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Basic Bioluminescence

Technical Report January 2014

DOI: 10.13140/RG.2.1.2855.9846

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 BASIC BIOLUMINESCENCE
www.photobiology.info

John Lee

Department of Biochemistry and Molecular Biology

University of Georgia, Athens, GA 30602
jlee35uga@gmail.com

On mention of the subject of Bioluminescence, most

people think of the tiny flying lights appearing among the
bushes and trees on warm summer evenings. Fireflies are
found in all parts of the world, and are so familiar because
they inhabit the same terrestrial environment as we do,
but in fact, bioluminescence is relatively rare on land
compared to the myriad forms that are found in the
ocean. For the firefly however, which is not a fly but a
beetle, there is a remarkable phenomenon that has
become a tourist attraction, primarily in S.E. Asia. In the
region of the Sengalor River in Malaysia, viewers will
encounter the Firefly Christmas Tree (Figure 1).
Hundreds of fireflies may settle on a tree and flash in
synchrony, all blinking on, then going dark over a several
seconds time period. It is as if their blinking is governed
by a single controller. A more accessible tourist site to see
the same display is in the Great Smoky Mountains
National Park in the Eastern USA. This phenomenon is one
among many impressive instances of "Living Light", i.e.,
from organisms that produce light. (see also the Historical
Vignette on Bioluminescence)

In this module we'll briefly explore the processes that

create this Bioluminescence, and the function that this
light performs for the creatures that produce it, as well as
the many applications of bioluminescence in scientific
research and commerce.
 Figure 1. The firefly "Christmas Tree" as
observed in regions of S.E Asia, from Malaysia
to Papua-New Guinea. It is due to the
synchronous flashing of fireflies.

What Is Bioluminescence?
Bioluminescence is defined as the emission of light from a
living organism that functions for its survival or
propagation. It is a "cold" light, resulting from a specific
biochemical mechanism involving chemical processes,
often specific for that organism. Bioluminescent organisms
occur mostly in the marine environment, and
bioluminescence is one of the major communication
mechanisms in the deep sea (1). Although less common
terrestrially, observations are naturally more frequent
there.

Bioluminescence can be thought of as a

chemiluminescence that is catalyzed by an enzyme. This
light emission from an organism needs to be distinguished
from other forms of luminescence, many also having
biological function, fluorescence, iridescence, diffraction,
etc. (2).

The Wide Distribution Of Bioluminescence

Bioluminescent organisms are found throughout the
biosphere, but only at levels below the mammals and
plants. The occurrence appears randomly distributed
among genera, and sometimes is found in some species
within a genus but not others, without evident reason.
Some 17 phyla and at least 700 genera contain luminous
species. Bioluminescence has been demonstrated in
cephalopods, copepods, ostracods, amphipods,
euphausids, and many fish, annelids and jellies, to name
but a few marine species. On land there are many types
of bioluminescent insects, fireflies, glow-worms, click
beetles, and some diptera, and there are many types of
luminescent fungi responsible for glowing wood. The
bioluminescent bacteria occur both terrestrially as well as
marine. In only a few cases have the bioluminescence
components from the various systems been characterized,
and the overall chemistry established. Many, if not most
luminous organisms in the deep sea, still remain to be
investigated.

Over the past several hundred years, many scientists

have been busy with the collection and classification of
bioluminescent organisms. In fact even earlier, Aristotle
(350 BCE) was probably the first to make systematic
observations of luminescent species and later, complete
and extensive descriptions of luminous organisms were
published by Pliny the Elder (23-79 CE) (3). One example
he described was the luminous mollusc, (Figure 2) a
Roman delicacy, the bioluminescence mechanism of which
is still not completely solved.

Figure 2. Left: The bioluminescent mollusc

Pholas dactylus, in the U.K. commonly known
as a "Piddock". It has worldwide distribution.
Right: Purple jellyfish, common in the
Mediterranean and described by Pliny the
Elder.
The discovery of bioluminescent organisms has been the
goal of many expeditions of ocean-going research vessels.
Marine submersibles are also deployed for the study of
bioluminescence in the deep ocean. Luminous coastal
organisms are more accessible, and can be usually be
collected by inexpensive methods.

Figure 3. Left: The soft coral, Renilla

reniformis, the "Sea Pansy" (about 30 mm
across), found in intertidal coastal areas.
Right: Bioluminescent mushrooms in the light
(top) and dark (lower); in the USA the
bioluminescent fungus is called "Foxfire".

Anatomic Distribution
The tissue distribution of the components of the
bioluminescent system within organisms, is quite varied.
The anatomic location of bioluminescence gives clues as
to the source of component synthesis, storage, transport,
and the functional role of the luminescence. One key
organ is the "photophore" or the light producing organ,
quite evidently seen in many luminous fish and very
vividly in cephalopods. Photophores are normally made up
of complex photogenic (light emitting) cells.

Bioluminescent reaction components have also been

detected in the stomach, secretory organs and liver of
some organisms (mostly believed to be there as a result
of synthesis or storage).
 Figure 4. A bioluminescent squid from the
deep ocean. Some squid can project luminous
clouds from their mouths, and also have
spectacular photophores (light emitting
tissue). From the Bioluminescence Web Page.

Geographic Distribution
Bioluminescent organisms are found world-wide, for
example the so-called "phosphorescence" in sea-water is
observed in all oceans, particularly densely in bays and
coral reefs, where high concentrations of nutrients
promote blooms of the responsible organisms. One
location in Puerto Rico named the Bioluminescent Bay, is
well known for spectacular displays of this dinoflagellate
luminescence. "Red Tides" are often blooms of
luminescent phytoplankton.

A great variety of firefly species are found in the

temperate to tropical regions of the Americas, in China,
and S.E. Asia. Several types of glow-worm have been
identified in North America, Europe, and Australasia.
Interestingly, some species of bioluminescent organisms
are luminous in one location and not in another, e.g., the
"Midshipman Fish", Porichthys notatus. The luminescence
of one population of this species has been postulated to
relate to the availability in that particular area of a dietary
source required for the light reaction. Around Japan, the
firefly squid (Watasenia) displays spectacular
luminescence, and is found in large numbers in restricted
localities. Small crustaceans such as ostracods, are also
found in abundance in Japanese coastal waters.

How Does Bioluminescence Work? All bioluminescence

reactions involve an oxygen oxidation of an organic
molecule (called the luciferin). The reaction is catalyzed
by an enzyme called a luciferase and in many cases, the
bioluminescence intensity is assumed to reflect the
velocity of the enzyme-substrate reaction, and this
intensity is used to analyze the kinetics on the Michaelis-
Menten model (Figure 5A). It was first a puzzle that the
bioluminescence of aequorin and subsequently of several
other like organisms, was found not to involve oxygen
kinetically, and these proteins were labeled
"photoproteins" (Figure 5B). It was eventually established
that the oxygen had already bound to the luciferin, and
the photoprotein therefore could be more accurately
thought of as a luciferase binding a stabilized reaction
intermediate, a peroxy-luciferin.

Many bioluminescent reactions in vitro require cofactors in

addition to oxygen, e.g., ATP and Mg2+ for the firefly,
Ca2+ for photoproteins (1, 2, 4). In the animal itself (in
vivo), there are additional proteins involved for production
and regulation, some called "accessory proteins",
examples being the fatty acid reductase group of enzymes
that produce the bacterial luciferin, a long-chain aldehyde,
and there are luciferin-binding proteins in the
dinoflagellate and Sea Pansy bioluminescence systems.
Also, there are "antenna proteins" that act to modulate
the color of bioluminescence, the famous Green-
fluorescent protein (GFP) in the jellyfish, and the
Lumazine Protein family in some types of bacteria (4).
These are named "antenna proteins" by analogy to
proteins of similar function in photosynthesis, except that
they act in a reverse sense.
 Figure 5. Reaction Schemes for a
luciferin/luciferase reaction (A), and for a
typical photoprotein reaction triggered by
calcium (B). The reaction product is the light
(hv) emitting species, the protein-bound
oxyluciferin or protein-bound coelenteramide.

To date, there are five known distinct chemical classes of

luciferins, namely, aldehydes, benzothiazoles,
imidazolopyrazines, tetrapyrroles and flavins. An
imidazolopyrazine derivative, aptly named
"coelenterazine", is the luciferin found in coelenterates
and many other marine bioluminescence systems (5, 6).

Physics: Characteristics Of The Light Emission

Bioluminescence results from a chemical reaction that
releases a large amount of energy which, instead of being
dissipated as heat as in a normal chemical reaction, is
channeled to populate the product molecule in its excited
electronic state. This excited state is the same one
produced in that molecule by the absorption of radiation,
so that the spectral distribution of the bioluminescence is
often the same as that of the product fluorescence. The
color of the bioluminescence however, is sometimes
"tuned" by the protein environment of the product excited
state, a property evolved to suit the function of the light
emission, that is for communication, defense against
predation, etc.

Visible radiation corresponds to light in the wavelength

range of 400-700 nm (Figure 6). Bioluminescence spectra
are broad bands with widths at half-height around 60-100
nm. The bioluminescence maximum of most marine
species falls within the range of 450-510 nm (7), whereas
terrestrial organisms have predominantly a yellow-green
bioluminescence color. In ocean water, blue to green
(400-500 nm) luminescence achieves maximum
transmission, whereas terrestrial species have their
maximum visual sensitivity for yellow light. Visual
pigments of most marine organisms are correspondingly
most sensitive in the blue-green region.
 Figure 6. The end-product of bioluminescence
is visible light. The visible part of the
spectrum is 400-700 nm, and the emission
maxima of most luminous marine organisms
falls within the range of 450-490 nm.

What Are Some Of The Functions Of

Bioluminescence?
As a result of its prevalence, bioluminescence plays an
important role in the ecology of the ocean. The function of
bioluminescence in the oceans is more clearly understood
in the context of the essentially dark environment below
about 200 m. The functions of bioluminescence are for:
Defense
Schooling of fish
Luminous lure
Feeding
Communication (in the dark)
Mating
Camouflage

Impact Of Molecular Biology And Bioluminescence

The cloning of various components of bioluminescent
systems has heralded major advances in biological
research. The calcium-dependent photoprotein aequorin
from the jellyfish Aequorea victoria was cloned in 1985 (8,
9). Because the intensity of its luminescence varies with
calcium concentration, aequorin has been used for
advantage in the monitoring of cell calcium. In 1985,
firefly luciferase was cloned (10). As an extremely
sensitive method for the assay of ATP, this
bioluminescence system has found wide application, e.g.,
to detect microbial contamination in foodstuffs, water
systems, etc. Living organisms contain ATP so its assay
detects the presence organisms that might lead to
spoilage or toxicity. Many other luciferases have been
cloned, including bacterial luciferase from Vibrio harveyi,
the luciferase from the sea pansy Renilla reniformis (11),
and the South American click beetle luciferase (12).

Current Applications Of Bioluminescence

The "Green-Fluorescent Protein" or GFP, is probably the
most famous protein in Biology (Nobel Prize in Chemistry,
2008). GFP was cloned in 1992 (13), and expressed in
various organisms in 1994 (14). Since that time the
number of literature citations has risen into many
thousands, as applications of GFP have increased. In
particular, GFP is now well established as an excellent
gene tag or protein tag. GFP can be fused to a protein of
interest, and fluorescence (and therefore the protein of
interest) can be tracked within a cell to study its
localization and behavior. GFP has outstanding structural
stability, and with the property of being able to form the
fluorescence in situ without the external addition of
substrate, GFP becomes an excellent tool for studying cell
and sub-cellular processes (15).

Rapid and effective diagnostic tests based on

bioluminescence are constantly evolving in the
marketplace. For example, "Microtox" for water
quality/toxicity testing employs the bioluminescent marine
bacteria Vibrio fischeri. When this organism is challenged
by a toxin, the respiration pathway is disrupted, resulting
in a decrease in bioluminescent intensity.

Some "fun" applications and ideas exist such as the

prospect of luminous Christmas Trees and walkways in
addition to luminescent beer and champagne. The use of
light sticks for night concerts and guiding aircraft to
airport gate positions are but a few every day applications
of this widespread phenomenon: luminescence.

Questions That Remain

Although bioluminescence is a spectacular phenomenon in
biology with a long history of investigation, questions
abound ranging from the biological advantage of light
emission to the animal, to the molecular mechanism of
efficient excited state population. Some fish possess a
photophore containing a culture of bioluminescent
bacteria, and use this to lure prey. The firefly flash is for
sexual communication, but why the synchrony of the
firefly "Christmas Tree", and what is the advantage of
luminosity to earthworms? How did the efficient
generation of biological light originate in various
organisms, and what is the evolutionary history? What is
the metabolic or dietary source of the luciferins, and what
are the control mechanisms for light flashing? At the
molecular level, the chemical-physical pathway that
generates the reaction product in its excited state, is an
outstanding current problem. Together with this, the
shifting of bioluminescence color via the luciferase binding
site environment perturbing the excited state energy
level, or by coupling to the excited state of antenna
proteins by some process, continue to be challenging
areas of current research (16).

Useful Websites
The International Society of Bioluminescence &
Chemiluminescence

The Bioluminescence Web Page

There is no Such Thing as a Jellyfish

Steven H.D. Haddock

The Weird, Wonderful World of Bioluminescence

Dr. Edith Widders TED talk

Five video lectures on Bioluminescence Mechanisms

Professor John Lee
 Bioluminescence, the Nature of the Light
John Lee, University of Georgia (2015)

References
1. Haddock, S.H.D., Moline, M.A., and Case, J.F. (2010),
Bioluminescence in the Sea. Annu. Rev. Mar. Sci. 2:443
493.

2. Campbell, A. K.(1988). Chemiluminescence: principles

and applications in biology and medicine. (VCH/Horwood,
Chichester).

3. Harvey, E.N. (1957). History of Luminescence.

(American Philosophical Society, Philadelphia).

4. Shimomura, O. (2006). Bioluminescence: Chemical

Principles and Methods. (World Scientific, Singapore).

5. Campbell, A. K., and Herring, P. J. (1990).

Imidazolopyrazine bioluminescence in copepods and other
marine organisms. Mar. Biol. 104: 219-225.

6. Thomson, C.M., Herring P.J., and Campbell, A. K.

(1997). The widespread occurrence and tissue distribution
of the imidazolopyrazine luciferins. J. Biolumin.
Chemilumin. 12(2): 87-91.

7. Herring, P.J. (1983). The spectral characteristics of

luminous marine organisms. Proc. Roy. Soc. Lond. B. 220:
183-217.

8. Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata,

T., Iwanaga, S., Miyata, T., and Tsuji, F.I. (1985). Cloning
and sequence analysis of cDNA for the luminescent
protein aequorin. Proc. Natl. Acad. Sci. USA. 82: 3154-
3158.

9. Prasher, D., McCann, R.O., and Cormier, M.J. (1985).

Cloning and expression of the cDNA coding for aequorin, a
bioluminescent calcium-binding protein. Biochem.
Biophys. Res. Commun. 126:1259-1268.

10. De Wet, J. R., Wood, K. V., Helsinki, D. R., and

 DeLuca, M. (1985). Cloning of firefly luciferase cDNA and
the expression of active luciferase in Escherichia coli.
Proc. Natl. Acad. Sci. USA. 82:7870-7873.

11. Lorenz, W. W., McCann, R. O., Longiaru, M., and

Cormier, M.J. (1991). Isolation and expression of a cDNA
encoding Renilla reniformis luciferase. Proc. Natl. Acad.
Sci. USA. 88:4438-4442.

12. Viviani, V. R., Bechara, E.J., and Ohmiya. Y. (1999).

Cloning, sequence analysis, and expression of active
Phrixothrix railroad-worms luciferases: relationship
between bioluminescence spectra and primary
structures.: Biochemistry 38: 8271-8279.

13. Prasher, D. C., Eckenrode, V. K., Ward, W.W.,

Prendergast, F. G., and Cormier, M. J. (1992). Primary
structure of the Aequorea victoria green-fluorescent
protein. Gene 111: 229-233.

14. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and
Prasher, D.C. (1994). Green fluorescent protein as a
marker for gene expression. Science 263: 802-805.

15. Zimmer, M. (2010) Green fluorescent protein: a

molecular microscope, on Photobiological Sciences Online
(KC Smith, ed.). American Society for Photobiology,
http://www.photobiology.info/.

16. Wilson, T. and Hastings, J.W. (2013)

Bioluminescence: Living Lights, Lights for Living. Harvard
University Press (Cambridge, MA).

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