Bioluminescence

Presented by:

Dr. Amarjeet Singh

Assistant Professor
Department of Zoology
Sri Venkateswara College
University of Delhi
 Introduction
• Three kinds of light emission from a living organism may take place:
• 1. Photosynthetic delayed light emission , also called delayed fluorescence or
afterglow. This is weak red light emitted by all green plants and algae. The
intensity is so low, and the light of such long wavelength, that we cannot see it, but
it is easily measured. It is due to reversion of the first steps of photosynthesis.
• 2. Ultraweak light emission takes place in all organisms. It is due to various
processes, mostly (but not always) involving molecular oxygen. It is regarded as a
by-product of metabolic activity and has no biological function in itself. It is even
weaker than delayed light emission, and although it is often of shorter wavelength,
it cannot be seen; rather sophisticated equipment is needed for its measurement. It
can be exploited for studying what is going on in cells in a noninvasive and
nondestructive way.
• 3. Bioluminescence is the best known of the biological luminescence phenomena,
mostly because it can be observed using only one’s eyes.
 Evolution and Occurrence among Organisms

• Although most species are nonbioluminescent, most phyla have bioluminescent

representatives. Among the exceptions are true plants and higher vertebrates (i.e.,
amphibians, reptiles, birds, and mammals).
• Bioluminescence has evolved from antioxidant systems.
• Thus bioluminescence occurs among bacteria, fungi, dinoflagellates, protozoa,
sponges, cnidaria, ctenophores (comb jellies), molluscs, annelids, crustaceans,
insects, bryozoa, echinoderms, and fi sh. The majority of bioluminescent species
live in the sea, although there are also many bioluminescent insects (all terrestrial),
especially beetles.
• It has been estimated that 60–80 % of the fish species in the deep sea are
bioluminescent.
• It is probable that bioluminescence first appeared during the “Cambrian
explosion”, when the evolution of eyes had made it meaningful.

• Molecular oxygen is required for all known bioluminescence mechanisms,

but the required oxygen partial pressure (or equivalent chemical activity) is
much lower than that of the contemporary atmosphere.
In those cases when we can clearly see a present-day biological role for
bioluminescence, we can divide the advantages gained into five main
categories:

1. Reproduction

2. Protection from predation (defence, camouflage, or aposematic signaling)

3. Food acquisition

4. Protection from reactive oxygen species (ROS)

5. DNA repair
1. Reproduction
• The best-known examples of bioluminescence having a role in the propagation of
the species are found among the beetles of the family Lampyridae (true fireflies
and glowworms), although bioluminescence also occurs in several other beetle
families.
• In glowworms only the female glows brightly (with a steady light) and by this
attracts the male, and the same is the case in some firefly species.
• In other fireflies, a sophisticated “light conversation” between males and females
has evolved, with a different “language” for each species. Males send out an
“interrogation” flash, and females respond.
• Species specificities are obtained both by the time course of the flash on a half-
second time scale and by the time delay between “interrogation” and “answer.”
• An intriguing phenomenon is the synchronous flashing of the males of some
fireflies. These males often collect in a tree, and the whole tree flashes “in step”.
• Another example of luminescence intensity being synchronized among individuals
is that of the cave-dwelling Tasmanian glowworm Arachnocampa tasmaniensis,
which exhibit 24-h rhythm even in the distant parts of the cave where no daylight
reaches.
• There is also a multitude of deep-sea animals which use bioluminescence for
finding a mate in the dark abyss.
• Dragonfish, uses light of a wavelength so long (maximum 702 nm) that it cannot
be perceived by other organisms on which the dragonfish preys.
• Bioluminescence has been reported in about 40 species of fungi, of which nearly
two thirds belong to the genus Mycena. Other genera with luminescent members
are Panellus, Armilariella, Lampteromyces, Pleurotus, Omphalia, and
Omphalotus. Panellus stipticus is a brightly luminescent fungus, common in North
America, which has served as material for several investigations. It has been
speculated that luminescent fungi attract insects which aid in dispersal of spores.
However, in many fungi, only the mycelium, and not the fruiting bodies,
luminesces.
 2. Protection from predation
• One of the best-known examples of bioluminescence, described by Aristotle, is the
“fire of the sea” caused by dinoflagellates such as Noctiluca and Gonyaulax. Its
survival value remained obscure for a long time, but it has now been shown that it
protects from grazing by copepods.
• Some squid, when attacked, give off a luminescent secretion which confuses the
attacker, and luminescent secretion from a shrimp may serve a similar purpose.
• A kind of marine annelid called a scale worm is covered on the dorsal side by scales
which first emit flashes when the animal is attacked and then are shed, still glowing.
• By aposematic coloration, we mean easily recognized bright color patterns like the
black-yellow banding of wasps, spots on ladybugs, and stripes on coral snakes, which
warn a predator of nasty consequences of an attack (and frequently are mimicked by
species which do not have any other protection). Bioluminescence of firefly larvae
serves a similar purpose. The bioluminescence of the millipede Motyxia sequoiae also
has an aposematic function. Luminescence of cockroaches is thought to be mimicry of
non-palatable click beetles.
• Bioluminescence can also be used for camouflage in two different ways.
• Fish can be either luminescent by themselves or can harbor luminescent bacteria.
Some fish of both categories use bioluminescence for counterillumination and
“disruptive illumination”, to avoid perception of their shape and size.
• Most fish are lighter on the ventral than on the dorsal side, and this can be
regarded as minimizing their visibility: from above, they look dark like the
background depth, and from below, they look bright like the sky above. This
cannot give complete protection; they still look rather dark against a bright sky.
But some fish, by bioluminescence, do match both the intensity and the angular
distribution of the downwelling surface light .
• Bioluminescent organisms such as the dinofl agellate, Noctiluca , respond to
predation, or even movement, by fl ashing, thereby increasing the visibility of
actual or potential predators to secondary predators, and thus protecting
themselves.
3. Food Acquisition
• Female fireflies of the genus Photuris reply to the “interrogating fl ashes” from
males of other fireflies, lure them to approach, and eat them
• Most sly and cunning of them all are the females of Photuris versicolor , who
know firefly languages sufficiently well to be able to prey on 11 different species,
but male Photuris, in turn, outsmart the females by mimicking their prey in order
to mate
• In addition to beetles, among bioluminescent insects, fungus gnats, members of
the order Diptera use bioluminescence. About a dozen of more than 3,000 species
in the family have larvae which use bioluminescence in different ways to catch
prey. Particularly famous are the larvae in the Te Ana-au caves on the South Island
of New Zealand. The gnat larvae sit on the roof and deploy luminescent, which
attract other insects which are caught and devoured
• Willis et al. (2011) found that using light to lure prey is an energetically “cheap”
predatory strategy compared to spiders’ web construction
• In many cases, marine animals are aided in their vision by their own
luminescence, which functions mainly in the service of food acquisition
• Several fish species, such as Aristosmonias scintillans and Pachystomias
microdon in the deep sea, and Photoblepharon palpebratus and Anomalops
katoptron in shallow waters, have luminescent organs in proximity to their eyes
4. Protection from ROS
• Coelenterazine, a common marine luciferin, and has its strong antioxidant
properties. It is proposed that during the early stages of their evolution, the
primary function of these systems would have been detoxification of oxygen and
other ROS at a time in which environmental oxygen levels were rising (modern
surface waters are rich in superoxide [SO] and hydrogen peroxide)
• Along the way, these organisms would have developed effective ways of handling
these potentially toxic and high energy systems, and as they colonized the less
oxidatively stressful deeper levels of the seas (less light, including UV, less
oxygen, and slower metabolism), their systems would be preadapted to produce
light for other purposes (all bioluminescent reactions are high energy and require
involvement of oxygen or its activated species)
• The studies have demonstrated that wild-type (=luminescent) bacteria of Vibrio
harveyi and other strains are able to survive UV irradiation or exposure to
hydrogen peroxide better than nonluminescent mutants and, further, that this wild-
type resistance can be conferred to E. coli
5. DNA Repair

• Many of the luminescent bacteria display a behaviour called “quorum sensing,” in

which the bioluminescent systems are not expressed until the colony reaches a
certain density
• A common interpretation is that this may deter predators from disturbing a colony
that has gained a beachhead in a food item. But many luminescent bacteria
luminesce at extremely low density, and in addition to possible detoxification of
ROS evidence now suggests strongly that this solo luminescence may function in
activation of photolyase enzymes in DNA repair
• In short, bacteria living in relatively dark levels of the ocean may be achieving
photo-reactivated DNA repair by producing their own “photo”
 Light Organs and Organization

• In most cases, the bioluminescence is manufactured within specialized cells called

photocytes
• Many higher vertebrates have a highly organized collection of these cells making
up a light organ or photophore
• In those fish that utilize the bioluminescence from symbiotic luminous bacteria,
the photophore is like a culture vessel
• Many other fish use a different bioluminescence system to produce light involving
either intra- or intercellular processes
• There also exist a number of highly specialized accessory structures to the light
organ, in some cases reaching a degree of complexity comparable to, and in fact
resembling, the vertebrate eye
 Origin
• One proposal for the origin of bioluminescence is that the first bioluminescence system
developed out of a biological chemiluminescence reaction involving an oxygenase enzyme
acting on a hydrocarbon. This mechanism is a free-radical one. The accumulation of a
suitably fluorescent metabolite, e.g., the highly fluorescent proteins that are found in the
bioluminescent bacteria and coelenterates, would then provide a means to sensitize or
amplify this light production at a minimal energy cost
• Higher on the evolutionary scale, selection was made of reactions that would produce highly
fluorescent product molecules directly in their excited singlet state, and with everything done
on only one enzyme
• Bacteria were the first organisms to "discover" bioluminescence and a niche in which it
could be of advantage. This is based on the facts that bacteria lie lowest on the evolutionary
scale, they can mutate and evolve rapidly if benefit is to be derived from it, and they already
possess the substrates for bioluminescence. Although luciferase, the enzyme needed to
generate the bioluminescence, is unique to the bioluminescent bacteria, it has been postulated
that luciferase might also have a terminal oxidase function in a respiratory pathway operative
at a very low oxygen level, i.e., one approaching anaerobiosis
 Origin & Function
• The alternative view held by most scientists is that bioluminescence arose independently in
many organisms. This is because the property is spread over such a diversity of organisms
that it seems a relatively recent event in evolution, and the luciferins often have very
different chemical structures, even among closely related organisms. Additionally, there is no
structural property of the luciferases that would suggest a common lineage
• Firefly system: The flash is a sexual signalling and is made up of a series of short flashes, a
"Morse code" that is characteristic of the species. The flying male flashes and may be
answered after a definite time interval, about 2 s, by the female. In one exceptional though
interesting case, the female of one species is insectivorous, feeding on the males of other
species and luring its prey to it by imitating the female response appropriate to the prey
species
• There is also a visually discernible difference in the colour of bioluminescence between
certain species of firefly. This appears to be an adaptation since the yellow bioluminescence
is from fireflies active at dusk, the yellow light contrasting against the green background of
foliage. The night-active fireflies emit at shorter wavelengths, the green light more efficiently
matching the visual sensitivity of their species
• It has recently been shown that starting with a mixed population of luminous and
nonluminous dinoflagellates and their predators under controlled laboratory
conditions, the luminous types show an increase in survival over nonluminous types.
The luminous flash probably deters the predator, conferring survivorship on the
species that possess this property
• Some luminous fish have a light organ that houses a symbiotic culture of luminous
bacteria. Some fish (the "flashlight" fish) have even adapted a lid to move over the
light organ to create a flashing of the otherwise continuous emission. Other fish
appear to contain a luminous system, i.e., the luciferins and luciferases cross-react.
The luminescence probably serves as a mating signal, or as a lure
• In the Australasian glowworm, which is the larva of a dipteran (fly), in contrast to the
firefly, which is a coleopteran (beetle), the luminescence has a lure function. The larva
hangs in the middle of a web and emits a blue glow. Small, winged insects are
attracted to the glow, ensnared in the web, and devoured
• Seliger summarized the biological functions of bioluminescence by the "four P's":
predation, protection, prenuptial (firefly communication), and perfidy (the
insectivorous firefly)
The substrate and enzyme molecules in bioluminescence are called by their
respective generic names, luciferin and luciferase. The emission colours of
bioluminescence range from the blue of the marine bacteria and deep sea fish to the
red of the railroad worm Phrixothrix
 Mechanism

• It was discovered by Boyle in the 17th century that air was required for
the bioluminescence of bacteria and fungi, and it is now known that
oxygen in some form is involved in all bioluminescence systems
• At the end of the last century, Dubois found that two components could
be extracted from the firefly light organ, one with hot water and the other
with cold water. The cold-water extract, which was heat-labile, he named
luciferase, and the hot-water extract, luciferin. He made a similar
observation with the clam Pholas but found that a cross-reaction between
clam luciferin and firefly luciferase, and vice versa, did not occur
• Most exhibit the classical enzyme (luciferase)-substrate reaction, but
some have a precharged system called a “photoprotein”
• Some systems only involve luciferase and luciferin with 02; others use
several proteins
• Some luciferases are small proteins, whereas others are large, multi-
subunit enzymes
• Also, each luciferase is specific for each type of organism
• Thus, the biochemistry of bioluminescence is analogous to the biological
potpourri remarked on by Harvey, i.e., each system has found its own
pathway to the final product, light emission
 Mechanisms of Light Production
• The different mechanisms require oxygen at some stage
• Many of them also involve a peroxide—either a hydroperoxide or a
cyclic peroxide. They involve catalysis by an enzyme called luciferase,
but luciferases from different organisms are different
• Luciferase action on a relatively low molecular weight organic
compound called luciferin results in an excited state of a pigment,
which either emits light directly or transfers excitation energy to
another emitter
• Bacterial luminescence is based on peroxidation of flavin
mononucleotide (FMN) and oxidation of a long-chain aldehyde to
carboxylic acid, so flavin mononucleotide can be said to be the
luciferin in this case
• Fungal bioluminescence
• In this, an emission maximum (on a photon per wavelength interval
basis) of about 525 nm was found
• The emission is caused by a reaction between hydrogen peroxide, a
lowmolecular amine, and panal (sesquiterpene aldehyde)
• Several other emitters have been proposed in fungal bioluminescence,
one of which is riboflavin
• Dinoflagellates have different luciferins depending on the species
• In the most studied organism, Gonyaulax polyedra , it is a
tetrapyrrole-like substance with an extra ring, clearly derived from
chlorophyll, but in another species, Pyrocystis lunula , it is quite
different
• The bioluminescence of Gonyaulax differs from that of most
bioluminescent organisms in that no peroxide seems to be involved
• For light emission by the firefly luciferin/luciferase reaction, prior
adenylation of the luciferin by reaction with ATP is required
• Both luciferin and luciferase are located in the peroxisomes in one part
of the cell, while another part of the cell is full of ATP-generating
mitochondria
• Although different beetles can produce light with different colours from
green to red, they all seem to possess the same kind of luciferin. The
differences in wavelength distribution are probably due to differences
in luciferase
• As for colour of emitted light, the most remarkable animal is the larva
in the beetle family Phengodidae ( Phrixothrix vivianii and Euryopa
species), the so-called railroad worms. These carry both red lanterns
(on the head) and yellow-green ones (on the sides) on the same
individual
• Among crustaceans the euphausiids are worth special mention because
of their strong light and sophisticated lantern optics. They are shrimp-
like animals but distinct from true shrimps and not members of the
group Decapoda
• Like the dinoflagellate Gonyaulax , they have a tetrapyrrole chlorophyll
derivative as a light-emitting chromophore, but the macrocycle of the
chlorophyll molecule is split open at another site
• Their lanterns are equipped both with a reflective backing and a lens
system to direct the light. Those lanterns which are located on the eye
stalks just above the eyes certainly serve as an aid to vision
• The structure of the luciferin typical of the coelenterates, i.e., cnidaria
and ctenophores (coelenterazine), has great similarities to the
prosthetic group of a light-generating chromoprotein, aequorin, of the
jellyfish Aequorea . The luciferin of the squid Watasenia is
coelenterazine with the hydroxy groups replaced by sulfate groups
• Earlier it was thought that the bioluminescence of Aequorea was
something in principle different, since the chromoprotein aequorin
extracted from it would glow in vitro when calcium ion was added,
without the need for molecular oxygen
• However, it is now realized that aequorin is an enzyme–substrate
(luciferase–luciferin) complex which requires oxygen for formation
and is stable in the absence of calcium ions
Thank You