change every week using Instant Ocean salt. Used Reef Crystals for the first year.
   - pH stable 8.0-8.2
   - Alkalinity stable at 3.5 meq/l
   - Calcium varies between 350-450ppm
   - Ammonia unmeasurable since week 4 of the tank
   - Nitrate was 5.0 ppm but has been 1.0ppm for past three months
   - Phosphate levels stable at 0.50 ppm
   - Specific gravity always maintained at 1.025
   - Temperature varies: 78°F in winter, 84°F in summer
Notes: this coral was stung by an anemone six months ago, and bleached four months ago. The condition appeared prior to either of these events. I also added a lot of sand to the tank three months ago and lost three other corals at the time.

This elegance coral was acquired from Joe's Fish Store in Memphis, Tennessee in June, 2003. It looked normal when I bought it, but developed a swollen disk and shrunken tentacles two weeks after I put it in the tank. I dipped it in freshwater, but it didn't change the conditions. I tried treating it with Maracyn, but again no effect. In August, 2003 the entire coral appeared shrunken, and a white web formed on the surface about two weeks after it began to shrink. The coral died in the first week of September, 2003. I purchased another one from Online Corals, Inc. (www.sickcorals.com) two weeks ago, and it arrived with the swollen condition. They told me this coral came from an exporter in Jakarta. It is still alive and its appearance is unchanged. I am sending this coral alive to you according to the information provided on the project page. I have emailed you photos of the coral taken on January 25, 2004 and provided the information requested. I will remove and send this coral to you on February 6, 2004 as per instructions.