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.

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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