Summertime (housekeeping)

A personal note. It's summer. A good friend tells of his three reasons for choosing teaching as a profession: Chtistms break, Spring break, and Summer vacation. It's summer again, with more time to spend to catch up on Millepora. This blog is about to be deconstructed and perhaps reconstructed---I haven't decided yet. I'm not sure a blog is the appropriate format for the purpose of THIS project. Even this post is out of place. I have thought about the wiki concept. Maybe something new, or something more traditional. Maybe a regular oldjavascript:void(0) web page. Speaking of which here are two ongoing www items of mine:

• Tank Beach Page
  
• Tank Beach Blog
  

My planned trip to Florida has been cancelled. So perhaps I can use the time to work on this project.

MIllepora day

Either yesterday or today is the day I expect Millepora platyphylla to release medusae here on Saipan.

Notes

Mangan mentions Duerden as describing the medusae of Millepora. Nothing is available of this paper on line, but J. Lewis did cite Duerden.

I do not have Duerden's paper at this time.

A bibtex citation:

@article{duerden1899zcb,
title={{Zoophyte Collecting in Bluefields Bay.}},
author={Duerden, JE},
journal={J. Inst. Jamaica},
volume={2},
pages={619--624},
year={1899}
}

Typescript of note on my 1995-1996 research.

I found the following on my hard drive as a scanned *.pnm file. Here it is, together with notes. I will edit it. I need to track this down, as it contains statements of mine that clarify issues that have become fogged over the years.

[Converted from a pnm file by tesseract, 15 March 2008, AED]
[Converted to tiff file: Millepora2.pnm -> Millepora2.tiff]
[This file was found on my system and run through tesseract on 15
March 2008. It is uncertain when the scan was made. I do not
recall where the original typescript might be located.]
[I have preserved the original typed text. Penned in corrections are
not here preserved, but it is planned to incorporate those
changes later on.]

Preliminary Results of a Study of Millepora sp. and its Zooxanthellae
======================================================================

From 1984 until 1986 I studied reproduction of Millepora spp. in Guam.
My initial interest was in determining whether reproduction is
seasonal. Millepora spp. have unique reproductive structures in the
calcium carbonate skeleta (hard parts), the ampullae, which surround
the developing medusa and gametes. These ampullae are only present
when the reproductive zooids, the medusae, are produced, and are
filled in within a matter of days after the medusae have been
liberated. Thus, the presence of ampullae in hard parts specimens is a
sign that the colony was reproductively active at the time of
collection, and their absence a sign, likewise, that the colony was
not reproductively active. I was determined to track reproductive
effort over time using these ampullae as markers. Later on, as the
study of Millepora spp. took on a life of its own, it indeed became
apparent that Millepora spp. are highly seasonal, and other aspects of
their biology, and that of associated species, piqued my interest.

Specimens of hard parts were collected at irregular intervals
beginning in September 1984 until fall 1986. Beginning somewhat after,
some specimens were decalcified, and tissues embedded in wax for
sectioning. Colonies at certain reefs were numbered and labelled to
enable samples to be taken, repeatedly from the same colony.

In both 1985 and 1986, Millepora platyphyla shed medusae about three
or four days after full moon in early April, in what appeared to be
the first event of a summer reproductive season. Each of these events
(except one, see below) was apparently highly synchronized on Guam,
with apparently substially all the ripe Millepora platyphyla colonies
on Guam reefs shedding medusae on the same night.

The first event of each of these seasons was striking to observe on
the reef. Each "spawning" was presaged by an abrupt change in the
appearance of a preponderance of Millepora platyphyla colonies on Guam
reefs: they turned a richer, darker brown. This change of coloration
seemed to be poorly correlated with reproductive condition; color was
apparently not correlated with reproductive condition in other
months. On many (but not all) of the darker colonies could be seen a
myriad of minute white rings about 1 mm in diameter, contrasting with
the background of dark brown. These white rings evidenced the opening
of the ampullae. Within approximately a week after this color change,
the colonies shed their burdens of medusae (bearing female and male
gametes) in a synchronous ”spawning” event. I observed shedding of
medusae in ziploc bags containing colony fragments at about sunset.
Each colony produced either male or female medusae, as Hickson
reported.


Swarming does not occur a uniform number of days after the full moon,
but usually somewhere between one and four days after full moon. Only
once were medusae observed to swarm on

[end of this pnm image]

The Zooxanthellae

Here I will collect the statements by Moseley and by Mangan regarding the zooxanthellae of Millepora spp.

Moseley's remarks bearing on zooxanthellae

[Space here for quoted text from Moseley]

Certain remarks of Mangan

A large section of Mangan's paper dealing with development of the egg and zooxanthellae:

In the material examined it was clear that the vacuolated substance of the ova in the free medusae, in all cases, contained numerous zooxanthellae; but they were equally absent from the ova of the numerous unliberated medusas that had been inspected.

However, after a long search some half a dozen medusae were found which, I think, afforded conclusive evidence that zooxanthellae pass in numbers from the manubrium into the ovum, and that this invasion may commence at a period prior to the liberation of the medusae.

The ovum, until it has attained a considerable size, is sharply
divided off from the vacuolated substance of the manubrium.
Eventually a period is reached when its compact cytoplasm becomes continuous with the central mesh work (fig. 8). This stage could be commonly observed, and zooxanthellae be seen round the margin of the egg, but never in its compact substance. Sometimes the egg cytoplasm displayed small incipient vacuoles.

The subsequent period, during which vacuolisation of the cytoplasm of the ovum occurs, and during which the zooxanthellae are incorporated, could be found only on one piece of Millepora, where half a dozen medusae had reached this stage (fig. 9). Their ova showed a variable amount of vacuolisation, which, so far as I could make out, is in great part inaugurated at the inner borders of the egg substance; as if in some manner the vacuolated portion of the manubrinm with its contained zooxanthellas were drawn in.

That the medusas are liberated at this stage can be concluded from
their mature appearance within the ampullae, and from the fact
that one free medusa (fig. 10) was met with which exhibited an
essentially similar structure.

The other free medusas examined, possessed ova, more completely
vacuolated, and with more numerous zooxanthellae. The egg-cells
showed various phases of an encroachment upon the manubrial
substance, which eventually was almost entirely reduced (fig. 12),
the ova at the same time becoming rounded off and similar to the
extruded ova that were examined.

During all this period the zooxanthellae exhibit their normal appearance, and it can be observed that they divide fairly frequently within the ovum. These cells have been figured by Moseley ('81) who was able to examine fresh material. He remarks that they closely resembled those of other Hydroids. They contained irregular granules of a bright gamboge-yellow colour,

gamboge
n 1: a gum resin used as a yellow pigment and a purgative;
2: a strong yellow color [syn: {gamboge}, {lemon}, {lemon
yellow}, {maize}]

the cell-contents frequently dividing into two, and sometimes, more rarely, into four (emphasis mine, AED). In the older portions of the colony the pigment; was of dark-brown hue.
I show their structure as displayed in stained specimens (fig. 18). The spherical nucleus exhibited a mass of closely packed chromatin granules. A pyrenoid was always present, the clear space around which, in most cases, gave the reaction for starch. The pigment-bearing granules varied in number and size, did not always stain to the same degree, and in some cases had a little starch associated with them. The cell membrane did not respond to cellulose tests. I observed in a few cases division of a cell into four. Their average diameter was
somewhat over 9um.

That zooxanthellae pass into the ovum from the parental tissues
appears to be undoubtedly the case from the foregoing evidence;
but the part these play in the future economy of the animal
remains to be solved. It may be that their enclosure is more
accidental than physiologically necessary, for the bulk of the
foreign cells in the canals and tissues of the colony may come
from the surrounding water at a subsequent period. An apparent
increase in the substance of a mature ovum (see below) might be an
indication of their activity. At all events, there is suggested,
an approach to a more complete symbiotic union than that which
exists in Convoluta roscoffonsis, for instance, where ifc has been
definitely shown ('07) that infection takes place after the animal
is hatched, for there the animal undoubtedly plays, in the long
run, the part of a parasite with respect to the alga, as under no
known conditions were algte found to escape alive from the body of
that turbellarian.

The Entry of Zooxanthellae into the Ovum of Millepora

Millepora platyphylla released medusae synchronously in early April in both 1985 and 1986, at dusk, four days after full moon. They continued to do so monthly, for a few months. This comprized the initial finding, and could probably have served for a Master's Thesis alone. The event was spectacular and beautiful---if one was focused on Millepora spp. to begin with: perhaps most divers are concerned mostly to avoid their powerful stings. Nevertheless it seemed bizaare to me that these events had not been widely photographed and films. In retrospect this is something to which I ought to have devoted greater attention---especially given my training as a photographer. Equally as interesting were findings observed in the laboratory.

Joseph Mangan's 1909 paper was entitled The Entry of Zooxanthellae into the Ovum of Millepora. My findings bore on this aspect of their biology. Moreover, the narratives of both Moseley, working aboard the Challenger, and Mangan, working with Hickson's material, provide clues to the resolution of another question that still, to my knowledge, remains unanswered, concerning the entry of zooxanthellae into the ovum of these animals.

When these observations of mine were made, I was working on Guam, far from even modestly useful libraries, and certainly from experts who might shed light on the basics of the systems I was observing (zooxanthellae, in particular). I will state briefly what I think I saw. Current efforts are directed toward making sense of extant material from 1995 and 1996.

-----------------------------------

I mentioned above my plans to utilize light microscopy (LM) in my studies. LM is reasonble in terms of cost and need for laboratory facilities, depending on the sophistication desired. At the time, I believe the only electron microscope on Guam was on a military base. I hoped to work in Chuuk Lagoon, also, which made even further demands for simplicity. I will mention that I was able to purchase at a steep discount, a monocular Zeiss Standard 16, custom made from parts in the shop of Zeiss in New York. Since that time, an anonymous benefactor has made more sophisticated optics available for that stand, as well as another stand---a 1950s Bausch and Lomb Phase Contrast Zoom stand with stock optics.

My prepared slides---probably 200 of them---were stolen from my classroom at Chuuk High School. What use will they be to anyone? Most of my literature collection was lost over my ten year residence in Chuuk Lagoon. Now, some 20 years after the original studies, I still have the paraffin blocks and hard parts specimens, thanks to my friends from the UOG Laboratory---particularly John Starmer and my Professor, Richard Randall.

Under the Microscope

I have to admit I had too little prior experience to prepare my samples. Before coming to Guam in August 1984, I had boned up on Neurosecretion. Perhaps a paragraph on this topic will be forgiven, and somewhat informative, perhaps, at least illuminating my reasons for pursuing the biology of Millepora spp.

I was mainly interested in Neurobiology, particularly, perhaps, Neuroethology. However, realizing I would be studying in Guam, where neither the laboratory essentials nor the expertise would be available to support such studies, I spent a good amount of time and energy during my final year at UCSB boning up on Neurosecretion. The techniques were simple enough that a good microscope lab would support it. And as far as I had found out, noone had discovered neurosecretion in control of gametogenesis of clams. What, I thought, if I could apply the microscopic techniques of Gabe to the giant clam?

On Guam, I immediately realized there would not be enough giant clams to support this kind of study. (In retrospect I ought perhaps to have looked at other bivalves). Within a few days, in conversation with Dick Randall---whom I prevailed upon to be my research advisor over the next two years---it became apparent that Millepora spp. have an important advantage for studies of the study of reproductive periodicity. They possess permanent markers of reproductive physiological state in their skeleta: the ampullae. As I related elsewhere, I began immediately to collect them.

Suffice it to say that in order to whet my curiosity about Gabe's microscopic techniques for study of neurosecretion: Chrome haematoxylin and phloxine---I experimented with this, and numerous other techniques. I also attempted silver impregnation---though I must say with little or no success.

My pragmatic nature impelled me to not put very many of my eggs in this basket: it was just a side interest as far as Millepora spp. were concerned.

------------------------

I began, at any event, with inadequate prior preparation, to embed my M. platyphylla tissues in paraffin wax. I applied dozens of techniques, with little or no prior concept or direction. Even my microscope skills were wanting. A Nikon photomicroscope stand in the lab seemed to have succumbed some time ago to fungi, although my untrained eye would not, regretably, know the difference. I spent quite a lot of time studying sections, fixed and stained in broad range of materials, but with little conception, either of how bad the sections were, or of how to interpret them.

Some vague ideas or findings:

• I suspect that Millepora dichotoma increases its production of nematocysts during reproductive activity. Stings at that time seemed particularly painful. I attempted to develop a water soluble mountant that would enable the rapid mounting of decalicified tissue pieces, with stains incorporated into them that would enable the rapid counting of nematocyts in situ, on the tissue. One or two stains seemed to bind preferentially to nematocysts, but the water soluble mountants made a mess of the whole thing, and the stains rapidly were leeched from those tissues.

• Early on, perhaps the first time I collected medusae, Professor L. G. Eldredge---my supervising advisor at the time---corroborated the observation that the medusae do have a velum. Mayer's early, encyclopedic work on the Medusae of the World (need reference) separated the "Medusae milleporinae" from all other hydromedusae on the basis of lack of a velum. The lack of a velum was one of the observations made by Sidney Hickson (reference). It might be added that Hickson's illustration of the medusa was incorporated into textbooks for many decades, until they were finally observed by myself and John Lewis, who illustrated one for one of his papers (need reference). Mangan published an important paper on medusae, using, I think, Hickson's material. The link to a pdf online is here. And here is the citation in BiBTeX format:

@article{JOSEPHMANGAN07011909,
author = {MANGAN, JOSEPH},
title = {{The Entry of Zooxanthellae into the Ovum of Millepora, and Some Particulars concerning the Medusae}},
journal = {Quarterly Journal of Microscopical Science},
volume = {s2-53},
number = {212},
pages = {697-710},
year = {1909},
URL = {http://jcs.biologists.org},
eprint = {http://jcs.biologists.org/cgi/reprint/s2-53/212/697.pdf}
}

• Mangan's paper also delved into the problem of infection of the egg by zooxanthellae. Or should I say capture and packing of the zooxanthellae into the egg by the animal? Or perhaps the felicitous marriage of the two? The most striking finding of my study was that of activity of the zooxanthellae at the time of gametogenesis of the egg, and what looked to be a correlation of this activity with the induction of the zooxanthellae into the egg. (Or should I say ... ? Or what?). A topic for another chapter.
  

In the field and in the laboratory

The first synchronous liberation event was observed on the fourth night following Full Moon, in April 1985. This suggested a hypothesis in answer to one of the important questions driving the study: is reproduction of Millepora platyphylla periodic? My interest was piqued.

I started a more systematic mode of collection, focusing on Toguan Bay: colonies were numbered by writing on their surfaces with plain graphite pencils. Numbered colonies were visited occassionally, I went to Toguan, and fragments collected from them. Toguan Bay is a leeward reef. I mostly dove at Toguan during the late afternoon or early evening.

The bottom at Toguan drops off rapidly. Millepora platyphylla, as well as the few colonies of Millepora dichotoma, resided mainly in a narrow band close to the reef margin. Corals were studied in the upper 10 or 12 feet. Swimming southward along the reef, most of the colonies could be visited along the way, sequentially, approximating a transect from the Toguan River toward Bile Bay, running probably not more than 200 meters (estimated 23 years later).

Numbered colonies were sampled sporadically, on days when the reef was visited. An attempt was made to visit the reef throughout the monthly cycle, Fragments of various sizes were broken off, marked with the colony number, and site, and dated, in pencil before placing them in large ziploc bags (probably Ziploc Freezer Bags, 1 gallon size). Alternately, each bag had a numbered tag, and the number and colony number written on a slate and recorded back at the lab or on the beach.

Back at the laboratory (the UOG Science Building had a well stocked microscopy laboratory) or the Marine Laboratory, colonies were prepared. Later in 1985, when I was able to utilize the microscope laboratory, many, if not all, specimens were broken, and a small piece placed in a fixative. Numerous fixatives were experimented with: 5-10% formalin in seawater; Bouin's fluid; Mercuric Chloride fixatives; Susa's; Dichromate---a large number. I had purchased a copy of Grays' Microtomist's Formulary and Guide, and I attempted to use as many fixatives and stains as possible.

Hard parts were filed separately, in plastic bags. They are still available in 2008, at the UOG Marine Laboratory. Fixed specimens were moved into various decalicfying agents. I experimented with Formic Acid, reasoning that the shock (or so I imagined) to the tissues/cells would be less, if the same basic functional groups were present. Bouin's Fluid has a decalicifying nature, and several changes were often made. A number of other agents were utilized. EDTA was used briefly, a time or two.

In decalicifying agents, the tissues readily separated from the Calcium Carbonate skeleton, floating up or resting on the dissolving skeleton, in a matter or hours or days, depending on the agent and its strength. These sheets were easily trimmed with scissors, and moved through a series of solutions, in the process of embedding them in wax blocks. They are little thicker than a sheet of tissue paper.

I also attempted to observe the living polyps in the stereomicroscope. Duerden had observed Millepora spp. in Jamaica, near the turn of the 19th to the 20th Century. He found it impossible to observe them in the laboratory, when he took freshly broken fragments into the laboratory from the reef: they would not open up. His solution was to break fragments off and leave them on the reef, for several days, or longer, allowing them time to heal.

I did not repeat Duerden's experiments. I attempted several times to see open polyps of Millepora platyphylla. Once I was able to do so. Only once. I observed a gastrozoid open. I applied intra vitam Methylene BLue, and observed the nerve ring located around the margin.

Next: Microscopical studies.

Earlier Work, continued

Since this is a blog, I guess I won't try to rewrite the earlier post, but I do need to get to the point rather immediately.

On the night I went with my Pingelapese fishermen friends to Toguan Bay, on the South West of Guam, a rather amazing event was observed. All of those months breaking off pieces of Millepora spp., filing them away (having written dates and places of collection on the fragments with an ordinary graphite pencil), I had expected to do some kind of vaguely imagined time series statistical analysis on the collection, in order to ferret out any periodic signals of reproductive activity. Millepora spp. exhibit ampullae when medusae are developing, so presumeably the presence or absence of ampullae would constitute a signal of physiological activities. Many questions remained unresolved, but the idea was to collect fragments and make the study later on. Nothing could be simpler. Right?

Well on that night I observed something I hadn't anticipated, even in my wildest hypothesizing: all along the reef, numerous colonies of Millepora platyphylla were found to be liberating medusae that night, in synchrony. I don't remember clearly (in Feb 2008) whether I collected some, but probably I did, and probably I saw medusae on that night in a ziploc bag. Whatever transpired on that night, the next day I visited several sites to check whether Millepora sp. on other reefs were also in the same condition.

I observed that the reproducing colonies had turned a darker brown. Little white circles were peppered all over them---marks of receding lips of the ampullae as they decalicified.

After this breakthrough, my focus shifted somewhat. I continued to monitor and collect, focusing almost exclusively on Millepora platyphylla. I enrolled in an independent study of microtechnique with Professor Doug Smith the following year, and was able to incorporate microscopical study of Millepora tissues during the 1995-1996 academic year, for seasonal reproductive events beginning in April 1986.

More recollections to follow.

AED