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

Moseley's Microscope

Moseley stated he used a Hartnack's Number 4 objective and No. 4 eyepiece. Here's a picture of a Hartnack with a Number 3 (10X) eyepiece.

I received the following response on the sci.techniques.microscopy group to my inquiry:

Hi

Not a historian but recently borrowed the splendid book by van Heurck
'The microscope' English translation by W E Baxter 1892 to form the
5th edition according to Preface.

There is a table of Hartnack's objectives on page 167 ...

The No 4 objective is listed as 'Equivalent focus ' 10 mm and
'Numerical Aperture' 0.5.
The mag of this would depend on what tube length microscope it was
used on. ca. 16x on a 160 mm

If it's the same no. 4 objective as the one the above author refers
to, he remarks on it on page 165: Quote:

"No. 4 is an excellent objective. It is very clear, and its powers of
definition and penetration are very remarkable."

If the Hartnack's eyepiece conforms to an earlier note in this book on
eyepieces but might not, p71: Quote:

"Thus the ocular 4 amplifies the image given by the objective four
times".

Me on Sidney Hickson

I have Googled Hickson, and found one of my own postings to a Microscopy mailing list about him:

-------------%------------- Sun Dec 2 23:02:29 2001

I am interested in gaining insight into the role of microscopic artifacts
in the history of biology. May I impose on list members to contribute
particularly glaring examples of misinterpreation of biological facts due
to improper microscopic technique? I apologize if this is off-topic or a
waste of bandwidth.

Let me provide the first example of a misinterpretation and request your
assistance in learning whether this was due to improper use of the
microscope, or perhaps even malfeasance: Sidney Hickson's early work on
Millepora spp. (Cnidaria:Hydrozoa) fire corals. It seems an incredible
lapse, a wholly fabricated natural history account, one that persisted for
a considerable period in the fabric of the mythology of biological
knowledge. I am interested because reproduction of Millepora platyphylla
is the subject of incompleted thesis research of mine.

Hickson published a report on reproduction of "Millepora" around the end
of the 19th Century. (Anong his other errors he synonomyized all species
of Millepora as ecomorphs of one, M. alcicornis.) In this report, which
I do not have available at this time, he included several plates of
drawings depicting a putative sequence of reproductive events in this
organism. We now understand that his depiction is not even close to the
way that Millepora spp. (which were later redesignated as proper
individual species through painstaking work by Boschma---notwithstanding
the issues recently raised by molecular work) reproduce. The depiction
involved dozens of drawings, and a sequence of events based on a
misinterpretation of what are apparently artifacts.

Hickson (of Cambridge University) worked extensively in the field,
including Indonesia and the Philippines. Was his microscopic work done
in the field? Are members of this list enlightened as to Hickson's
methods?

Hickson's erroneous drawings of the medusae of Millepora lived on for over
3/4 of a century in virtually every Invertebrate Zoology textbook
published until the late 1980s or 1990s. His erroneous description of the
medusa of Millepora as lacking a velum led to the designation of a
separate branch of hydromedusae by Mayer, as the only hydrozoan medusa
without a velum. My unpublished observations in the 1980s as well as
published observations by John Lewis of McGill University showed that the
medusae of Millepora spp. clearly possess a velum.

I apologize for monopolizing the bandwidth. I hope this is as fascinating
a topic for others as for myself, and not considered off-topic.

-----------------%----------------------- END

Millepora: what's in a name (or a picture)

"Millepora"---just a quick note here on violence done to Millepora spp. names and pictures by Sydney Hickson, and by history.

This genus has been treated poorly. Professor Richard Randall at the UOG Marine Lab had few if any kind words for Hickson, the Don of Invertebrate Zoology at Cambridge, at the turn of the 19th to the 20th Century. In particular, Hickson decided that all species of Millepora spp. were synonyms of Millepora alcycornis. Somehow, either because of this fiasco, or for whatever reason, the name "Millepora" stuck, and still does today. Even among those who should know better. My sense of the English language makes it sometimes problematical to recite the whole name of any particular Millepora species. It took the great Hulbrand Boschma a very long time, and a lot of work, to sort out the species, years later.

The illustration of "Millepora" likewise suffered violence at the hands of Hickson. I observed medusae of Millepora platyphylla several times in 1985 and 1986: little did they resemble those drawn from Hickson's illustrations early in the 20th Century, or late in the 19th. And Mayer even had it wrong in his medusae of the world, a copy of which exists at the U. of Guam. He called them Medusae Milleporinae, and stated that they differed from all other hydrozoan medusae, in not having a velum. We confirmed that the medusae of M. platyphylla do indeed possess a velum.

John Lewis of McGill University subsequently published illustrations of medusae of caribbean Millepora sp. that did resemble what we saw.

Beginning a Narrative on my earlier Research on Millepora platyphylla, 1984-1986

As a graduate student at the University of Guam Marine Laboratory, between 1984 and 1964, I came to study Millepora platyphylla reproduction, as well as other aspects of their biology. In this blog I will reiterate that research. I plan to continue with further research as well in directions suggested during that time. My research of those years was left incomplete, because I left Guam to go to Chuuk Lagoon to attend the birth of my first son, Forrest who as of Auguest 2007, is now 20 years old. The work I did at that time is still relevant today, and I intend to return my attention to it. My bread and butter is earned as a full time as a secondary science teacher, at Kagman High School, so my initial efforts will be done in my spare time, as I prepare, I hope, for more focused study in the near future.

Professor Richard Randall at the University of Guam had pointed out that Millepora spp. bear markers of reproductive activity in their hard parts. Therefore, by collecting fragments of the coralla (skeleta) of colonies data would be produced on reproductive condition. I was interested in reproductive periodicity, especially in higher invertebrates that exerted neurosecretory control over gametogenesis. Bivalves seemed interesting, perhaps giant clams. It was apparent, however, upon arrival at Guam, that Tridacnids were anything but abundant there---insufficient to enable any meaningful study. It could not hurt to start a collection of hard parts of Millepora spp., since I had been snorkeling quite alot on various reefs of Guam. Since Prof. Randall was recruiting a student for study of coral reproduction, and these particular corals possessed certain remarkable qualities, I set about right away to begin collecting fragments of Millepora platyphylla---the most common species where I had been snorkelling during the first month or so on Guam. I also collected a few fragments of M. dichotoma; however, it was not as convenient as the former species.

In fact, I had been intersted in neurological/neurosecretory control of gametogenesis. I had read methods by Gabe, involving what promised to be relatively straightforward light microscopy methods, simple and cheap compared to other kinds of laboratory studies. Or so I reasoned. During the first year, I began, then, a collection, even though I had not formally selected a research topic for my thesis.

A few specimens each time I snorkeled: that was relatively easy, and I figured I could do statistics, once I had a large enough collection, and generate some kind of picture of periodicity. Studies of neurosecretion are multi-disciplinary studies, suggesting a need for ecological data (for example, lunar cycle, tidal cycle), as well as microscopical study of neurosecretory structures. I didn't know that Millepora spp. would have neurosecretory cells, but that was far from my thoughts as I commenced my study in August and September, 1984.

Along about late May and Early April of 1985, toward the end of the second semester at U. of Guam, I was invited to go fishing with Ahser Edward, a student at the Marine Lab, and a colleague. I accompanied Ahser and his brothers and cousins to Toguan Bay, on the SouthWest coast of Guam, between Umatac and Merizo. I did not fish. I carried my flashlights and studied the reef, collecting Millepora platyphylla along the route, Southward from Toguan Bay, S. of Toguan River.

During my time at UOG Marine Lab. a seminar by George Barlow Prof. Barlow, from UC Berkeley, an offhand comment by him had a profound effect on my philosophy. Barlow studies fish behavior, and is something of an eclectic---something I appreciate. He made the point that Kenneth D, Roehder, a physiologist, had discovered a relationship between bat echolocation and avoidance by Notuid moths. Professor Barlow's point was that Roeder's approach to biological research was to focus on one species. By doing so, connections emerged between that species and other species. Something like that happened with my Millepora spp. research..