Entry of Zooxanthellae into the Ovum of Millepora spp. and Skeletal events at liberation

Methods and concepts:  

cursory description

I began collecting fragments of Millepora spp. in August, 1984, with the intention of eventual statistical analysis of the times of reproductive activity.  Observation of a liberation event of medusoids, on an evening in early April, 1985, removed any question of synchronous spawning: study of Millepora platyphylla turned a corner.  I would collect and preserve tissue specimens to pair with the hard parts fragments, to ferret out the sequence of events---development of medusae and gametes, opening of ampullae, and liberation of medusae.   At that point, it became. feasible to focus collection within certain meaningful time frames.  

The Moon that night was 4 or 5 days past Full.  I had reason to believe that this was a synchronized event; if this was the case, it would be useful to focus future collection effort on the days leading up to this night of the Moon.  A question remained: was this a synchronous event among populations on Guam?  Immediately on the next morning, I made forays on other accessible reefs, all on the Western Coast of Guam.  Other colonies demonstrating similar appearance were observed elsewhere, suggesting that this was a synchronous event on a scale greater than a single reef.   

Since I had been focusing my attention on this single species complex, I recalled that a few days prior to this liberation event, an unusual spectacle was observed among a number of colonies at Gun Beach (Fafai), North of Tumon Bay.  Some proportion of colonies had turned a darker brown color; and tiny white rings--less than 1mm in diameter---had appeared, densely scattered irregularly on various parts of colonies.  The stark white of the rings presented a very pleasing contrast against  the dark backgrounds of these colonies.  I suspected the darker colored colonies might betray proliferation of symbiotic dinoflagellates (probably Symbiodinium sp., here called zooxanthellae), or an increase of photosynthetic pigments.   

Since Millepora spp. are widely reported to exhibit vertical transmission of symbionts---ova being imbued with zooxanthellae before their fertilization---proliferation of zooxanthellae might actually be required to generate a supply sufficient for infection of the egg.  Remarkably, as of this writing, 37 years later, I have neither met nor heard of anyone else who has observed this remarkable event.  This points to the importance of focusing field studies on a single species (pointed out by George Barlow as a watchword of the biologist Karl Roehder).  It is one of the most remarkable exhibitions I have ever witnessed.

At this point, due to a previous interest in gametogenesis, field collection took on a new aspect.  Follows a cursory description of field and laboratory treatment of specimens.

• Fragments of Millepora platyphylla (and sometimes M. dichotoma) were broken off from identified colonies with a masonry hammer, and immediately collected into zip loc bags with a good quantity of sea water.  Each specimen---or perhaps specimens from the same colony---was isolated in its own bag.   Each specimen was marked in graphite pencil with date and place of collection.
• On the beach, or perhaps in the laboratory, part of each specimen was fixed in one of various solutions, and matching part was immersed in a tank of Calcium Hypochlorite (swimming pool chlorine) for a few days.  
• Fixation: some specimens were fixed in an acidic fixative, like Bouin's, or Formaldehyde with Formic Acid and other solutions.  After a few days, others were immersed in various decalcifying solutions.  
• The tissue of Millepora spp. comprises as thin sheet, <=1mm thick, that lifts off of the CaCO3 skeleton in the dish during decalcification.  These sheets were cut with scissors into smaller sheets that could be handled, for dehydration and embedment in paraffin wax.  
• Some blocks were sectioned on a rotary microtome and slides prepared. 
• Casson Trichrome was relied upon for staining, for the most part.  

I left Guam and moved to Chuuk, in late 1996.  I did not have a microscope, but had a number of the the slides I had prepared on Guam.  These slides mysteriously disappeared from my classroom at Chuuk High School, a year or two later.  

Thanks to a friend, my remaining blocks and some of the matching skeletal material was recovered some years later, and brought to Saipan.  I have observed and collected a few skeletal fragments on Saipan, between 1999 and 2001.  A spring timing similar to that on Guam, and similar lunar synchrony seemed to be indicated by these sparse observations.  I did not have the wherewithall to preserve tissue specimens during those years. 

Thanks to the interest of coral pathologist Esther Peters, a number of blocks were sectioned, slides produced and stained, which I am still studying some 20 years later.

In Berkeley, a opportunity arose to section about 4 blocks, out of the nearly 200 that remain.  Out of the kindness of a laboratory manager, slides were then stained with Hematoxylin and Eosin.  I had almost randomly selected blocks to section, based on my hunch that darker tissues represented darker colonies that could be endowed with medusae in a state of near readiness for exodus from their home colonies, and spawning.   The results of this   selection seem to support the supposition that darker colonies are reproductively active: at least three blocks yielded sections displaying medusoids.  

 

I hypothesize that reproduction is seasonal on Guam, that the first reproductive event  happens in late March or early April, and that medusoids are released 3 to 5 days after Full Moon (with some exceptions), just after Sunset.   This evening is the first of the lunar cycle when a short period of full darkness happens after the setting of the sun, before the rising of the Moon.  This mode of reproductive timing seems common among marine animals.

 Induction of Zooxanthellae into the Ovum

Here are presented   images  from the ongoing study of existing slides, portraying stages in the approach and incorporation of zooxanthellae into the egg.

Cluster of Zooxanthellae in Immature Medusoid

Above, within a medusoid---apparently flattened during sectioning---zooxanthellae are dividing.  One possibility seems to be that zooxanthellae proliferate during the events leading up to and during the process of entering the medusoid and ovum.

Early movement of zooxanthellae toward developing ova

Phase contrast image.   Note zooxanthellae crossing the field of lipid droplets.

Among the presumptive ova, only three in each medusa, develop into mature eggs.  As seen below, other cuboidal cells provide nutritional support for the growing ovum.  These are termed here "nurse cells." 

Zooxanthellae are nestling up to the Ovum and Nurse Cells.

In the above image, the larger cell at the bottom is one of the row of rectangular cells that are potential ova, but are here called nurse cells, because they nurture the successful ovum,  coalescing with it.  The nuclei of the nurse cells are not as distinctive as the large nucleus of the ovum.  Small zooxanthellae are embedded in the matrix of Lipid Droplets, and are apparently approaching the egg.  They are not yet incorporated.  The definition of "ovum" seems unclear: are the lipid droplets part of the ovum, or not?

Zooxanthellae swarming and dividing

Ovum at a more advanced stage, with zooxanthellae well along in the process of invasion.  
Numerous zooxanthellae are dividing, even within the egg.  

The above image is a brightfield focus stack (with a 40X objective; scale to follow) showing a medusa well along in maturity.  At top and bottom are nuclei of nurse cells; the nucleus of the ovum is distinctive, and larger.  Zooxanthellae at the bottom left, outside the medusa, are not as actively dividing.  

Below, the same subject is shown in Phase Contrast.  

Phase Contrast, Focus stack by Helicon Focus

Opening up of Ampullae

Medusoids---medusae that are incompletely developed---develop within closed pits, the ampullae.  In order for the medusoid to escape, the covering of an ampulla must give way, or (thought it seems unlikely) be broken open by a flailing medusoid.  In the first image below, several ampullae appear to be fully open. 

The following image presents a conundrum, in that one ampulla still retains its covering, while an open one is seen farther to the right.  This begs the question whether a subsequent release may happen.  I have not made sufficiently detailed inquiries to determine whether medusoids may be released over more than one day in, as I presume, a monthly event.  This specimen has been cleaned in

Above, the encircled ampulla is intact, possibly slightly eroded in center.  The depressed condition was identified by Boschma as a diagnostic character for Millepora platyphylla.  On the right, the ampulla is open.  Smallest pores are dactylopores; they are arranged in a "cyclosystem" around the somewhat larger gastrozoids.  Boschma also noted that M. platyphylla cyclosystems are irregular, not neat encirclements as in at least some other species.