2004 Deep-Scope Expedition Where is That Light Coming From?

FOCUS TEACHING TIME Bioluminescence One 45-minute class period, plus time for student research GRADE LEVEL 9-12 (Chemistry & Life Science) SEATING ARRANGEMENT Classroom style or groups of 3-4 students FOCUS QUESTION What is the basic chemistry responsible for bio- MAXIMUM NUMBER OF STUDENTS luminescence, and how does this process benefit 30 deep-sea organisms? KEY WORDS LEARNING OBJECTIVES Chemiluminescence Students will be able to explain the role of lucif- Bioluminescence erins, luciferases, and co-factors in biolumines- Fluorescence cence, and the general sequence of the light-emit- Phosphorescence ting process. Luciferase Students will be able to discuss the major types of Photoprotein found in marine organisms. Counter-illumination Lux operon Students will be able to define the “lux operon?” BACKGROUND INFORMATION Students will be able to discuss at least three Deep-sea explorers face many challenges: ways that bioluminescence may benefit deep-sea extreme heat and cold, high pressures, and organisms, and give an example of at least one almost total darkness. The absence of light poses organism that actually receives each of the ben- particular challenges to scientists who want to efits discussed. study organisms that inhabit the deep ocean envi- ronment. Even though deep-diving submersibles MATERIALS carry bright lights, simply turning these lights on ❑ None creates another set of problems: at least some mobile organisms are likely to move away from AUDIO/VISUAL MATERIALS the light; organisms with light-sensitive organs ❑ (Optional) Images of deep-sea environments may be permanently blinded by intense illumina- and organisms that use bioluminescence (see tion; even sedentary organisms may shrink back, Learning Procedure) ceasing normal life activities and possibly becom- 1 2004 Deep-Scope Expedition – Grades 9-12 (Chemistry and Life Science) Focus: Bioluminescence oceanexplorer.noaa.gov

ing less noticeable; and small cryptic organisms When the electrons return to their original lower- may simply be unnoticed. In addition, some energy orbitals, energy is released in the form of important aspects of deep-sea biology simply visible light. can’t be studied with ordinary visible light. Many marine species are known to be capable of pro- Chemiluminescence is distinctly different from ducing light, and it is reasonable to suppose that fluorescence and phosphorescence, which ability to produce and detect light might be par- occur when electrons in a molecule are driven ticularly important to organisms that live in near- to a higher-energy orbital by the absorption of total darkness. light energy (instead of chemical energy). Both processes may occur in living organisms. Atoms The primary purpose of the 2004 Ocean of a fluorescent material typically re-emit the Exploration Deep-Scope Expedition is to study absorbed radiation only as long as the atoms deep-sea biological communities using advanced are being irradiated (as in a fluorescent lamp). optical techniques that provide new ways of Phosphorescent materials, on the other hand, looking at organisms that make their home in the continue to emit light for a much longer time after blackness of the deep ocean. These techniques the incident radiation is removed (glowing hands are based on a number of basic concepts that on watches and clocks are familiar examples). can be summarized under the general heading of Chemiluminescent reactions, on the other hand, “bioluminescence.” produce light without any prior absorption of radiant energy. Another light-producing process Bioluminescence is a form of chemiluminescence, known as triboluminescence occurs in certain which is the production of visible light by a crystals when mechanical stress applied to the chemical reaction. When this kind of reaction crystal provides energy that raises electrons to a occurs in living organisms, the process is called higher-energy orbital. bioluminescence. It is familiar to most of us as the process that causes fireflies to glow. Some of us The production of light in bioluminescent organ- may also have seen “foxfire,” which is caused isms results from the conversion of chemical ener- by bioluminescence in fungi growing on wood. gy to light energy. The energy for bioluminescent Bioluminescence is relatively rare in terrestrial reactions is typically provided by an exothermic ecosystems, but is much more common in the chemical reaction. The light-emitting molecules in marine environment. Marine organisms producing bioluminescent reactions are known collectively bioluminescence include bacteria, algae, coelen- as luciferins. The light-emitting reactions typically terates, annelids, crustaceans, and fishes. involve a group of enzymes known as luciferases. Several different luciferins have been found in The fundamental chemiluminescent reaction marine organisms, suggesting that biolumines- occurs when an electron in a chemical molecule cence may have evolved many times in the sea receives sufficient energy from an external source among different taxonomic groups. Despite these to drive the electron into a higher-energy orbital. differences, almost all marine bioluminescence is This is typically an unstable condition, and when green to blue in color. These colors travel farther the electron returns to the original lower-energy through seawater than warmer colors. In fact, state, energy is emitted from the molecule as a most marine organisms are sensitive only to blue photon. Lightning is an example of gas-phase light. chemiluminescence: An electrical discharge in the atmosphere drives electrons in gas molecules

(such as N2 and O2) to higher-energy orbitals. 2 2004 Deep-Scope Expedition – Grades 9-12 (Chemistry and Life Science) oceanexplorer.noaa.gov Focus: Bioluminescence

LEARNING PROCEDURE has higher energy than light at the red end of 1. If you want to include demonstrations of chemi- the spectrum (including infrared). luminescence, bioluminescence, fluorescence, and phosphorescence, see the ‘Cool Lights” 3. Tell students that their assignment is to investigate lesson plan for suggestions. The following web the chemistry of bioluminescence, and prepare a sites are useful resources if you want to show report that answers the following questions: images of deep-sea environments and organ- • What is the role of luciferins, luciferases, isms that use bioluminescence: and co-factors in bioluminescence, and what http://www.biolum.org/ is the general sequence of the light-emitting http://oceanexplorer.noaa.gov/gallery/livingocean/livingocean_coral. process? html • What are the major types of luciferins found http://www.europa.com/edge.of.CyberSpace/deep.html in marine organisms? http://www.europa.com/edge.of.CyberSpace/deep2.html • Different groups of marine organisms have http://www.pbs.org/wgbh/nova.abyss/life.bestiary.html different luciferases and co-factors. http://biodidac.bio.uottawa.ca/ • How much light is emitted by biolumines- http://www.fishbase.org/search.cfm cence, and how long is a single burst of light emission? 2. Ask students to describe characteristics of deep- • What is the “lux operon?” sea environments (depth = 1,000 meters or • What are three ways that bioluminescence more). You may want to show images of vari- may benefit deep-sea organisms? ous deep-sea environments and organisms that use bioluminescence. 4. Have each student or student group present and discuss the results of their research. Points that Focus the discussion on light in the deep ocean. should emerge during these discussions include: Students should realize that light is almost com- • The role of luciferins, luciferases, and pletely absent. Ask whether plants and animals co-factors in bioluminescence, and the are ever able to produce their own light. Most general sequence of the light-emitting students will be familiar with fireflies, and may process: mention bioluminescence in other species. The generalized steps in a bioluminescent reaction are: Review the basic concept of chemilumines- – Release of a large quantum of energy cence, and contrast this process with fluores- involving oxide or hydroperoxide radicals; cence and phosphorescence. Students should – Transferance of this energy to a substrate understand that every light-producing process (known collectively as luciferins), causing requires a source of energy (chemical, electri- an electron to be driven to a higher-energy cal, mechanical, or light). Tell students that bio- orbital (a less stable state); and luminescence is a form of chemiluminescence – Return of the electron to its original orbital that occurs in living organisms, but do not (a more stable state), accompanied by explain the details of bioluminescence at this release of energy as a photon of light. point. Students may ask about incandescence, in which light is produced by combustion reac- These reactions are typically catalyzed by tions (thermal energy). Review the concept enzymes known as luciferases. In some spe- of the visible and near-visible light spectrum. cies, the luciferin and luciferase are bound Students should understand that light at the blue together in a “photoprotein” which is acti- end of the spectrum (including ultraviolet light) vated by a co-factor (such as calcium ions). 3 2004 Deep-Scope Expedition – Grades 9-12 (Chemistry and Life Science) Focus: Bioluminescence oceanexplorer.noaa.gov

• The major types of luciferins found in Reports vary. Light emission from dinoflagel- marine organisms: lates is on the order of 6 x 108 photons in – Bacterial luciferin involves a long chain a flash lasting only about 0.1 second. The aldehyde and a reduced riboflavin phos- intensity of bioluminescence from dinoflagel- phate. lates is strongly dependent on the intensity – Dinoflagellate luciferin (also found in of sunlight the previous day; the brighter the euphausiid shrimp) is a tetrapyrrolic com- sunlight, the brighter the flash. Jellyfish are pound whose molecular structure is similar reported to emit about 2 x 1011 photons per to chlorophyll and may have been derived second for periods measured in tens of sec- from chlorophyll. onds. Light emission from a single biolumines- – Ostracod luciferin (known as vargulin, and cent bacterium is on the order of 103 to 104 also found in some fishes) is an imidazolo- photons per second. By comparison, the light pyrazine consisting of modified molecules of emitted from a 100-watt light bulb is about , and . 1018 photons per second. – is the luciferin found in cnidaria, ctenophores, squid, copepods, • The “lux operon:” decapod shrimp, mysid shrimp, and some A group of genes containing the information fishes. This luciferin is an imidazolpyrazine needed to synthesize enzymes needed for consisting of modified molecules of two tyro- bioluminescence. sines and a phenylalanine. • Ways that bioluminescence may ben- In contrast to the marine luciferins, firefly efit deep-sea organisms: luciferin is a relatively simple benzothiazole Some organisms seem to use biolumines- assembled from the amino acids tyrosine cence to locate other members of the same and cysteine. species, and we infer that this would be use- ful for mating activities. Almost any biolumi- • Different groups of marine organisms nescent organism may be inferred to derive have different luciferases and co-fac- this benefit. tors: Marine luciferases are complex proteins con- Bioluminescence may also be useful for feed- sisting of very long chains of amino acids. ing. Some organisms (such as the angler Amino acid sequences have been determined fish) use bioluminescence to attract prey spe- in luciferases from a bioluminescent bacte- cies. Others (such as fishes in the malacos- rium, a firefly, a jellyfish and a dinoflagellate teid family) have a “floodlight” system that and they are all very different structures. allows them to see nearby organisms. These Required cofactors differ as well. Cnidaria fishes have organs that produce red light (an require calcium ions to trigger biolumines- exception to the “blue only” rule), as well cence, while dinoflagellates require a change as eyes that can detect red light. Since most in pH, and at least one marine annelid worm other species (so far as we know) cannot requires ferrous iron. see red light, the malacosteids can sneak up on their prey without being detected. • The amount of light emitted by biolu- minescence, and how long is a single A third potential use for bioluminescence burst of light emission: is camouflage. It may not be immediately

4 2004 Deep-Scope Expedition – Grades 9-12 (Chemistry and Life Science) oceanexplorer.noaa.gov Focus: Bioluminescence

obvious how emitting light could make an to many organisms) and can be programmed organism less visible, yet this is the strategy to acquire time-lapse images (one minute of involved in counter-illumination. You can recording every fifteen minutes) over several illustrate this by holding a white index card days. The video recording system can also be against a window in a darkened room. The programmed to respond to bioluminescence, so card will block out light coming through the that whenever a bioluminescent event is detect- window and be visible as a darker object ed, the recorders will start to capture additional against the bright background. If you shine bioluminescent images, then will turn on the red a flashlight on the card, the illumination on illumination to capture an image of organism the “dark” side of the card will be closer to producing the bioluminescence. that of the background, making the card less visible. Counter-illumination could thus be Other observations will use ultraviolet light a useful strategy to a swimming organism to search for fluorescent organisms that may (such as a squid or fish) trying to be less vis- be less visible under “white” light. A related ible to a potential predator swimming below. study will measure the spectral reflectance of captured organisms, to determine what kinds Bioluminescence may be used for defense of illumination will make these organisms most in a different way. Some tube-dwelling visible to observers. Yet another series of stud- worms (such as the volcano worm) spew out ies will investigate whether polarized light is clouds of glowing blue material when they used by deep-sea organisms, how polarized are threatened. The strategy is similar to the light is changed by these organisms and deep- fear scream of monkeys or birds, which are sea water, and whether these changes can be intended to attract the attention of higher detected and recorded. order predators that may attack the threat- ening predator. So a glowing cloud pro- THE BRIDGE CONNECTION duced by the worm exposes the threatening www.vims.edu/bridge/ – Click on “Biology” in the navi- invader and makes the invader vulnerable to gation menu to the left, then “Plankton,” then attack by a higher order predator. A similar “Phytoplankton” for resources on ocean food glowing cloud might be used by swimming webs. Click on “Ecology” then “Deep Sea” for species to temporarily blind an attacker. This resources on deep sea communities. strategy is used by squid, shrimp, and some fishes. THE “ME” CONNECTION Have students write a short essay on how biolu- 5. Discuss how light-producing processes and the minescence might be potentially important to their ways they are used by deep-sea organisms own lives. could be useful to scientists exploring deep ocean environments. The 2004 Deep-Scope CONNECTIONS TO OTHER SUBJECTS Expedition will use several techniques to make English/Language Arts, Earth Science, Life observations that have never been made Science before. A new deep-sea observatory called Eye-in-the-Sea can be placed on the bottom and EVALUATION left alone to observe sea life without interfer- Written reports prepared in Step 3 provide ence from a submersible vessel. The observa- opportunities for assessment. tory is capable of capturing video images using only red light (which should be invisible

5 2004 Deep-Scope Expedition – Grades 9-12 (Chemistry and Life Science) Focus: Bioluminescence oceanexplorer.noaa.gov

EXTENSIONS NATIONAL SCIENCE EDUCATION STANDARDS 1. Have students visit http://oceanexplorer.noaa.gov to Content Standard A: Science as Inquiry find out more about the 2004 Deep-Scope • Abilities necessary to do scientific inquiry Expedition and about opportunities for real- • Understandings about scientific inquiry time interaction with scientists on current Ocean Exploration expeditions. Content Standard B: Physical Science • Structure of atoms 2. Bioluminescence can be demonstrated with • Structure and properties of matter several organisms. Dinoflagellates are widely • Chemical reactions used; see http://siobiolum.ucsd.edu/Biolum_demos.html and http://www.lifesci.ucsb.edu/~biolum/organism/dinohome. Content Standard C: Life Science html for sources and demonstration ideas. • Interdependence of organisms Fotodyne, Inc. offers kits for demonstrating • Matter, energy, and organization in living bacterial bioluminescence (see http://www.foto- systems dyne.com/education/safelumi.php) • Behavior of organisms

RESOURCES Content Standard E: Science and Technology http://www.geocities.com/bioluminus/how2detect_bioluminescence.html • Abilities of technological design – Web page on how to detect and record • Understandings about science and technol- bioluminescence of small animals photo- ogy graphically Content Standard F: Science in Personal and Social http://www.lifesci.ucsb.edu/~biolum/ —The Bioluminescence Perspectives Web page • Natural resources

http://www.nightsea.com/ – Web site offering products for FOR MORE INFORMATION studying fluorescence underwater Paula Keener-Chavis, National Education Coordinator/Marine Biologist http://www.flinnsci.com – Web site for Flinn Scientific, NOAA Office of Exploration Inc., source for materials for demonstrating Hollings Marine Laboratory chemiluminescence; Phone 1-800-452-1261 331 Fort Johnson Road, Charleston SC 29412 843.762-8818 http://www.biolum.org/ – Harbor Branch Oceanographic 843.762-8737 (fax) Institution Web site on bioluminescence [email protected]

http://ice.chem.wisc.edu/materials/light/lightandcolor7.html – Web ACKNOWLEDGEMENTS site with links to other activities involving This lesson plan was produced by Mel Goodwin, fluorescence and phosphorescence PhD, The Harmony Project, Charleston, SC for the National Oceanic and Atmospheric http://oceanexplorer.noaa.gov/gallery/livingocean/livingocean_coral.html Administration. If reproducing this lesson, please – Ocean Explorer photograph gallery cite NOAA as the source, and provide the follow- ing URL: http://oceanica.cofc.edu/activities.htm – Project Oceanica Web http://oceanexplorer.noaa.gov site, with a variety of resources on ocean exploration topics 6