by Edith Widder
*4 Several years after our return, she resumed teaching full-time at the University of Massachusetts Boston.
*5 Only one because nobody can agree on what the plural of “platypus” should be.
*6 This glow is sometimes mistaken for bioluminescence but is in fact sunlight-induced fluorescence.
*7 I wasn’t “narced,” i.e., suffering from nitrogen narcosis, which can result in such euphoria that afflicted divers have been known to offer their regulator to a passing fish that looks in need of oxygen. I was only at twenty feet and have made much deeper dives without experiencing that syndrome.
*8 One of the stories I grew up on was about how, after receiving her B.A. in mathematics and a University Gold Medal for high honors in mathematics, my mother went home to help out on the farm. One day she was in the fields, working a team pulling a binder, when the harness broke. The neighbor in the next field saw she was in trouble and rushed over to help, but by the time he got there she had gotten control of the team, repaired the harness with a piece of hay wire, and was back on the binder. He looked at her and said, “Well, I guess it’s all right for a girl to study mathematics as long as you can still do something useful.”
Chapter 3
FIRST FLASH
When I got home that evening, after sharing with David the surprising result of my job interview, I looked up bioluminescence in our recently purchased Encyclopedia Americana. It was a short entry—less than a half page of text—that defined it simply as “chemical light produced by living organisms.” There was very little explanation of how the light was produced, except that it could manifest as a steady glow or in flashes, and that it involved a substrate and an enzyme and it was thought that these chemicals were different in different species. Living light producers on land included the well-known fireflies and less well known worms and fungi, the latter illustrated by a picture of green-glowing bell-shaped toadstools. Also mentioned, but not shown, were deep-sea fish with glowing lures used to attract prey, as well as squid and crustaceans that can spew luminous secretions, just like an octopus releasing an ink cloud as a defense against predators. Additionally, there were microscopic light emitters that included bacteria as well as dinoflagellates like those I saw in Beazy’s flask. At the bottom of that encyclopedia entry, I was impressed to see that its author was Beatrice M. Sweeney.
As I would later learn, bioluminescence was a field in which both Beatrice Sweeney and Jim Case were superstars. Beazy had done groundbreaking research on circadian rhythms using bioluminescent dinoflagellates to learn how temperature and light affected their internal clocks, while Case had focused on the neurophysiological control systems of fireflies. Their idea that P. fusiformis*1 might make a good model organism for studying bioluminescence had come out of recent pioneering work by a scientist named Roger Eckert, who had managed to stick an electrode in a very different bioluminescent dinoflagellate, one with an equally wonderful name: Noctiluca scintillans, which means “sparkling night light.” While the notion of getting a microelectrode inside a dinoflagellate sounded challenging, I had the reassurance of knowing it was possible, at least for N. scintillans, the largest of all dinoflagellates.
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Just because dinoflagellates are single-celled organisms doesn’t mean they’re simple. In fact, their diversity and peculiarities can be a bit overwhelming. “Dinos” occupy a range of habitats, including marine, freshwater, and estuarine waters, although they are predominantly (about 85 percent) marine. Some species live on snow and sea ice, while others are parasitic on animals like crustaceans and fish, and still others grow as endosymbionts in corals, bestowing upon their hosts both life-sustaining energy and a resplendent palette of colors. For reasons unknown, they contain more DNA on a per-cell basis than humans do, in some cases almost one hundred times more.
Some dinoflagellates produce toxins and can occur in such profusion that they turn the water red, earning the label “red tide,” and if their toxins accumulate in shellfish or fish that are eaten by humans, the result can be potentially deadly outbreaks of paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhetic shellfish poisoning (DSP), and ciguatera. In fact, lowly dinoflagellates are responsible for ten times more human fatalities each year than shark attacks.*2
Dinoflagellates are so highly varied that it sometimes stretches the imagination that they are related, ranging in size from twenty to two thousand microns and in shape from smooth, round, and unarmored to spiked, armored,*3 and sporting two beating flagella—lashlike appendages used for propulsion. It is the latter form that is most typical and from which the dinoflagellates’ name derives, after the Greek dinos, which means “whirling,” and the Latin flagellum, “small whip.” It is this derivation that spurred purists like Beazy to insist the correct pronunciation is DEE-no, not DYE-no, to distinguish it from a word like dinosaur, which is derived from the Greek deinos, meaning “terrible,” and sauros, “lizard.” Dr. Case, unmoved by Beazy’s classicism, insisted on DYE-no, forcing me to switch pronunciations depending on my audience or, when they were both in the room, to either avoid the word or mumble through it.
That some dinoflagellates produce light adds to their mystique; the light-producing dinoflagellates are known as sea sparkle. Blooms are responsible for breathtaking light shows as flashes are elicited by the slightest touch or disturbance in the water, creating eddies of molten light and cold blue fire in every wave cap. Charles Darwin provided a vivid description of the appearance of one such bloom witnessed from the deck of the HMS Beagle as it cruised off the coast of Uruguay:
While sailing a little south of the Plata on one very dark night, the sea presented a wonderful and most beautiful spectacle. There was a fresh breeze, and every part of the surface, which during the day is seen as foam, now glowed with a pale light. The vessel drove before her bows two billows of liquid phosphorus, and in her wake she was followed by a milky train. As far as the eye reached, the crest of every wave was bright, and the sky above the horizon, from the reflected glare of these livid flames, was not so utterly obscure as over the vault of the heavens.
Such phenomena are more common than most people realize. Unfortunately, artificial lighting used on boats and human habitation along shorelines, where dinoflagellate blooms are most likely to occur, overwhelm bioluminescence. As a result, modern sailors have less poetic encounters with living light, often stumbling upon it for the first time in the ship’s head (a.k.a. toilet), which is flushed with unfiltered seawater. The result is that many a seasick sailor who was so toilet-huggingly sick that they neglected to turn on the lights may have thought they were having a religious experience while “talking on the porcelain telephone to God.”*4
Blooms, which can reach concentrations of millions of cells in a cup of water, are largely unpredictable but often appear after the introduction of nutrients, as may occur with rain runoff. There are a few very special places called bioluminescent bays, where bioluminescent dinoflagellates are present in high abundance year-round. It takes a unique combination of characteristics to sustain such densities. Requirements include a tropical climate and a shallow bay with a narrow channel and small tidal flux; a dense stand of healthy mangroves around the edges of the bay; and prevailing winds that work to increase the residence time of the dinoflagellates in the bay. These magical places, not surprisingly, attract a lot of tourists, often to the detriment of the bays. Many have been negatively impacted by a variety of anthropogenic stressors like light pollution, sunscreen chemicals, motorboats,*5 coastal development, and pollution runoff from construction sites, roads, and parking lots.
Dinoflagellate bioluminescence was well known to early mariners, though its cause was not. Aristotle (384–322 b.c.) described the light, which appears when the ocean is agitated at night, as akin to lightning. Some two thousand years later, Benjamin Franklin (1706–1790)
drew a similar analogy. In fact, he believed the sea was the source of lightning. Based on his observations of sparkling seas, he assumed that the light was a kind of electric fire resulting from friction between water and salt. However, excellent scientist that he was, he began to question this view when he conducted experiments demonstrating that a sample of sparkling seawater in a bottle would produce light when first shaken but would lose the capacity over time. He also found that if he added sea salt to freshwater, he could not produce any light. Based on these results, he said, “I first began to doubt of my former hypothesis, and to suspect that the luminous appearance in sea water must be owing to some other principles.” This new perspective was reinforced by a letter Franklin received from the then governor of Massachusetts, James Bowdoin (1726–1790). A keen observer of nature himself, Bowdoin described how he discovered that the sparkles in seawater could be removed by filtering the water through a cloth, and he suggested to Franklin that “said appearance might be caused by a great number of little animals, floating on the surface of the sea.” Franklin concurred and accordingly discarded his original hypothesis that lightning came from the sea.
Dinoflagellates aren’t animals, but they aren’t plants, either; they are part of a large grouping of mostly single-celled life-forms called protists (eukaryotes that are not a true animal, plant, or fungus; examples include amoebae, paramecia, and algae). About half of all known dinoflagellates behave like plants and get their energy from photosynthesis, while the other half behave like animals and get their energy from consuming other organisms. N. scintillans is one of the animal-like dinoflagellates, while P. fusi*6 is plant-like. Part of the point of my thesis research was to determine how different or similar they might turn out to be in terms of their light-producing abilities.
This is the kind of research that often mystifies nonscientists. Why does it matter? Answering that question comes down to the difference between basic science and applied science. With applied science, there is a specific problem that needs to be solved, like how to prevent polio, how to cure cancer, or how to build a bigger bomb. With basic science, there is a curiosity-driven question to be answered, like How does a living creature make light? In the latter case, there is no specific application in mind. It is simply driven by the fundamental human desire to understand how things work. Some of the greatest scientific discoveries ever made were generated by basic science, and all applied science is built on the foundation of basic science.
When I began my thesis research, the luciferin and luciferase involved in light production were as yet undescribed in dinoflagellates. All that was known was that the light originated from organelles (membrane-bound structures inside cells) called scintillons. When you bump a dinoflagellate, its scintillons flash. The question was how?
Every living creature has some means of responding to environmental changes. The organ or cell or system that effects (i.e., brings about) that change is called the effector system. When you absentmindedly lift the lid on the casserole dish that minutes ago you removed from a four-hundred-degree oven, but this time without benefit of hot mitts, a remarkable sequence of events occurs before the cursing ensues. You actually pull your hand away before your brain registers the pain. To accomplish this, a sensory neuron detects a potentially damaging stimulus and transmits a nerve impulse to the spinal cord, where a relay neuron transfers the signal to a motor neuron. The motor neuron then sends a nerve impulse back out to the effector, in this case the muscle, causing it to contract and pull your hand away. At the molecular level, the muscle cells contract because two large molecules called actin and myosin ratchet past each other. As in most effector systems, these large molecules are poised to act when triggered by the introduction of very small charged atoms, called ions.
The flash of a bioluminescent dinoflagellate is another sort of effector system, in which a mechanical stimulus, like bumping the cell, initiates an electrical signal that somehow triggers light emission from the scintillons. Just like the withdrawal reflex that protects us from dim-witted interactions with hot casseroles, it’s a response that occurs without any conscious effort, but in this case it all occurs in a single cell.
Life throbs, pulsates, and scintillates because of excitable membranes. We owe our mobility, our thought, our very existence to the ability of a cell to transmit an electrical signal, but this is a very different electrical transmission than occurs in electronics, which are so named because they transmit electrons. The electrical signal in cells is the result of a flow of ions across membranes.
In a classic neuron, which sports a long, slender projection called an axon, the opening and closing of sodium and then potassium ion gates ripple down the length of the axon in less than the blink of an eye. Although that sounds fast, it’s actually glacially slow compared with the speed of an electrical signal, but in evolutionary terms, all that’s required is sufficient speed to evade one’s predators. Take, for example, the giant nerve fiber of a cockroach, which has a conduction speed of ten meters per second.*7 That’s twenty-eight million times slower than electricity running through a 12-gauge wire, but it’s plenty fast enough to evade your attempts to stomp on it.
There is no axon in P. fusi, but there is an excitable membrane, and I wanted to study the linkage between its electrical excitation and the flash it produced. But in order to do that, I first needed to figure out how to piss on some fence posts.
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The Case Lab was a large one. It occupied about one-quarter of the first floor of the multistory Bio II building, which sits on a bluff overlooking the ocean. The main lab had a warren of small rooms branching off it, most of which contained different setups for recording the electrical activity of either nerve bundles or individual neurons. These rooms were chock-full of fantastic toys: oscilloscopes, amplifiers, high-voltage power supplies, photomultiplier tubes…It was tech heaven. There was just one catch. As the newest member of the lab, I was at the bottom of the pecking order and, as such, eligible for only the dregs in terms of equipment and space. My fellow lab mates (all twelve of them) were super friendly and helpful, except when it came to what constituted the primary currency of the lab: the gear, which was conspicuously branded with their initials, along with warnings like TOUCH THIS AND DIE.
During his career, Jim Case produced an impressively long string of graduate students who have gone on to highly productive science careers. He attributed his success in this regard to what he called his policy of benign neglect. His students were left to their own devices to a much greater degree than occurs in most labs. We were thrown into the deep end of the pool, where we’d either sink or swim. After showing me around on my first day in the lab, he rather offhandedly suggested that I could get started by sharing a rig with Linda, a graduate student who was working on the electrophysiology of bioluminescence in fireflies.
Her rig had all the gear I needed for the kinds of recordings I wanted to make, but sharing a rig is like sharing a car with someone who adjusts the seat and mirrors differently than you do. Linda was understandably none too pleased with the arrangement, but she managed to be gracious, so long as I could make myself invisible by working entirely around her schedule and returning all settings on the amplifiers and oscilloscope to her preferred positions, the micromanipulators to her preferred locations, the chair to her preferred height, and the microscope to her preferred focus.
Scheduling was the biggest challenge, because P. fusi wasn’t bioluminescent anytime I wanted to work on it. It produced flashes only at night. Fortunately, I didn’t have to become nocturnal, because it was possible to maintain the cultures in incubators on a reverse light/dark cycle so that the cells were fooled into being luminescent during the day, when I much preferred to work on them. However, if they were exposed to too much light during their dark phase, their bioluminescence would shut down, so I was forced to do all my microscopic manipulations under red light. This reduced their
tendency to shut off their luminescence but made it very difficult for me to see what I was doing.
Also, I was finding it difficult to measure consistent flashes. They seemed highly variable. The first time I ran up against this problem, I was doing a series of measurements to test membrane excitability. I was called out of the room briefly, and when I came back, the first stimulus I applied resulted in a flash that was literally off the charts. The flash was so bright that it saturated the amplifier. You expect some recovery from fatigue, but this wasn’t just a whole lot brighter; it seemed to be a whole lot faster as well, with both a more rapid onset and a shorter duration—P. fusi was jamming more light into a shorter period. It was so unexpected that I initially thought there might be something wrong with my light measurement system, but when I observed a cell through the microscope there was no question: That first flash was a whopper.
I began to wonder what kinds of flashes a cell would produce if it hadn’t been stimulated previously within a given night phase. I didn’t realize it at the time, but this was going to become a theme throughout my career in bioluminescence—how to observe the light-emitting abilities of organisms so that the act of observing didn’t influence the outcome.
To run proper tests, I needed a dark room, someplace where nobody else was going to walk in and flick on the lights. Luckily, there was some lab reorganization going on that freed up a small room right off the main lab. All it needed was some way to enter and leave without letting light in. Over a weekend when the lab was largely unoccupied because of an out-of-town science meeting, David and I went to work. We constructed a light-tight antechamber just inside the door with a solid-wood frame and heavy-duty black plastic sheeting for the walls. Thanks to David’s construction and design skills, it was an architectural marvel that included a sliding door on rollers that I could open and close after shutting the main door, preventing light leakage.