“Bioluminescence” is a little understood, but fundamental and widespread phenomenon in the marine biosphere. The water column provides few places to hide and wherever the sun shines there is the risk of being seen by a predator. Indeed, the largest migration in the animal kingdom is the daily escape by a great variety of organisms into deeper, darker waters to hide from those that would eat them. If all marine life were dark, or transparent and thus invisible, then a highly developed sense of sound would be the only useful adaptation. However, creating and receiving sound is a more recent and complex adaptation – sound is more diffuse and travels more slowly, thus it has only really been adopted by the top of the marine food chain. Even famous echo-locators like sperm whales are believed to use their weak sense of sight to spot their squid prey’s bioluminescent trails (19) and may even attract squid by displaying some light from symbiotic organisms living near their mouths.
For millions of years, on the other hand, the vast majority of life in the ocean has been able to produce and manipulate light. Marine organisms typically do this using chemicals they have stored in their bodies, and less frequently via symbiotic bacteria that produce their own light (7). While “phosphorescence” is the result of absorbing and re-emitting photons from the sun, bioluminescence is a biological form of chemical luminescence, where organisms are able, through a variety of means, to create their own light-producing reactions.
A great quantity and diversity of marine animals and microbes possess bioluminescent abilities – so many in fact that bioluminescent trails were famously used to spot U-Boats and aircraft carriers by friends and foes in WWII (7). What is more, the production of biological light is an almost exclusively marine phenomenon, with a few terrestrial exceptions, and is the primary form of illumination beneath the (two hundred meter deep) photic-zone, in the cold, dark expanse that comprises the vast majority of our world’s ocean. Unlike freshwater bodies, the ocean has a much longer evolutionary history, less sedimentation, and a depth-induced darkness that renders it an unexplored cosmos of its own. This star-scape, however, is lit not by celestial bodies, but by myriad living creatures in constant interaction. Bioluminescence is in fact incontestable evidence that life on our planet has a quintessential need to communicate in order to feed, reproduce, and navigate. Furthermore, like a sportsman in a heated match, communicating in order to trigger offensive and defensive maneuvers is an important part of winning the game of survival in the sea (24).
The Japatella octopus attracts mates with a bright, bioluminescent ring.
The importance of bioluminescence is highlighted by the prominent theory that it has evolved independently, no less than, and probably more than, forty times in extant (non-extinct) organisms alone. Luciferin, in biochemistry, refers to any of several organic compounds whose oxidation in the presence of the enzyme luciferase produces light. Luciferins vary in chemical structure, and the luciferin of luminescent bacteria, for example, is completely different from that of fireflies. Amazingly, a 19th century scientist, perhaps named after the master painter of light himself, Raphael Dubois, mixed glowing firefly beetle abdomens with hot and cold water to discover that oxygen, sufficiently present in the cold water, was needed to create bioluminescence. Indeed, Raphael was correct as science later proved that it was metabolic oxidation powered by adenosine triphosphate (ATP), the product of cellular respiration, that was driving the fireflies’ internal reaction. But, the requirement for an enzyme to physically interact with luciferin in order to produce bio-light is not universal, and no binding of luciferin to an enzyme occurs during the light-emitting reaction of the boring clam Pholas dactylus. Other marine animals, such as the crustacean Cypridina hilgendorfii, can actually be ground into instant light powder, and are able to illuminate without the need for ATP.
That said, bioluminescence is most commonly produced via the oxidation of light–emitting molecules, or Luciferins, as Dubois dubbed them, together with a catalyzing enzyme such as luciferase, or another photo-protein. In order to date the occurrence of the primitive adaptation of bioluminescence, we can thus look back through the genetic fossil record for the crucial luciferin enzyme, which is shared by all non-symbiotic bio-illuminators. Furthermore, we can set the clock back to the first mechanisms of sight in marine predators, for without a form of biological light reception there would be little need to produce light in the first place (24). By tracing the divergence of two, even lineages of ostracod crustaceans, Halocyprida and Myodocopida, each of which uses a different type of luciferin, we could suppose that their singular bioluminescent adaptations evolved up to four hundred million years ago in the Devonian, when we might presume that God said “let there be light!” (Genesis 1:3). Today, there is no part of the ocean where bioluminescence can’t be found, and all the major marine Phyla are represented by at least one bioluminescent species. Notably, the largely planktonic phylum Chaetognatha, the arrow worms, are nearly devoid of the adaptation, whereas the colonial-cnidarian siphonophores are almost universally lit. The arrow worms, originating in the Cambrian, are so ancient that they may even pre-date the adaptation. Indeed, dietary linkages suggest that some extant luminescence is almost certainly a post-Cambrian development (24). Also in support of this theory, we have thus far detected only four types of luceferins – all steadily maintained across Phyla, though it is also likely that more of these molecules exist undiscovered.
My own experiences with bioluminescence have involved swishing my hand through the sea at night to create a disturbance-induced light show. From the window of a deep sea submersible, on the other hand, it is easier to perceive the true, carefully controlled nature of bioluminescence in its natural state. Unicellular organisms are triggered by physical distortion, but more complex organisms control their bioluminescence through the use of a neurotransmitter, such as noradrenaline in some fish. In fact, bioluminescent signals have, since the 19th century, allowed us to literally see the velocity with which a species’ sympathetic nervous system functions, and this is not the only thing that our understanding of bioluminescence has helped illuminate. Research on the hydromedusa Aequorea’s fluorescent protein in 2008 had three scientists, and more pioneering corporations, seeing green, with a $1.4 million Nobel Prize granted in return for a new genetic-marker protein that has become invaluable to emerging biomedical and technological research. From collecting thousands of jellyfish from a rowboat, to using cutting edge technology in the lab, studying bioluminescence made it possible to insert fluorescent proteins into cells, even producing monkeys that glow green under UV illumination (9).
Studying bioluminescent organisms behaviors is also a difficult task, as it necessitates our not disturbing them and accidentally triggering any responses to our actions. For this reason and others, we are just beginning to crack the ocean’s code of light. Although the mechanisms and expressions of bioluminescence vary significantly between organisms, there are certain recurring trends that we have been able to observe. For example, measurements at various depths have shown that the light that bioluminescent organisms emit is concentrated in the blue-green region of the light spectrum, which is logical as these are the most visible colors underwater. In other words, a wavelength of around 470nm transmits furthest in water and looks blue to the human as well as to the deep-sea eye. As a result, most organisms even lack receptors to be able to absorb warmer colors associated with longer wavelengths or cooler colors from shorter wavelengths.
Remarkably, the Malacosteid family, or loose-jawed fishes, have capitalized on this widespread trait by developing a series of chemical filters that allow the fish to emit long wavelengths of 705nm from a special organ. So, along with a regular blue warning light, they also have a specialized red spotlight, like an airplane at night. This allows them to spot their prey without being seen and to navigate under the radar of predators, like having a set of a night vision goggles. The independence of these adaptations from other fish families is underlined by the way the Malacosteus genus perceives the reflection of its own light. Lacking photoreceptive pigments in the eye, it possesses instead a special molecule related to chlorophyll which absorbs red light and transfers its energy to blue-green pigments that the fish can see.
Humans, meanwhile, can just barely perceive these infrared wavelengths with the dark-adapted eye. On the other hand, we can easily detect most regular bioluminescence as it comes primarily from an abundance of dinoflagellates in flashes of about 0.1 of a second, and with the strength of about 6e8 photons per flash. Because these unicellular organisms borrow the sun’s energy for their chemical reaction, greater exposure to sunlight the day before will increase the intensity of their flash (16). Radiolarians, Copepods, including all species of the metridinidae, ostracods, and nearly all species of euphausiids, along with an enormous variety of cnidarians, ctenophores and pelagic tunicates are all abundant and wide-spread bio-illuminators that we can see, for example when churned up in the bioluminescent wake of a ship cutting through the sea night.
Dinoflagellates and radiolarians both practice intracellular bioluminescence. This is why in the case of single celled organisms, impacting the cell membrane by driving a propeller through the water causes them to glow. If we were to examine an individual animal on a microscopic level, the water pressure around the organism is actually initiating an action potential that releases protons from the acidic vacuole, which excites “micro sources” scattered throughout the cytoplasm (25). Disturbance is not necessary in all organisms, however, and most multicellular organisms are in close control of the reaction. Some copepods, for example, have luminescent glands on their tail that release light-producing chemicals into the water. The pattern and intensity of the releases can be altered to confuse a predator in a cloud of “ink,” or to carefully seduce a potential mate. Euphausiids on the other hand, possess complex light organs called photophores, which are located on their undersides and “emit a relatively steady glow that is a near perfect match for the intensity, color and angular distribution of the down-welling light field (25).” In this case, the bioluminescence foils predators by camouflaging the silhouette against a backdrop of tiny points of light.
In colonial tunicates, each zooid has two luminescent patches on either side of it body, and thus the amount of light produced is a function of the size of the colony. A 30m colony is not uncommon, and could potentially attract a lot of minute food with its intense and steady glow (25). Furthermore, some invertebrates with light receptors even practice “empathetic” luminescence, where photic excitation from an external source, such as another bioluminescent organism triggers light emission. In this way, two jellies can have an actual “conversation” through exchanges of light (16). It is difficult to know how each organism obtains luciferins, because they are present in both luminous and nonluminous marine animals, and are thus relatively easy to obtain through diet and chemosynthesis. Whereas the catalyst luciferases and photo-proteins clearly come from many unique evolutionary lineages, the presence of luciferins such as Coelenterazine and Cypridina, sharing the same basic structure and producing light via the same mechanism, but having no amino acids in common, suggests that they derive from a common origin in the food chain (18).
Coelenterazine is the light emitter in at least nine very different phyla, including protozoans, jellyfish, crustaceans, mollusks, arrow worms and vertebrates, so it could not come from one food source, but is likely passed along from small creature to big like in the way heavy metals bio-accumulate. Supporting the dietary connection theory, researchers have found in the gut of some deep-sea fish large quantities of the specific luciferin that they use to illuminate. Clearly, the luciferin in question must persist in order to be of use to the consumer, and the greater stability and thus lifetime of Coelenterazine, may be another clue as to why it is so widespread (18). Luminous bacteria, although they use a different molecular process for bioluminescence, are also common in the oceans. A minority of eukaryotic species, namely sepiolid and loliginid squid and monocentrid, leiognathid, apogonid, and morid fishes, even form symbiotic relationships with bacteria that are given a home in return for the use of their glowing properties. But, these relationships are not representative of the vast majority of bioluminescent organisms. Unlike eukaryotes, bacteria that luminesce do so consistently after being stimulated through a density dependent response trigger. Bacteria can even form large light-emitting surface slicks which can last for days, a phenomenon that is likely linked to previous or simultaneous phytoplankton blooms (24). These rarely seen events are called “milky seas,” and have been prominently recorded in the Indian Ocean where monsoon-induced, warm sea surface temperatures concur uniquely with high productivity from upwelling, creating a substrate that is highly conducive to plankton blooms and microbial life.
Due to its sensitive nature and the black environment in which bioluminescence is most intense, studying it can be like taking a “shot in the dark.” Moreover, despite the previously mentioned extravagances like giant, glowing tunicate spheres and “milky seas” visible from space, research has shown that most organisms use bioluminescence rather more conservatively. The way we came to appreciate this was to monitor bioluminescence from a neutrally buoyant submersible that had no impact on the surrounding water. These studies were done in the deep-sea region of Monterey Canyon, a mile deep trench with high nutrient availability off the coast of California. From observing swaths of constant light while the submersible was in motion, to almost total darkness when it stopped and became neutral, it was clear that bioluminescence created a veritable minefield for predators and prey alike. The next step was to examine the nature of this luminescent minefield, and elucidate the spatial, temporal and taxonomic characteristics of a highly variable and partitioned water-column. This was something like re-inventing the telescope to map the intricacies of space beyond what we can see with the naked eye.
Early studies on bioluminescence were conducted using tubes called bathyphotometers, consisting of a light detector in a light-tight chamber, where planktonic bioluminescence was stimulated by some turbulence-generating mechanism. The “photon flux” recorded by the bathyphotometer depended on four key factors, “the detection chamber volume, the flow rate through the chamber, the method of stimulation and the amount of pre-stimulation resulting from mechanical stimulation in the light baffling (25).” Because of these determinants, results tended to be instrument specific, and furthermore the sampling was biased for weaker swimmers like dinoflagellates. Nevertheless, researchers did prove that the upper portion of the sea was generally permeated with bioluminescent organisms. They also discovered important ecological trends, such as seasonal variability, vertical migration of many luminescent species, circadian, or 24hour rhythms in the luminescent capacity of many dinoflagellates, and photic inhibition of bioluminescence in organisms such as some heterotrophic dinoflagellates and ctenophores. By recognizing these inherent control mechanisms and chrono-biological patterns, we began to comprehend that bioluminescence might be the key to the ancient ecological mystery of the sea (25).
More questions arose from these early findings, such as, “how much is photic inhibition in unicellular organisms versus diel migration patterns in copepods, euphauciids and ostracods, responsible for variations in intensity and flash rate of bioluminescent organisms between day and night?” (Batchelder, 1992) suggests that photo-inhibition in dinoflagellates is the more decisive factor in bioluminescent capacity in the northern Sargasso Sea, a confluence of currents forming a sub-tropical gyre in the North Atlantic. Here, measurable, stimulable bioluminescence is four to ten times greater at night (26). This would perhaps imply that bioluminescence in heterotrophic organisms, such as the diely migrating copepods, co-evolved with autotrophic plankton in the photic zone. Seeing as prey is vulnerable to being detected by bioluminescent plankton, and that bioluminescence has been shown to deter grazing on individual dinoflagellates via the “burglar alarm theory,’’ the relationship is at least significant on a trophic basis. Furthermore, by playing a crucial role in the food chain, bioluminescence facilitates and inhibits the cycling of vital biotic and abiotic constituents of seawater. Because “marine snow,” or detritus that falls from the photic to the benthic zones, “contributes significantly to the transport of particulate carbon and macronutrients,” in the marine system, its relationship to bioluminescence is of consequence. In other words, bioluminescence is not just a tool that weird fish use to lure prey, but a general attribute of marine life that affects even the most diminutive creatures, such as diatoms, planktonic larvae and bacteria (11).
It is even possible that bioluminescence evolved from trophic interactions between bacteria and consumers. In this scenario, bacterial cells may have been making use of luciferins for their use in naturally scavenging damaging free radicals from oxidation reactions already taking place, and to increase their metabolic capacity. Moreover, bioluminescent bacteria’s ancestors, who would have favored life inside the gut of a fish, may have been selected towards greater production of luciferins, which made them more attractive to consumption. The fish would then manipulate the luciferins to their own adaptive advantages, commencing the great ecological light show in the sea today (24). Some scientists believe that luciferases were “accessories derived from a variety of precursors in the eventual adaptation of weak chemiluminescence to biologically useful light production.” The many theories and paths for the evolution of diverse forms of bioluminescence certainly serve as a reminder that chemiluminescence is actually widespread in biology. Even in our own cells, the oxidation of many organic molecules such as proteins and lipids produces light. With these reactions already occurring in ancient organisms, there exists a viable foundation for more intense mechanisms to evolve through parameters such as yield, kinetics, spectrum and localization (23).
What is sufficiently illuminated, is the effectiveness of bioluminescence as a tool for ecological signaling. Depending on the conditions, a bioluminescent flash can be seen from tens to hundreds of meters away, and a 0.5 mm long dinoflagellate signaling to a large fish 5 m away is the equivalent of a 2m tall human being able to communicate over a distance of 20 km. Its uses for an individual organism would likely be many and variable in nature, making the reproduction of all natural scenarios in a lab an unrealistic aim. Our predictions about the uses of bioluminescence are thus hypothesized, and based on the concept that organisms would either want to attract attention or repel it, depending on the circumstances, such as the inter or intra species relationship and the range of the signaling. A flashing light in the distance might attract your focus, but a flashlight in your eyes will surely cause you to look away (23). Dinoflagellates are thought to flash in order to startle their predators, and to indirectly warn other prey by acting as a burglar light. Crustaceans, meanwhile, use counter-illumination, or light present on their ventral side, because in the sea light comes from above. This is a useful adaptation in avoiding the notice of predators like the Spookfish, Dolichopteryx longipes, which uses biological mirrors inside diverticular eyes, split in half to spot the silhouettes of prey above and its own predators below (21).
Another predator that may use counter-illumination to its advantage is the cookie cutter shark, Isistius brasiliensis. Instead of having to seek larger, faster prey, the shark uses a falsely smaller bioluminescent silhouette which may camouflage it in a school of squid to attract unsuspecting guests. It is so aptly named for taking bite-sized pieces out of much larger fish, seals, cetaceans and even people, with its massive teeth and melon-baller like mouth (22). Crustaceans may also create a smoke screen effect, as previously mentioned, by releasing a luminescent cloud. The vampire squid, unlike its diurnal relatives that secret black ink, releases glowing ink as a diversion tactic. Jellies on the other hand, may be warning off organisms that could damage their fragile bodies with colorful bioluminescence in the same way that other aposematic animals such as blue ringed octopus offer warnings of their deadly poison. Another interesting stratagem, used by pelagic sea cucumbers, is to loose a glowing body part called a “sacrificial tag” in order to make an escape. The lost body part is of little consequence to the evader, but becomes sacrificial when it continues to glow in the stomach of the foiled and commonly transparent predator. Remarkably, this has led to increased red pigments in the gut of predators, which help to absorb the exposing blue light after it is ingested.
Predatory mechanisms are perhaps more recognizable, with glowing lures hanging unambiguously off the grisly countenances of sharp teethed Angler Fish, and on the long and treacherous tentacles of squid. The effectiveness of this technique is clear, in that there are only two scale-less stomiids without bioluminescent lures, or “barbels,” out of 25 genera. Other fish, like flashlight fish and dragon fish, spotlight their prey, and/or confound it with light. Some researchers even believe that large deep-sea predators like Sperm Whales and Megamouth Sharks collect symbiotic bio-illuminators around their white-fringed mouths to attract squid and increase plankton consumption respectively (24). If prey can be enticed, then surely so can potential mates in a large and lonely sea. One species that is thought to do this seasonally is the Japatella Octopus, which forms a ring of light on its ventral side. This is a critical adaptation for a semelparous animal, or one that has only has one reproductive cycle in its lifetime (20). Other species that take this to a new level are the pony-fishes, which produce synchronized group displays and have evolved luminescent-based sexual dimorphism, which would presumably help distinguish a mating partner from a competitor.
Because the distribution of bioluminescent organisms in the oceans correlates with regional biomass and physical forcing, such as upwelling events and water currents, it is likely that future technology will allow us to further use its ubiquitous nature to help map the marine environment. This mapping has and will include abiotic information, such as chemical composition of the water column, as well as highlighting organismal diversity, thus providing a much-needed tool to observe and understand the dynamics of planktonic communities in the ocean (24). Understanding bioluminescence on the microscopic level has already proved invaluable. Osamu Shimomura, a Japanese molecular and marine biologist who witnessed and survived the atom bomb in Nagasaki, was the first person to isolate the green fluorescent protein (GFP) from the Crystal Jellyfish, Aequorea victoria. His dedicated and genuine interest in the bioluminescent mechanisms of this abundant denizen of Puget Sound, Washington, led him on a quest to collect and squeeze out, through rayon gauze, the luminescent substance from thousands of jellies that villagers helped him collect by the bucket-load. The green bioluminescent “squeezate” he was after, comes from small photo-organs located on the rim of the jelly’s umbrella. By examining this substance, Shimomura found that Aequorea releases calcium ions which then bind to a protein that he called aequorin, releasing blue light in the process. The blue light is then absorbed by green fluorescent protein, which in turn gives off the characteristic green light (8).
Some years later, in 1987, a scientist named Douglas Prasher, after examining Shimomura’s work, realized the potential of GFP as a “tracer molecule,” or a molecule that might show us when a protein was being made in a cell, through its relatively small and luminous properties. Even electron microscopes cannot detect the infinitesimally minute proteins that are such an important part of our chemistry. For example, hemoglobin is a vital protein that carries the oxygen in our blood. When our bodies need to make more of it, our genes provide a code to start and stop our molecular machinery. Prasher believed that it would be possible to insert the GFP gene at the end of the hemoglobin gene, right before the stop codon, which halts production, so that the cell would switch over to making GFP, like turning over a piece of paper that has just been printed and re-inserting it into the active printer before the job is through. Indeed, he had chosen the right protein and his experiment was a success.
By first cloning GFP, it was then possible to attach it to other cells, such as that of the bacteria E. Coli, which would then visibly glow under ultraviolet light. These studies were collaborations with other scientists, which eventually led to the discovery of more bioluminescent proteins in other lit and unlit marine organisms, such as the sea anemone Anemonia majano. Finding a fluorescent protein in a non-bioluminescent cnidarian came as a surprise, but perhaps should not have been so long overlooked given the abundance and diversity of bioluminescent mechanisms in the sea. In 2007, fluorescent proteins were even found in Lancelets, some of the most ancient marine chordates.
A veritable revolution in biotechnology and biomedicine was born from these discoveries, and thanks to the work of Nobel Prize winning scientists such as Roger Tsien, we now have an abundance of artificial fluorescent proteins with almost as many colors as magic markers, and with comparable names like tangerine and banana. These can be used to light up organisms from inside out, revealing mechanisms that would otherwise be impossible to see. Among the many uses discovered for fluorescent tracer molecules, scientists have devised a cellular thermometer, a cellular timer, a molecular stress detector, and a way to detect the progress of cancer cells in lab animals. The list goes on and is surely just beginning, as is our understanding of bioluminescence, the silent language of the sea (17).
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