Technical questions regarding grafting/implantation

[Mostawesomecoffee]

I have a few questions regarding nucleus implantation. It is my understanding that implanting a nucleus in a salt water mollusk (South Sea or otherwise) is difficult relative to implantation in a freshwater mollusk. Why is this? Is it because gonad implantation is difficult relative to implanting in a recipient mantle? Along with this, aparently once the technique is learned, it takes quite a while to sufficiently master it so that a quality pearl is produced. I'm guessing that this aspect has to do with proper mantle graft implantation. Is this true?

Why is it that there are different implantation techniques for salt water and fresh water mollusks? It's my understanding that regarding salt water nuclei, they are implanted in the gonad with a piece of donor mantle tissue. Aside from mabe pearls, this is the only way that things are done.

With freshwater mollusks, one nucleus, along with donor tissue, can be implanted in the gonad, or a piece of donor mantle can be implanted in the recipient mantle, or a piece of donor mantle along with a nucleus can be implanted in the mantle. Why are there so many options for freshwater mollusk implantation that don't exist for salt water mollusk implantation?

Why is a piece of donor mantle used for gonad implantation? Why isn't the mantle cut from the same mollusk into whose gonad it's implanted? Is this a question of trying to reduce overall injury to recipient mollusks, or something else?

If trying to create a pearl in mantle tissue, why use donor mantle? Why can't the recipient mantle create its own nacre? Why not just implant some irritant, instead of using foreign mantle?

How is it that mantle tissue from another mollusk of the same species implanted into either the gonad or the mantle isn't rejected, the same way a heart transplanted from one person to another would be rejected?

I know that this is a long post, but I think that if I can come to an understanding regarding these issues, I'll have a far greater understanding of pearl culture. Thanks a bunch!

 

[GemGeek]

Where will you be growing these mollusks?

 

[Caitlin]

1. have a few questions regarding nucleus implantation. It is my understanding that implanting a nucleus in a salt water mollusk (South Sea or otherwise) is difficult relative to implantation in a freshwater mollusk. Why is this? Is it because gonad implantation is difficult relative to implanting in a recipient mantle? Along with this, aparently once the technique is learned, it takes quite a while to sufficiently master it so that a quality pearl is produced. I'm guessing that this aspect has to do with proper mantle graft implantation. Is this true?
2. Why is it that there are different implantation techniques for salt water and fresh water mollusks? It's my understanding that regarding salt water nuclei, they are implanted in the gonad with a piece of donor mantle tissue. Aside from mabe pearls, this is the only way that things are done.
3. With freshwater mollusks, one nucleus, along with donor tissue, can be implanted in the gonad, or a piece of donor mantle can be implanted in the recipient mantle, or a piece of donor mantle along with a nucleus can be implanted in the mantle. Why are there so many options for freshwater mollusk implantation that don't exist for salt water mollusk implantation?
4. Why is a piece of donor mantle used for gonad implantation? Why isn't the mantle cut from the same mollusk into whose gonad it's implanted? Is this a question of trying to reduce overall injury to recipient mollusks, or something else?
5. If trying to create a pearl in mantle tissue, why use donor mantle? Why can't the recipient mantle create its own nacre? Why not just implant some irritant, instead of using foreign mantle?
6. How is it that mantle tissue from another mollusk of the same species implanted into either the gonad or the mantle isn't rejected, the same way a heart transplanted from one person to another would be rejected?

I want to try to answer a couple of your questions. I know if I say something incorrect, someone will post to let me know, so here goes
Some of your questions have more than one part.
1. Yes, the technique is very subtle and takes practice and a delicate touch, probably something like the skills needed to play a piano, it takes talent and time to get the technique.

2. I believe the insertion of a piece of mantle into the mantle is a far easier technique than into the gonad. I don't know why they don't just wound the pinctadas in the mantle, that is how natural pearls are formed. Natural pearls are actually always found in the mantle. The culturing process into the gonad can also start a keshi making process in the mantle, so that there are often itsy-bitsy pearls in the mantle when they remove the one from the gonad.

The reason a piece of mantle tissue is implanted is that a bit of the edge of the mantle where the nacre making cells are is the only way to get a pearl growing.

So it is not strictly an irritant, but anything that damages the mantle a bit so that some nacre making cells can get inside the mantle where they keep on producing nacre will make a pearl. The trick is to get those edge cells into the mantle whether by fish bite, parasite, or another slight wound that carries the nacremaking cells into the mantle. This is not as easy to explain by just saying "an irritant" or PearlGoddess forbid, "a grain of sand". Sand won't do it because the mollusks of all kinds can easily get rid of sand with mucous. Douglas has a YouTube video that demonstrates it.

3. The freshwater mussels are big and easy-going. They can tolerate a lot of tiny implants. The gonad thing was invented, I think, as a way to get a round pearl. It appears to be far more difficult to get a good pearl from a mussel gonad, so the technique has been worked on for a couple of decades, but has only given enough smooth pearls to sell very recently. Before, all the gonad pearls in mussels produced mainly ripple pearls.

So there are three pearlmaking techniques, the mabe, the mantle and the gonad. The natural equivalent of mabe are blister pearls, which often encase a parasite or cover a hole a parasite made, so only the gonad is a new manmade technique, the others happen in natural pearls..

4. They sacrifice one mussel to produce enough mantle tissue for many implants. In FW pearls, if they took all the mantle tissue off the same guy they want to implant, it would be sorely wounded, because there can be dozens of implants in each mussel.

5. I think you may know the answer to this already, from my previous answers. There must be some nacre producing
cells INSIDE the mantle to get the pearl sack growing. It is a lot easier to implant a foreign piece of mantle than try to irritate the mussel enough to carry some cells inside the mantle. That is a very hit and miss technique, which is why natural pearls are so rare.

6. You are not the first to think of this, but I don't believe it has been done, though we once had a faker who said she had done it- transgrafting; she faked us all out for awhile, LOL!

I'll come back to this. I am in shock about the hurricane hitting Guaymas as we speak. Facebook has the details.

 

[Mostawesomecoffee]

thanks Caitlin, that cleared up a lot!

So, for anyone just jumping into the post, it seems that I/we are down to two qustions of the original six:

1. why is foreign mantle tissue implanted into the recipient mantle? Can't the recipient mantle create a pearl on its own with the help of an irritant?

2. why isn't the foreign mantle tissue rejected, as an organ transplant between people would be?

 

[Lagoon Island Pearls]

I don't believe it has been done, though we once had a faker who said she had done it- transgrafting; she faked us all out for awhile, LOL!

Xenogeneic grafting is well known. It's the prerequisite in most natural pearls, especially where parasites are concerned.

Homogeneic and Xenogeneic Implantation in pearl mussel surgery.

 

[Caitlin]

Thank you Dave! That is a fantastic article. I hope you can answer some of Mostawesomecoffee's questions. So, our faker was only lying about sponsoring such a project, not about transgrafting, itself. I knew she wasn't creative, but merely borrowed all her info and presented it as her own.

MAC, Dave works with many unusual species of mollusks and retrieves their natural pearls as he encounters them He may even know the species you are interested in.

Question 1. Because so many little pieces of mantle tissue are implanted in the mussel, if they all came from their own mantle tissue, they would be severely injured. However in natural pearls, the pearl reacts to any irritant with a pearl sack,only IF cells from its own mantle get into the meaty part of the mantle.

Also in natural pearls, many of the tiny ones are imbedded in the edges of the mantle, which sometimes must get pretty irritated to produce them. We have some phots somewhere, maybe they are Douglas'.

2. I don't know why similar, but foreign tissue is not rejected, but my guess is that since the nervous system is missing in mollusks, that tissue rejection has not yet evolutionarily emerged in mollusks, but will show up in more complex and recent animals.

According to the research paper link Dave gave us, different species can be transgrafted into each other. Though the early results have a low success rate, no doubt if there were a commercial application, there would be people doing it.

 

[Lagoon Island Pearls]

Question 1. Because so many little pieces of mantle tissue are implanted in the mussel, if they came from their own mantle tissue, they would be severely injured. However in natural pearls, the pearl reacts to any irritant with a pearl sack,only IF cells from its own mantle get into the meaty part of the mantle.

The first part is correct. It serves no purpose to graft a mollusk's own tissues elsewhere within it's anatomy. It extends the convalescence period if not raising the mortality rate. From a farming standpoint, there is no viability in the procedure.

As a rule, the second part doesn't happen. There can be exceptions, but it's rarer than rare. For a predator, the easiest point of entry is the periostracial mantle, afterall it's merely soft tissue directly exposed to the water column. Mollusks are generally adept at detecting and responding to this stimulus, hence the shell closes and/or the foreign body is sloughed in mucous. Some predators however, have hooks or beaks and perforate the epithelium. Some predators, such as Polydora take the indirect route and drill through the shell, often causing blister pearls. These can be classified into a category known as extrapallial pearls. Grafted pearls are not extrapallial, they are intravisceral.

This is where the term "irritant" becomes irritating to the point of ad nauseam. Pearls will form and grow faster in the absence of irritation. In the case of a boring predator or physical shell damage incidents, they essentially "split" the epithelium. The adjacent surrounding cells reproduce rapidly to encapsulate the intruder or bridge the gaps. To form a pearl with decent appearance, the epithelial cells must reproduce quicker than the predator itself can grow, otherwise it will burst the sack. Sometimes, it's a vicious circle, sac bursts, sack encloses, sack bursts, sack encloses and we'll end up with some funky looking pearls. This is very common in blisters.

Also in natural pearls, many of the tiny ones are imbedded in the edges of the mantle, which sometimes must get pretty irritated to produce them. We have some phots somewhere, maybe they are Douglas'.

The edge of the mantle this year, will not be the edge of the mantle next year. With the mantle extended, individual epithelial cells generally remain at the same distance from the heart. As the mollusk grows, new cells divide and multiply at the edges, occupying new space in the water column or substrate.

Blaire (GemGeek) touched on this in her talk at this year's ruckus. She spoke to apoptosis aka programmed cell behavior. Newly formed cells produce mainly proteins (conchiolin) which provides a water-tight barrier from the environment. Once the barrier is extruded, the role of the cell changes to mineralize calcite, constructing prismatic lathes, which serving to thicken and harden the shell. After a few months to a year, the role changes yet again to nacreous, foliated or columnar (depending on species), where spherules extruded from the cells lay up the classic orthorhombic forms of aragonite. Senescent (old age) cells revert to producing calcite. Calcite is the bane of pearl culture and collection, occluding the deep luster and giving a chalk-like appearance which greatly reduces their value. This is why farmers prefer to produce pearls from juvenile growth. Peeling inferior pearls will often result in smaller, higher quality pearls within.

In nature, shell damage is commonplace. Logs, tumbling stones, feet etc. can break shells. During this trauma, the mantles are "split". Any foreign body, including the proverbial grain of sand can become lodged in these splits and form pearls. Not because they are a "graft" but because there are already epithelial cells present. Graft technology as we know it, is something completely different. It introduces foreign epithelial cells to a part of the anatomy that otherwise has none, but can be sustained by blood supply.

2. I don't know why similar, but foreign tissue is not rejected, but my guess is that since the nervous system is missing in mollusks, that tissue rejection has not yet evolutionarily emerged in mollusks, but will show up in more complex and recent animals.

Mollusks have nerves, but not brains. Cephalopods have a crude brain in the form of a ring surrounding the esophagus. Rejection is largely physical from muscular contortion. "Irritated" cysts often contain necrotic cells (dead tissue) from both predator and host and granular tissue (scars). These will very often become septic then burst and drain or they may not, but generally will not form pearls because healthy epithelial cells are not present.

As a rule, epithelial cells DO NOT appear spontaneously out of the blue. Either they are present naturally or they are introduced artificially. A few years ago, I posted an image of a thousand pearls in one mussel. Without a doubt, those pearls were not individually created by the processes I just described, but by a blood-borne condition. Hormonal disorders, blood acid/base imbalance or protozoan (or other organisms) infection can cause pearls to form. In the blood stream, pearl sacs indeed form, but by RNA/DNA or other aseptic factors at levels few understand.

I have just spoken about three distinctively different pearl types. Extrapallial, intravisceral and autoimmune. All to often, aspects of one are needlessly confused with the aspects of another, hence the perpetual wheels of myths are kept turning. To an expert, the myths are off-topic and to the layperson or someone trying to learn, it's misleading.

And finally to the OP.

Without a lot of groundwork on species, tenure, anatomy, biology, oceanography, etiology, government, marine engineering... and a whole lot more, do not preoccupy yourself with a graft procedure. Cross that bridge only when you get there.

If I may offer an anaIogy, I can tell you where to buy a Stradivarius violin. I can teach you how to read music. However, I doubt you'll be joining the New York Philharmonic by the end of the week based on what I've done for you.

No farmer, no expert, no scientist can tell you how and what to do because what works for them may not necessarily apply to you. They are not in the business of enabling your failures, no less stepping on their own toes or those they are associated with. Today, production from most farms are at a bare minimum of traditional harvests. Please remember you are asking for what is often proprietary information, unpatented and premised upon years of research, trial and error. While Pearl-Guide is a terrific resource, it's neither a school nor a free-for-all service under demand. Technology is borne of education, hard work and sound business relationships and collaborative efforts with trustworthy colleagues. I have been passionate about pearls since I was six years old and have nearly forty years of intensive field research and development under my belt. Although I enjoy successes and continue to do experimental production runs, I am only just arriving at "procedures" to build a business model around.

In all honesty, had it not been for biomedical research, public health and safety monitoring, natural pearl collection, cataloging, film making and other diversified maritime duties, I'd never have a hope of succeeding as a pearl farmer.

 

[CortezPearls]

Hey! Got here and Dave has all the correct answers! Really happy with the answers and don't feel like I would have to add anything else.
Kudos Dave!

 

[GemGeek]

Great information, Dave.

 

[Caitlin]

Thank you Dave. I love it when you correct me! I think you should write an article that we can direct people to, when they ask how pearls are formed.

Meanwhile, I got my info from Strack, page 115, if I remember correctly without looking it up. She had diagrams of each step as I described it in layman's terms. Cells from the epithelial tissue must be introduced into the body of the mantle to form a pearl sac. And, I was only describing that one process, even though I know pearls form in other ways too, ones we understand and ones we don't understand.

In my personal experience I learned that only 1 of 500 radiatas (Persian Gulf) has a good pearl. But a far higher rate of mollusks have tiny seed pearls. Almost all that they open, especially in certain beds, have tiny clumps of nacre. Probably only half of those are drillable, but the ones that are yellowy and odd shaped are/were not considered good pearls and not counted.

I know that other processes also work to form pearls and whatever they are, it happens to a great many oysters in the Gulf, esp. around Bahrain. That area also has a lot of freshwater springs rising directly into in the bed of the Gulf. My grandfather was able to tap one on Umm' a Sabaan, and it had so much fresh water, they can't use it all- or couldn't in 1950.

I never asked, but now I wonder if some element such as too much freshwater passing over the beds can cause mass reactions in the mollusks, which produce good pearls at a higher rate than anywhere else in the world. Could that at least be why there are so many seed pearls in Gulf radiatas?

Is Strack wrong? Since Mostawesomecoffe is a beginner, I tried to keep it simple, but how wrong am I?.

 

[Caitlin]

How do keshi form in the culturing process? Is it a kind of systemic reaction to a gross violation of their gonads?

There are a lot of questions yet to be answered about how mollusks respond to the environment by forming a pearl. Why don't most natural pearls have a bug in the center? It appears to me that the overwhelming majority of those x-rayed have nothing in the core.

 

[Lagoon Island Pearls]

Okay, before I go into detail, I'll make a disclaimer. These are not to scale, nor are they meant to provide information on definitive techniques used by farmers. It's a generalization of cell behavior based upon my field observations, peer review and common knowledge fundamentals of invertebrate zoology.

Let us begin. Fig. 1 describes basic shell structure in most mollusks, I'll detail a few marked differences between species later.

A shell grows into the water column or substrate by expanding at the level of the periostracum. The inner surface of the middle fold creates a pair of thin sheets of conchiolin to provide a barrier to water. Soon after extrusion, this barrier turns almost 180 degrees, creating a pocket. As new epithelial cells grow, they excrete calcite into the new space. After a time, the cell refines itself to produce spherules which then form into tablets of aragonite. There is some discussion whether this process is entirely intracellular or some crystallization is extracellular, but we'll leave that for another time.

When we observe the mantle, we can see several things. A muscular structure, mucous glands and epithelial cells. There are two types of epithelial cell, inner and outer. The outer mineralizes, the inner does not. Instead forms a skin to protect the mantle and viscera from the outside environment.

Fig.2 represents a hypothetical graft donor selection. The technician will trim away the outer folds, but retain the flat juvenile growth behind it. The purpose is to be rid of extraneous tissue and concentrate juvenile epithelial cells. When a mantle is out of the water and dissected, much the water falls away, making the graft much thinner than normal.

Fig. 3 is a cross-section of the select donor tissue. A suitable graft must have both epithelial layers, mucous cells and some musculature to hold it all together. To have any chance at becoming a pearl sac, all three elements must be present. I realize you've asked this question before Caitlin, this is why simply nicking and cutting the mantle at random does not give rise to keishi pearls.

Fig. 4 demonstrates orientation of the graft, bead and blood stream. For a pearl sac to grow, it relies on adjacent cells to continue to grow by multiplication and division only. Epithelial cells will not appear spontaneously.

How do keshi form in the culturing process? Is it a kind of systemic reaction to a gross violation of their gonads?

No. While there can be no doubt the autoimmune response to lesions of the gonad is substantial, the process itself is not necessarily any different than pearls in the mantle. For the most part, the pocket surrounding the gonad provides an ideal space to grow a bigger, rounder pearl. Mantle grafts generally develop flat spots, baroque shapes and are much smaller. Chinese hybrids were bred to produce thicker mantles, creating more space for larger, multiple pearls.

Fig. 5 is a generalization of random mis-orientation of the graft. One shows a proximal graft where the outer epithelium no longer contacts the shell bead nucleus. While the cells are healthy, there is no stimulus to form a sac around the bead, instead forming a sack around itself. The other demonstrates and example of separation, but distal to the heart. This effect can do one of three things. A- fail, B- form a keshi or C- become bridged to the bead, forming what we know as "fireballs". Again, I emphasize this is not in every instance, but only to give a general description.

Fig. 6 is a scenario where a predator (in yellow) has perforated the outer epithelium. In this case, the cells have reproduced enough to encapsulate the intruder. If the predator is too large or becomes necrotic, a sac cannot form. Instead a fluid/dead cell cyst is likely to occur if not subjected to mortality.

There are a lot of questions yet to be answered about how mollusks respond to the environment by forming a pearl. Why don't most natural pearls have a bug in the center? It appears to me that the overwhelming majority of those x-rayed have nothing in the core.

Fig. 7 demonstrates perforations in the outer mantle epithelium. This can happen in many ways. Attacked by a predator, split by cracked shells etc. Healthy epithelial cells become isolated from the existing structure, hence remaining viable. In this case, a sac can grow, possibly even two sacs. Usually the outer one, will over-rule the other. I have observed this in many of my pearls several times, a pearl within a pearl if you will, however a single sac, natural pearl in this case will often reveal nothing at the nucleus. Attacked by predators in this manner can give rise to multiple pearls in the mantle, even though no bug itself was encapsulated. All they need do is bite it, for lack of a better term. From my field notes, only about 5% of natural pearls actually have the bug inside. It's like amber, if I may compare to another gem.

Fig. 8 shows a rare occurrence, but documented nonetheless. Sometimes, especially if a shell becomes dry, the outer periostracial layer shrinks slightly. When this happens, the inner layer can prolapse during the 180 degree turn. This can perforate the epithelium and give rise to natural pearls at the edge of the mantle. It's usually a single event, creating a single pearl.

Fig. 9 One footnote I'd like to add is the "purple" we often see in pearls. While the research is incomplete as to exactly why purple, one common factor in every case is a dual-outer epithelium. This occurs where two shells, shell and pearl or even two pearls are very close to each other. It is micro-thin and mineralizes from both sides, unlike normal mantles which mineralize from one side.

One other footnote. Pearl oysters in general have retractable mantles and gonads contained as a single organ. Mussel differ in that they do not retract, but instead are attached to the shell at the level of the prismatic layers and no singular gonad, but a series of gonoducts that share the same space as the mantle. Anything I graft into mantle also has the effect of grafting into a gonad.

I never asked, but now I wonder if some element such as too much freshwater passing over the beds can cause mass reactions in the mollusks, which produce good pearls at a higher rate than anywhere else in the world.

A good question. We get a lot of rain in winter. I have a little saying... It only rained twice last week, once for three days, once for four days.

All living things need calcium, lots of it. During periods of low salinity, mollusks are very adept at recovering calcium from their own shells. Yes, you heard me correctly... the role of the epithelial cells reversed themselves, instead to excrete acids which creates a calcium-rich slurry they can reabsorb into their tissues. This process of give and take, can give rise to extraordinary quality pearls in some cases. the previous surface is removed slightly, then a newer... more juvenile layer can be laid up later. Too much freshwater can cause shells or pearls to deteriorate. Although susceptible to freshwater diseases, freshwater helps to control saltwater diseases and predators and provides for a healthier environment overall.

 

[Lagoon Island Pearls]

Let us touch on xenogeneics for a moment. My octopus pearl is a perfect example.

We all know octopuses do not have shells. We do know they are green blooded creatures (copper based as opposed to iron in mammals), as are all other mollusks.

My specimen had a visible scar from a predator attack. One and a half legs were amputated. Octopus recover upwards of 25% of what they eat in body weight, hence grow fast and eat lots.

The consensus is this. The octopus escaped the attack alive and went on with his routine gathering food. Because the proximity of the pearl from the beak was a mere inch, it stands to reason a small piece of mantle tissue from the octopus's meal ( a clam, mussel, oyster or cockle perhaps) drifted off to become lodged in the wound. The octopus's own blood stream supplied the "graft" and the epithelial cells continued to grow, forming the pearl.

It's really quite simple.

 

[Caitlin]

Wow. I don't see the figures, but that was an impressive amount of explanation presented in language I understand. This is great! Have you ever written an article for a peer reviewed journal? You could write peer review quality articles, and then give us the layman language summary with the article as an attachment for those who want to go deeper like coffee. Or something.. You have wonderful examples. You write well and your information is unobtainable for most of us without a whole lot of research and maybe paying to read.
There are no attachments to this article, yet. I hope you can put them up soon.

 

[Lagoon Island Pearls]

Wow. I don't see the figures... I hope you can put them up soon.

Oops, the attachments linked but didn't load correctly... I'll re-do those now.

 

[Caitlin]

MACoffee- I can't find the right thread to make this comment on, but if you are still there, and not discouraged by those who have done it, I wonder why you want to grow mollusks. When did you embrace the idea? There are several ways to go with your idea. The first would be to experiment and see what happens. Another would be to be an armchair pearl farmer (at first) and study the foundational articles of information on your species of mollusk. There is no telling what info you will find. It may be that some of the writers kept them in tanks in labs or something. Research has already been done on your species, though you may have to go to a University library to access it.

 

[lisa c]

Caitlin, have you stickied this thread yet?

 

[Lagoon Island Pearls]

Have you ever written an article for a peer reviewed journal? You could write peer review quality articles, and then give us the layman language summary with the article as an attachment for those who want to go deeper like coffee. Or something.. You have wonderful examples. You write well and your information is unobtainable for most of us without a whole lot of research and maybe paying to read.

No and I'm too much of a bucket scientist and enjoy the field work too much.

 

[Caitlin]

Well, this kind of writing and explanation is like an article and we are the beneficiaries. I just saw the diagrams and think this is an important thread. I will sticky it, but I hope it also get promoted to being an article. The forum can do that now, but it hasn't happened yet that I know of. I am also starring each of those posts. They each represented some good thinking and some hard work writing. Thank you.

Another option for everyone that should on the bottom edge of each post is to blog the article to your personal pearl blog. I am not sure if this button is in general release yet. I think I will try it with these posts. This blog can serve as a kind of personal bookmark system for all to keep track of the posts that are important to them. It just does each post, I think, but I haven't used it yet.

 

[lisa c]

Oh, I see that gizmo now!