Clustering proliferation of dividing symbiotic dinoflagellates in Millepora sp. medusa/egg

 I will post this image for now, and get back to it.  These symbionts infect or are drawn into the medusa/egg before liberation from the mother colony.  As far as I have seen, all medusae of a given colony are either male or female.  The larva is pre-charged with it's symbionts.   The order and nature of events during the infection process are of great interest.  

From a stack of 9 images.

Annotated image to demonstrate the dividing cells with two nuclei.  Other cells, more visible in the image above, are near enough that perhaps they are just recently divided.

Ongoing imaging explorations

Millepora platyphylla: interface between medusa and ampulla. 

Phase contrast

 

I was exploring phase contrast with a new--to me--used Zeiss Plan Ph2 40/0.65 160/0.17 objective.  This slide was stained using Cason Trichrome stain.  

This shows a part of a medusa, with scarcely developed ova.  I think most of will contribute themselves to the growth of the three, or possibly, sometimes, four ova that eventually develop.  These may be called nurse cells?  

A meshwork of vacuolated material---lipid droplets---is being traversed by some zooxanthellae.  The eggs of Millepora spp. are hosts to symbiotic dinoflagellates (zooxanthellae) before their release and fertilization.  This is part of the process.

 

 The ampulla is a hollow vessel that holds the medusa as it develops.  The tissue outside the medusa is apparently related to the ampulla.  Intense yellow indicates calicoblastic tissue.  I am interested that in this image the yellow resolves into green and yellow particles; I wonder, does this point to two separate structures involved in producing---and in this species sometimes dissolving?---Calcium Carbonate.  To my knowledge the structural changes involved in generation of ampullae, in conjunction with growth of the medusae and development of the gametes in both male and female medusae has not been worked out in its entirety.  The reproductive events, per se, have been studied closely.

 

 

 

Photographs: structure of Millepora platyphylla skeleton

The Tabulae are apparent in the following photographs of Millepora platyphylla Skeletons

 

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I was attracted to the study of Millepora platyphylla because of their hard parts, their skeleta.  My overriding interest was in reproductive timing.  The Milleporid  Hydrozoan corals had me from where I learned that their reproductive status is heralded by certain characters in their hard parts, the ampullae, which are exhibited only when they are reproductively active: the ampulla is a protective cove in the skeleton that houses the medusae, the reproductive individual that carries forth the gametes.  I reasoned that information regarding seasonality of reproduction could be learned by studying time series of their the hard parts.  

I have to admit that I have learned little about the hard parts of Millepora.  Much more could be learned.  

 

Recently I have been awakened to a seriously interesting aspect of the biology of Millepora spp.: the degradation of the tissues, and the zooxanthellae, especially in the lower layer of the living tissue.   This, in turn, has turned my attention to another aspect of the morphology of their skeleta:  as the tissues lay down more and more layers of the Calcium Carbonate---the skeletal tissue---the polyps produce a sequence of new basement plates.  Akin to the sequence of corallites in scleractinian corals, these plates leave behind a sequence of consecutive chambers, evidence of the progressive accretion of the massive skeletal structure, the corallum. 

These plates are termed the tabulae.  Interestingly, tabulae are also described for the extinct rugose corals, in the _Treatise on Invertebrate Paleontology_.  

 

I note, in part:

• The distance between successive tabulae varies.  One wonders to what extent growth rate of the corallum is determined by, or alternatively determines, the gaps between deposition of these tabulae.  
• The surface of the colony is planar.
• Structure is organized at several levels: (1) gross morphology (tabulae and apparent palisades forming a corallite wall-figure 5);  (2) orthogonal crystalline (trabulae?)--figure 3; and over the broken surface may be seen grains, as in figure 1 and others.
• thick bands orthogonal to the tabulae are visible 

 

Photographs

The following photographs are presented.  It is planned to continue to capture other views of the hard parts of Millepora spp.

 

1

2

3

4

5

Much remains to be learned from studies of the skeletal remains of Millepora platyphylla.

These images were made with a Canon EOS M50, with the 28mm EF-M macro lens.  This is the maximum magnification with this lens, without additional add-on supplementary lenses.  This lens does not accept an extension tube.

 

 

Note to me: Processes during development of the Ova in the medusa

My interest in Millepora spp. (or just Millepora) begins with reproductive timing.  My interests range into seasonality and annual cycles, lunar cycles, daily (circadean) cycles.  I started paying attention to Millepora when I learned that the Ampullae were present in the hard parts (the rock, the skeleton) during reproduction; thus, if I collected a few pieces of their skeleta every time I went to the reef, I would eventually have a record of reproductive activity, enabling deeper understanding into the seasonality of this hydrozoan coral.  I was actually interested in neurosecretory control of gametogenesis,and had hoped to study giant clams.  But these charismatic and delicious macro-molluscs were too few and too far between for such a study on Guam.   Millepora was an interesting option.  Although I did not intend this to be my thesis topic, thse proved to be an intensely interesting organisms.

One night, I was invited to tag along on a night fishing foray with  friends from Pingelap Island.  That night, in early April, 1985, I was thrilled to observe Millepora shedding medusae!   The next day, I drove around the Western side of Guam and sampled Millepora at several sites: these sites also proved to be reproductively active. 

So, it was actually possible to observe reproduction in real time.  My study caught fire at this point.  I started collecting tissue specimens along with the pieces of skeletal material.  I made a point of collecting paired specimens from each colony, and I marked the colonies. 

I have some 200 specimens, collected from different colonies at different times over two seasons on Guam.  Much of my second year at the University of Guam was spent working on these specimens.  Over 200 tissue samples were fixed and embedded in paraffin.   John Starmer rescued them from Guam, when they were about to be thrown out.  

To make a long story short, my interest in Millepora has been focused at the microscopic level.  While I've been trying to grok tha variables involved in attaching a camera to a microscope, I have been focusing on primarily events at the level of developing medusae.  Millepora  hydrocorals are hydroids, that produce ephemeral medusoids (short lived free swimming medusae that exist to carry gametes off and spawn).  

Is it not obvious that the timing of the development of these medusoids is part of the complicated dance that led to the synchronized liberation of medusae observed on the reef that night, at Toguan Bay, Guam.

Here I will post some photos made with a cell phone of a slide from a specimen collected on 9 April 1986, the night of the New Moon.  I have made some assumptions about the timing of reproductive events in Millepora.

 Assumptions (for Millepora platyphylla)

1. Liberation of Medusae is sychronous, on the same night.  At several sites on Guam, liberation was evident on the same night, as far as I could tell.
   
2. Medusae are released/liberated (or alternately break free) on the fourth or fifth evening after Full Moon.
3. Reproduction is seasonal.  The season is about three or four months long.  The first event is in early April or late March.
   
4. Reproduction happens each month during season.  

 

Millepora dichtoma was reproductively active later in the Summer.

 

 

Some cell phone micrographs (not recent anymore)

I have been studying slides of Millepora platyphylla more closely recently.  Currently, effort has been expended toward getting a higher quality camera setup for my microscope.  More photos will be posted in the near future.  

This is a remarkable organism.  In just one slide, so many discoveries!  I still have not mastered the anatomy of this hydrocoral, so at this point, it's about exploration.  

Here are some photos I made tonight with my cell phone, a OnePlus 8t+.  I used the Pro mode, set to Raw.  This camera does not produce useful "raw" photos, though.  This mode works decently, but in these photos can be seen the kind of blurring I typically see with this camera.  

I cannot explain any of them well at this point. I post them now with the intention of commenting later. 

Here is a blank hole where a zooid belongs, or a medusa?  Probably a zooid.  Notice the nematocysts, so this may be at the top level.  This section is tangential along the extent of the thin tissue (c. 1 or 2 mm thick).

A zooid.  I am curious about the very small cells at what I am tempted to call the floor of this.   Some nematocysts.

The following structures are seen, on this slide, as probably part of the dactylozoids.  They are scattered around in a similar pattern, around the apparent gastrozoid.

The edges, as on the first image, above.  

Another similar structure as above.

Fiddling Around with Microphotography

I'm hoping to have a new camera hooked up on my refurbished scope, soon.  And I also look forward to, at some point, having more slides to study.   More about that later.   

 As for now, this blog has languished for a long while.  Recently, though, I have been diving into study of Millepora spp.   And, most recently, into microscopy and photography.

 Today I was messing around in The Gimp.  The following bits result from my fooling around.  

Recently, I've wondered about the distribution of the medusae on the surface of the colony.  We might ask many questions.  A few:

1. Where do the medusae originate in the scheme of things, on a non-reproductive colony?   
2. Where are all the Gastrozoids and Dactylozoids in this shot?  
3. What is the density of Medusae/Ampullae?
4. This eventually leads to a singular question: where, when, and how do medusae first begin to differentiate?

I have also been noticing the patterns on these slides.  Esther Peters seems to have preferentially sliced these sections parallel with the surface.   I hadn't thought of that!  I had thought that cross sections would be more revealing, so that is what I mostly had done.  I was right, but I think Esther was righter.   It takes a tremendous amount of time to work through the number of cross sections that would be required to cover 1cm2.

I was/am right too, because some processes are better revealed in cross sections---albeit they be more laborious to uncover.   For example, one of the biggest questions is this: What is going on at the surface, where the ampullar tissues and the ectodermis connect  SOMETHING is happening to dissolve CaCO3, I assume, to make it possible for the Ampullae to break their way out.

 

Micrographs without Microscopes

Yesterday I set up to photograph a slide with a macro lens on my Canon EOS M50.  It's a pretty good macro lens, the 28mm EF-M macro lens.   It was a laborious process.

 

The plan was to photograph the slide in the highest possible resolution, on a background of glossy photopaper.  Two ikea desk lamps were used to light from both sides (more would be better, or a ring light), to knock out the shaddows; the previous day my results were horrible, with stark shaddows, as the sections were on top of the slide, shadows being thrown through the couple of millimeters of glass.  This was not as bad, but far from perfect.  

Next I will try a scanner, 1200 or 2400 pixels.

Then I edited to "clean up" the images.  I haven't gotten far, but here are some shots:

With edge detection, inverted.

Cleaned up the background.

 

 

 

Edge Detection: are some medusae paired up?

More Slides

I am posting slides without annotations, for the time being.  Some file names and brief IDs.  All samples collected at Toguan Bay, Guam, by Alan Davis.  Fixation and calcification varied.

All slides prepared by Esther Peters. 

Medusae (Date unknown)

 Slides from Toguan Bay, Collected 7 April 1986

Slides of Millepora platyphylla collected 15 April 1986

 

 

Esther Peters's slide # 3, Toguan BE 530 B 15 April 1986

 

 

Photographed with a Canon 1000SD point and shoot 

The green structures are a puzzle.  Are these the structures Moseley referred to when he stated that he saw zooxanthellae dividing into four parts?

 

Scans of Slides

 

Cropped 311 IID TogD #4 12 April 1986

 

Cropped 548 #4

Some loose ends

 

Mystery Red Cluster: better visualized elsewhere.

Esther's slides #9-18.  (Where are # 1-8?)

Moseley's drawing of a cross section (gastrodermis?)