BIOLUMINESCENCE Q&A

The following questions were posted on the Discovery Channel Web Page following the airing of "Ocean Currents", which included footage shot during a Bioluminescence Department research cruise to the Gulf of Mexico. The answers were provided by E. Widder and T. Frank.

WHICH OCEAN ANIMALS ARE BIOLUMINESCENT?
Because bioluminescence is rare on land, people sometimes assume that the same thing must be true in the oceans. It's not. Some of the most abundant creatures on our planet are bioluminescent oceanic inhabitants. For example, the most abundant vertebrate on earth is a little bioluminescent fish called the benttooth bristlemouth (Cylcothone spp.). There are also enormous numbers of lanternfish (Myctophidae), squid, decapod shrimp (such as the oplophorids and sergestids) krill, copepods, ostracods, amphipods and gelatinous zooplankton. The list is very long and growing longer. Until recently the only way to study the life in the oceans was to bring the animals up into our world in nets. This destroyed many of the most fragile creatures like the jellies and exhausted the bioluminescent capacities of most of the others. However, as we gain greater access to the oceans with submersibles and remote operated vehicles (ROVs) we are discovering many animals have the capacity to make light. The most recent published list of all known bioluminescent creatures, both on land and in the sea, is "Systematic distribution of bioluminescence in living organisms" by Peter J. Herring published in the Journal of Bioluminescence and Chemiluminescence vol. 1 pp 147-163 (1987).

WHAT ARE THE PRACTICAL USES OF BIOLUMINESCENCE?
Given the enormous numbers of animals in the ocean which are bioluminescent it obviously plays a very important part in the marine ecosystem. To understand life in our oceans we need to understand what part bioluminescence plays in helping animals survive. Marine scientists are also seeking new, improved methods of sampling animal distribution patterns in the ocean. Measurements of bioluminescence may prove to be a very useful indicator of such distribution patterns.

Bioluminescence is also a useful tool in scientific research. For example, the light emitting chemistry of fireflies has been extracted and used to measure ATP, which is a very important molecule used to store chemical energy in our bodies. Bacterial bioluminescence is used a lot to test for contaminants in the food and health industries. Chemicals from jellyfish are very much in use at the moment in genetic research where they enable researchers to see when a gene is activated.

WHAT IS THE CHEMISTRY?
Animals make light with a chemical reaction and though the substrate is called luciferin and the enzyme is luciferase, these are just generic terms because different animals use different chemicals. Bioluminescence has apparently evolved many (possibly as many as 30) different times in evolutionary history. This degree of convergent evolution is an indication of just how important the ability to make light is to survival.

WHAT USE IS BIOLUMINESCENCE FOR ANIMALS?
Different animals use bioluminescence for different reasons. Many, like the fireflies, lanternfish and sea firefleas (ostracods) use their light to help attract mates, others, like many of the shrimp and squid use their bioluminescence for defense, (for example by squirting a cloud of light into the face of a predator as they escape into the dark depths), and many like the flashlight fish and angler fish use their light to find food.

WHAT COLOR IS BIOLUMINESCENCE?
Bioluminescence in the ocean is usually blue because that is the color that travels farthest through seawater. In coastal waters, where there is more particulate matter in the water, most bioluminescence tends to be blue-green and on land most bioluminescence is yellow or green. However, bioluminescence comes in all colors, red, orange, yellow, green, blue and violet.

HOW DO ANIMALS MAKE LIGHT FROM THEIR BODIES?
Have you ever seen a light stick? When you mix the chemicals together inside the light stick you make light. Animals make light the same way, by mixing chemicals inside their bodies. Most animals make the chemicals they need out of the food they eat. A few animals, like the angler fish, grow bioluminescent bacteria in their light organs. The fish supplies the bacteria with the food they need to grow and the bacteria provide the fish with the light it needs to attract prey. When a fish or shrimp comes to nibble on the angler fish's glowing lure it finds itself engulfed by a mouth full of needle-sharp teeth.

WHAT ARE THE UNITS OF MEASUREMENT FOR BIOLUMINESCENCE?
In terms of figuring out what different animals can see, the best units of measurement when talking about light energy are photons per second. Measurements in lumens or footcandles are only appropriate when you are talking about what humans can see. A single bioluminescent bacterium will emit 1000 to 10,000 (103 to 104) photons per second. At the peak of a flash, a single bioluminescent dinoflagellate will emit 1010 to 1011 photons per second. A 100 W light bulb emits about 1018 photons per second. Because it is possible to pack millions of bacteria into a very small volume, some of the brightest bioluminescent animals in the ocean, like the flashlight fish, use bacterial bioluminescence.

CAN EXPERIMENTS IN BIOLUMINESCENCE BE DONE IN A CLASSROOM?
It is easy to grow bioluminescent dinoflagellates in the classroom. You can order them from Carolina Biological Supply Co. (West U.S. call 800 547 1733 and East U.S. call 800 334 5551). The cost is $5.00 per culture. Try Pyrocystis fusiformis Cat#15-3304.

These cells need to photosynthesize in order to make their bioluminescent chemicals so you will need to set them up under fluorescent lights. Although they are usually only bioluminescent during the night you can fool them by having the lights on during the night and keeping them in the dark during the day. Then if you take them out and shake the container you will see them flash. Be careful not to let them get too warm. They grow best at about 68 degrees F. You can blow a fan over them to help keep them cool. You can keep the cultures going indefinitely by transferring them to sterile sea water with some added nutrients. You can sterilize seawater in a microwave if you can adjust the temperature so the water doesn't boil. Put 100 ml of seawater in a 250 Erlenmeyer flask with 0.05 ml of Micro Algae Grow (Aquaculture Supply 5532 Old St. Joe Road, Dade City FL 33525 Tel. 904 567 8540 A 200 ml bottle cost $4.20). Put a 50 ml beaker upside down on the flask. Microwave the sea water for 20 minutes at 180 degrees F. Don't let it boil! Allow the sea water to sit for at least a day before inoculating the culture. Transfer a little of the old dinoflagellate culture to the flask by lifting the beaker but holding it over the mouth of the flask.

Once you have a few flasks going, try growing them under different light/dark cycles and look at the cells to see how different they appear in their day phase compared to their night phase. See what happens if you keep one of the cultures in constant darkness. What about under constant light? How long does it take for the bioluminescence to turn on when you place a culture in the dark? How long to turn off when you place it in the light? Can you mechanically stimulate the cells to exhaustion, so they don't bioluminesce any more? Do they recover during their night phase? How about after their next day phase? Are there other ways to stimulate bioluminescence than mechanically? Place some of the culture in a test tube and add a drop or two of 10% acetic acid or if you don't have any laboratory acids try vinegar. What happens? Can you get any more bioluminescence out of the cells after they have been chemically stimulated?

You can learn more about bioluminescence by purchasing the "Secret Lights in the Sea" DVD or "The Bioluminescence Coloring Book" available in our store.

IS BIOLUMINESCENCE JUST A VESTIGIAL ADAPTATION?
It has often been suggested that bioluminescence in certain animals must be vestigial because there seemed to be no possible function for the light production - see for example the next question about the bioluminescent tube-dwelling worm Chaetopterus. In fact bioluminescence is so energetically costly it is highly unlikely that evolution would permit the persistence of such an adaptation that no longer had survival value. The environment that many of these animals inhabit is so alien to our light-filled terrestrial world that it is difficult for us to imagine how many of these seemingly strange adaptations must function. However, just because I can't guess at a function for a particular type of bioluminescence doesn't mean that I believe it has no function. There are unquestionably some wonderful stories out there, yet to be discovered.

WHAT USE IS BIOLUMINESCENCE TO AN ANIMAL THAT LIVES IN A TUBE,
LIKE CHAETOPTERUS?
Chaetopterus is also known as the volcano worm because of its ability to spew out billowing clouds of blue light from the end of its parchment tube. This is what is known as a burglar alarm. Some creature trying to invade its tube is now suddenly exposed and consequently made easy prey for some higher order predator. The cloud of light is similar to the fear scream of monkeys or birds. It is intended to attract the attention of higher order predators that may attack the primary predator thereby affording the prey an opportunity for escape.

WHAT DO ANGLER FISH EAT?
The stomachs of angler fish are generally found to contain small fish and invertebrates such as shrimp and amphipods. Different anglers have very different looking lures and it was once thought that these different lures were meant to attract different types of prey. However, stomach content analyses seem to show pretty much the same diet for these different anglers. Now it is believed that the different shape of the lure may be a sexual signal to help males find conspecific females. For many species of angler fish the males are very small and parasitic on the females. When a male finds a mate he latches onto her flank and her blood stream grows into his body. He no longer has to feed and basically becomes a little sperm sack, ready to fertilize her eggs when the time is right. Since deep sea angler fish are solitary and don't school it is difficult for males and females to find each other and this is one solution to the problem of securing a mate.

WITH SO MANY FISH IN THE WATER, HOW DO YOU KNOW WHAT'S THERE?
The best way we have of learning what fish inhabit our oceans is with net sampling. We drag large nets behind oceanographic vessels and then examine the catch. There are experts called taxonomists that can identify most of the fish that are caught. They use a variety of characteristics to identify them, such as the position, structure and number of fins, pattern of scales, size and shape of the body/head, and, for the bioluminescent species, the pattern of photophores (light-emitting organs) is often a key characteristic. If we find a fish we can't identify we send it to a taxonomist. If he or she doesn't know what it is and can't find any description of it anywhere in the scientific literature, then the fish has to be given a new scientific name.

If you collect fish from what is known as the mesopelagic zone, the depth range between 200 and 1000 m, you will find that most of these fish (often between 80% and 90%) are bioluminescent.

HOW DO DEEP SEA CREATURES COMPENSATE FOR PRESSURE?
In the ocean, pressure increases by 1 atmosphere for every 10 m you descend. Animals in the very deepest parts of the ocean are living at pressures of more than 1000 atmospheres. Many deep-sea animals lack any gas filled spaces in their bodies, like lungs or swim bladders, and since the liquid that their bodies are composed of compresses very little under pressure, they experience very little effect from pressure changes. We have found that we can bring many of these animals to the surface alive if we just keep them cold. At the depths these animals live at, the water temperature is cold (usually around 40 degrees F) so if we bring them through warm surface waters, unprotected, they are cooked. However if we catch them in a container that is not a pressure vessel, but is thermally insulated, then we can bring them up alive. Some deep sea fish have swim bladders that involve remarkable gas exchange systems that allow them to pump out gas against the very high pressures that they live at. If you bring these fish to the surface then the gas in their swim bladder expands too rapidly and they essentially explode. However, if you bring them to the surface very slowly, allowing them time to adjust the pressure in their swim bladders, then even some of these creatures can be brought up alive.

HERE CAN I FIND MORE INFORMATION ON ANGLER FISH?
A good place to find more information about angler fish as well as a lot of other great deep sea creatures is in the book "Deep-Sea Biology" by N.B. Marshall, Garland STPM Press, New York and London, 1979. If your library doesn't have it, maybe they can borrow it for you.

WHAT IS THE RELATIONSHIP OF MARINE BIOLUMINESCENCE TO FIREFLY CHEMISTRY?

Luciferins
The luciferins are the substrates in the bioluminescent reactions. The firefly luciferin is a benzothiazole which is assembled from the amino acids tyrosine and cysteine. It is a very different molecule than is found in marine systems. For example in ostracods the luciferin is an imidazolopyrazine which is assembled out of 3 modified amino acids, tryptophan, isoleucine and arginine. Jellyfish also have a luciferin that is an imidazoloyrazine but it's a very different structure assembled out of 2 tyrosines and a phenylalanine. The bacterial system involves a long chain aldehyde and dinoflagellates have a luciferin that is a tetrapyrrolic compound which is probably a metabolic product of chlorophyll.

Luciferases
The luciferases are the enzymes in the bioluminescent reactions. They are complex proteins formed of very long chains of amino acids. Thanks to genetic cloning we know the amino acid sequences of luciferases from a bioluminescent bacterium, a firefly, a jellyfish and a dinoflagellate and they are all very different structures.

Co-factors
Firefly bioluminescence is unique among luminescent chemistries in that it requires ATP to trigger the light emitting reaction. Coelenterates (jellyfish) require calcium ions to trigger the light emitting reaction and dinoflagellates require a change in pH. Bioluminescent earthworms require hydrogen peroxide as a cofactor while the marine worm, Chaetopterus requires ferrous iron.

In other words there is a remarkable number of different ways to make light!

HOW DO DIFFERENT FISH USE BIOLUMINESCENCE?
Different fish use bioluminescence for different purposes. Angler fish use their bioluminescent lure to attract food to them. Many fish like the flashlight fish have light organs next to their eyes which they use just like a flashlight to help them find food in the dark. But there are other uses of bioluminescence besides finding food. One of the most common uses is as camouflage. Animals in the open ocean have no trees or bushes to hide behind and those living in sunlit surface waters (down to depths of 1000 m) are easily seen as a silhouette by predators swimming below them. To make their dark silhouettes less conspicuous many fish have light organs on their bellies that produce bioluminescence that exactly matches the color and intensity of the sunlight or moonlight filtering down from above. This use of bioluminescence is called counterillumination and it is very, very common. Not just fish but even shrimp and squid use bioluminescence in this way. Another way that bioluminescence is used to defend against predators is by spewing out a cloud of blinding light into the face of an attacker and then escaping into the darkness. This line of defense is most common among squid and shrimp but there is even a fish, called the shining tube-shoulder, that spews bioluminescence out of a tube on its shoulder (hence the name). Bioluminescence is also used for finding mates. There are many examples of gender differences among bioluminescent animals where the female has one sort of light organ and the male has another sort, and presumably these are used to produce a kind of "come hither" signal.

HOW DIFFERENT ARE DEEP-SEA FISH FROM THOSE THAT LIVE IN SHALLOW WATER?
There are many differences. Since shallow water fish live where there is plenty of light to see by, many have brightly colored patterns on their bodies to make themselves more visible for attracting mates. Deep water fish don't usually show these color patterns and instead depend on bioluminescence. Shallow water fish generally have higher respiration rates and metabolisms than deep water fish. Many deep water fish have weak muscles and conserve energy by sitting and waiting for food to come to them (like the angler fish) rather than using energy for swimming and hunting. Shallow water fishes (0-200 m) generally reach a large size, grow fairly rapidly, have a long life, and have fairly high reproductive rates. Very deep-sea fish (below 1000 m) also attain fairly large sizes (by large, we mean large compared to the size of anchovies and sardines, not marlins and swordfish) and grow rapidly, but they have shorter lives, and often have very low reproductive rates. The fish we call midwater fish (20-1000 m) tend to be small, experience fairly slow growth, have fairly long lives, and fairly high reproductive rates. You might ask why the really deep-sea fish (deeper than 1000 m) tend to be bigger than the midwater fish, and it appears that these deep-sea fish put most of their energy into growth, while the midwater species put more of their energy into reproduction. Also, a lot of the midwater fish are vertical migrators, meaning they move up into surface waters to feed at night, and drop back down to deeper waters during the day to hide from predators, and they use a fair amount of energy for these migrations as well.

ARE THERE ANY BIOLUMINESCENT SQUID?
There are examples of bioluminescent octopus (Order Octopoda), cuttle fish (Order Sepioidea) and squid (Order Teuthoidea). The cuttle fish use bioluminescence for defense - they squirt a cloud of glowing mucus into the face of an attacker. Many of the squid are studded with jewel-like light organs which they use both to attract a mate and as camouflage against the down-welling surface light. The octopods, Japatella and Eledonella, have strange looking green light organs which encircle their mouths. Since these light organs are only found on females they are probably used for attracting mates. There is also a wonderfully strange deep-sea cephalopod called the vampire squid (Order Vampyromorpha) which has a large pair of light organs at the base of its fins as well as tiny light organs scattered over the rest of its body.

HOW DO YOU GET SO CLOSE TO THE FISH?
Good question. I think some of the fish freeze in the lights of the submersible - in fact they are probably blinded by them. For many deep sea fish the best way to avoid detection is not to move, because every time you move you risk stirring up some bioluminescence and making yourself visible to potential predators. Sometimes the fish you see on nature shows have been caught in nets and filmed in an aquarium, where it's really easy to get close to them.

WHAT IS THE SOURCE OF BIOLUMINESCENCE THAT YOU SEE
IN SHALLOW WATERS?
The bioluminescence that you see in shallow waters at night is probably from dinoflagellates. Dinoflagellates are single-celled algae and some species are bioluminescent. The bioluminescent flash which a dinoflagellate produces when it's disturbed acts as a defense against predators. The primary predators on dinoflagellates are tiny shrimp-like creatures called copepods. When a copepod sees the dinoflagellate flash it stops feeding and swims away. This is because the flash may have attracted the attention of something bigger, like a fish, which might eat the copepod.

ARE THE NEON JELLIES THAT I SEE NEAR MY DOCK BIOLUMINESCENT
COMB JELLIES?
You are right that the neon jellies you saw are comb jellies (ctenophores), probably the lobate ctenophore Mnemiopsis, which is often found in high concentrations in coastal waters. The multi-colored light that you saw is not bioluminescence but in fact is just the iridescence of the comb plates that propel them through the water. You would have to look at these comb jellies in the dark to see their bioluminescence, which will appear as lines of blue light running near their comb rows. Their bioluminescence is used as a burglar alarm defense.

I THINK YOU HAVE THE BEST JOB IN THE WORLD. HOW CAN I BECOME
A MARINE BIOLOGIST?
I agree it is the best job in the world, which is why I have difficulty knowing what to say to students who ask me what they need to do to become a marine biologist. There is no question that we need marine biologists. This is an ocean planet that we live on and we need to understand and care for our oceans in order to keep our planet healthy. But there is very little money to support marine biologists and many students graduating with degrees in marine biology can't find jobs. My best advice is don't just take marine biology courses, but get lots of training in the basics, especially math, physics and chemistry so that you can maximize your employment options.

COULD BIOLUMINESCENCE EVER REPLACE LIGHT BULBS?
It will probably be a pretty long time before bioluminescence gets used as a substitute for the electric light bulb. For the present at least, electric light bulbs are a lot cheaper and more efficient than trying to maintain any kind of biological system. Bioluminescence also has the disadvantage of being many orders of magnitude less bright than a 100 W light bulb (see the answer under measurement units).

WHERE CAN I READ ABOUT LUMINOMETERS AND THE LUX OPERON?
A good source of information for such questions is the book "Chemiluminescence: Principles and applications in biology and medicine" by A.K. Campbell published by Ellis Horwood Ltd. Chichester England, 1988.

I PROPOSED TO MY WIFE ON A BEACH IN BERMUDA AND JUST AFTER SHE SAID
YES WE SAW ALL THESE BEAUTIFUL GREEN LIGHTS IN THE WATER. COULD THIS HAVE BEEN BIOLUMINESCENCE?
Did you propose just after sunset on a night just after the full moon? If so the bioluminescence you saw may have been the mating dance of the fireworm, Odontosyllis. To attract the male, the female fireworm swims up out of her burrow on the bottom, and secretes a green luminescent trail as she swims in tight circles. The males, attracted by this display, discharge their sperm into the female's luminous wake at the same time that she discharges her eggs.

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