lesliecantley-final+paper

Leslie Cantley

__//** The Chemistry and Ecology of Bioluminescence: **//__

__ Introduction: __

Luminescence is characterized as a type of light that is emitted from a source without the creation of heat, and is sometimes called “cold light”; the name of this phenomenon arises from the Latin word “lumin”, meaning “light”. [1] The first scientific study involving luminescence was carried out by a French physiologist named Raphael Dubois, whose basic method is still being used in experiments involving luminescence today. In 1885, Dubois made a temporarily luminescent solution by mixing cold water with the ground up abdomens of the Elateridae beetle, yet did not obtain a luminescent solution at all when hot water was used. The luminescence was emitted from the hot water solution only once the water had cooled and was added to the extinguished cold-water solution. He concluded that the hot water had destroyed a necessary component of the light-producing reaction, what we now know to be the enzyme luciferase, yet had not destroyed the component that had been used up in the cold water solution, now called luciferin. [2] In addition, this type of light was first defined by Eilhard Wiedemann, a German science historian and physicist, in 1888. Presently, the definition of luminescence is light arising from a spontaneous emission of radiation from an electronically or vibrationally excited species that does not exist in thermal equilibrium with its surroundings. [1]

Similarly, bioluminescence is defined as this type of light that is emitted from a living organism, and has evolved independently multiple times. [3] It is used by a multitude of organisms, from a plethora of very different species, and is used in nature for a variety of purposes. Several types of chemical reactions are responsible for luminescence in organisms, yet the general chemical reaction is similar throughout various species. A substrate, called luciferin is oxidized by molecular oxygen and forms a product molecule in an electronically excited state (named (P)* in Figure 1). This entire reaction is catalyzed by an enzyme called luciferase. There are many chemically different luciferases and luciferins found in nature; the components of the reaction depend on the bioluminescent species in question. [4]

In this paper, I will examine the mechanisms of bioluminescence in several familiar organisms, as well as explain a few of the ways humans are using bioluminescence for their own benefit, especially in the various fields of scientific research.

__ Evolutionary Origins of Bioluminescence: __

As there are many luciferases and luciferins with different chemical structures, varying throughout all bioluminescent species, it is very likely that bioluminescence evolved separately, perhaps as many as thirty different times, across species. It is evident that bioluminescence holds some sort of selective advantage for organisms, even if the purpose for their emission of light is not readily apparent. [4]

Just as the chemical structure for luciferin and luciferase varies across species, so too does the mechanism for control over bioluminescence. For example, the emission of light in bacteria is continuous and cannot be shut down by the organism; in contrast, some squid and fish harbor special organs designed to produce light, and are controlled through the use of shutters. Yet more organisms, such as the marine-dwelling //Vargula//, produce luciferase and luciferin separately and can squirt the chemicals outside of their body to mix and react in the seawater. These varying mechanisms for control over light production is also evidence that bioluminescence evolved separately throughout species. [4]

Despite the very likely differing evolutionary origins for luciferins, luciferases, and the bioluminescence reaction across species, there are some cases where molecules similar in structure are utilized by distantly related species. Sometimes there is a genetic cause for this, and sometimes there is not. An alternative hypothesis suggests that DNA may have been laterally transferred between organisms that are distantly related, such as the mechanism for gene transfer by symbiosis. This has occurred with biological traits other than bioluminescence, and so is a plausible hypothesis for the evolution of light emission by vastly different organisms. Interestingly, bioluminescence may have also evolved differently even amongst very similar organisms; for instance, mollusks and fish possess very different luminescence mechanisms and are likely not to have come from the same evolutionary origin. [4]

The various systems for bioluminescence reactions do share some common features, a fact which helps to explain the frequency and diversity of light producing species, despite the fact that the mechanism evolved independently. For instance, all luciferases are oxygenases. In addition, it is hypothesized that more advanced bioluminescence systems evolved relatively late, after the development of vision, for example. This may also be the cause of the various and divergent light producing mechanisms by many different organisms. [4]

Now that the evolutionary origins and fundamental chemical reaction of bioluminescence have been explained, it will be helpful to learn how this information applies to specific species, especially those with which many people are familiar. I will therefore begin with what is quite possibly the most famous light producing organism, and one that has be extensively studied by scientists.

__ The Firefly: __

While there are multiple species of arthropods that produce light, the family of insects known as Lampyridae, or fireflies, is arguably the most well known bioluminescent organism. These beetles, over 2000 species of which are known to science, are crepuscular and communicate with each other through flashes of visible light for mating purposes and to attract prey. The wavelengths of the light produced from their lower abdomens range from 510 to 670 nanometers, and can be yellow, green, or red. [5] One of the most commonly studied species of firefly is //Photinus pyralis//, the common North American firefly. //P. Pyralis// is able to initiate a luminescence reaction with a high quantum yield with respect to luciferase (0.88); the light it produces has a yellow-green color and has a peak emission at 560 nanometers at pH 7.5 to 8.5. [6]

The chemical reaction involved in the bioluminescence of //P. Pyralis// is very similar to the general luminescence reaction detailed in Figure 1, and like many biological reactions, is mediated by ATP, or adenosine triphosphate. The substrate D-luciferin reacts with the enzyme luciferase in the presence of Mg2+ and molecular oxygen, forming oxyluciferin in an electronically excited state. [7] The generally accepted mechanism for the firefly's light producing reaction is as follows: D-firefly luciferin reacts with ATP-Mg2+ (adenosine triphosphate bound to a magnesium ion), forming luciferin-AMP (luciferin bound to adenosine monophosphate), and finally reacts in the presence of molecular oxygen to form the product oxyluciferin in an excited state. [8] A schematic of the complex of the enzyme luciferase binding with luciferin is shown in Figure 2. [9] The color of the resulting luminescence of the reaction can be shifted if different ions are used instead of magnesium, as well as with a shift in the pH of the reaction. [7]



Fireflies are a common and evolutionarily successful group of insects with which many people are familiar, and which use bioluminescence to great effect.

__ Fungi: __

Fungi, although perhaps not as well known for being bioluminescent as fireflies, are another group of organisms that sometimes use luminescence during their life cycles, and there are a wide range of species that are bioluminescent found all over the world. Presently, there are 71 out of the 100,000 categorized species of fungi that have been confirmed to be luminescent, and these species are distributed among four related lineages. All known fungi that exhibit bioluminescence belong to a mushroom-forming group known as Agaricales. [10]

For many decades, the chemical mechanism for luminescence in this group of fungi remained unproven, and it was unclear whether or not a luciferase enzyme was present to mediate the reaction, as in the general luminescence reaction depicted in Figure 1. Recently, it was proven that not only is luciferase present in the reaction of these fungi, but the components of the reaction taken from each of the four lineages can be cross-reacted with those taken from the other lineages. This suggests that a single bioluminescence system is shared by all four fungal lineages and may have the same evolutionary origin. [10]

The fungal system of bioluminescence is similar to the general mechanism for luminescence, and involves a luciferin, a NAD(P)H-dependant retuctase, NADH or NADPH, and a luciferase. [10]

There have been several proposed reasons for the evolution of fungal bioluminescence, and as one can imagine, the advantages for fungi that produce light are very different from that of fireflies. One of the hypothesized ecological reasons for fungi luminescence is as a mechanism for spore dispersal. Studies have shown that arthropods are attracted to the light given off by luminescent fungi and therefore may congregate around the mushrooms while spores are emitted, helping the fungi disperse its spores over a wider area. However, there has been some disagreement about this hypothesis, as some arthropods feed on the fungi, and it therefore may be a disadvantage for the fungi to attract insects and other predators. [11]

Another hypothesis regarding the ecological reasons for the bioluminescence of fungi is that the light produced is simply a byproduct of respiration. It has been noted that the bioluminescence reaction requires very little energy, and therefore may be a method for the fungus to the expend energy created by enzyme mediated oxygenation reactions, expending light instead of heat. Furthermore, it is possible that undergoing bioluminescence is advantageous for the fungus in order to avoid negative effects of reactive oxygen species produced by mitochondria during respiration. [11]

The ways in which terrestrial organisms such as the firefly and certain species of fungi use bioluminescence are certainly interesting, especially when taking into account the divergent evolutionary pathways that distant species used to develop biological light emitting reactions. However, there are many more species that live in marine environments that also make use of bioluminescence.

__ In the Ocean: __

There is a whole host of marine species, both vertebrates and invertebrates, and especially those that reside in the deep parts of the ocean, that make use of bioluminescence; indeed bioluminescence is more common in marine organisms than those that are terrestrial. Bioluminescent organisms exist at all depths of the ocean, but are the most common in the upper 1000 meters of the open ocean due to the unique challenges put forth by this type of environment, such as the near-complete darkness of this portion of the ocean. Emitting light is of key importance for these types of organisms in finding a mate, camouflage, and in luring prey. [12] The maximum intensity of light in the deep portions of the ocean is found in the blue to green region of the electromagnetic spectrum, specifically about 475 nanometers. Therefore, not only have the eyes of the primary number of organisms that exist in this environment evolved to see best in this range, but much of the bioluminescence produced by organisms living in the deep sea is emitted at this wavelength as well. [13] One of the most well known species of bioluminescent fish is the deep sea anglerfish, or //Oneirodes acanthias//. Female anglerfish exhibit a luminescent organ called an esca, which is a modified first dorsal ray. This organ is used both as a lure to attract prey as well as a signal to attract males when the female fish is ready to mate. There had originally been some contention over the origin of the luminescence produced by anglerfish, but studies have proven that the light from the esca is produced not from the fish itself, but from a colony of symbiotic luminescent bacteria growing in the lure. Unfortunately, when biologists isolated and cultured bacteria from the esca in female anglerfish, the cell cultures did not produce light. This suggests that the fish is able to provide some sort of nutrients required to make the bacteria luminescent. [14]

Another notable bioluminescent organism that resides in the deep ocean is the comb jellyfish, or ctenophore, which belong to the phylum Ctenophora. These jellyfish are characterized by a pliable and transparent body; the refractive index of their tissues is nearly exactly the same is the surrounding water, making these organisms very difficult to detect. [15] More than 90% of the species that belong to this type of jellyfish are known to produce light. The jellyfish emit light by making use of coelenterazine, a type of luciferin, and proteins that are activated through calcium ions. Many comb jellyfish produce light from internal structures, although some eject luminescent particles as an escape mechanism. [16]

Comb jellyfish of the species //Beroë cucumis// also have eight rows of cilia that run along their bodies; these are used for locomotion. The areas of the animal just below the rows of cilia are the primary portions of the organism that emits light, causing the cilia to appear iridescent and exhibit nearly the full spectrum of visible colors as well as some wavelengths in the ultraviolet range; this also makes the jellyfish more noticeable in the dark environment of the deep ocean. More specifically, the bioluminescent organ of the comb jellyfish emits light at a wavelength of 489 nm. The bioluminescence of this type of jellyfish is particularly interesting from an ecological and evolutionary standpoint due to the fact that the production of light, which makes the organism more noticeable, not only appears alongside the iridescent cilia that are used for locomotion, but also that the bioluminescence is coupled with the transparency of the rest of the animal, which is used for camouflage. [15]

It is evident that a large number of marine dwelling species make use of bioluminescence for a variety of purposes, and that these creatures can often be vastly different from each other from an evolutionary and biological standpoint. By studying these creatures, as well as terrestrial organisms that also make use of light emission, human beings have applied what is known about biological light emission to advance the various fields of scientific research.

__ Human Uses: __

The study of bioluminescence and organisms that produce light has resulted in the development of a number of methods that humans have used in their own favor, especially in the various fields of scientific research. There are far too many notable experimental techniques involving biological light emission to innumerate here, but an explanation of a few of them will help the reader get a sense of the usefulness and diverse applications of bioluminescence in scientific research.

One of the most widespread and versatile ways biological light emission has been incorporated into a laboratory setting is by the wide range of uses of Green Fluorescent Protein, or GFP. More accurately, GFP is a family of proteins that occur naturally and are encoded by a single gene. GFP itself was the first representative of this family of proteins that was identified; it was described in a small Pacific jellyfish named //Aequorea victoria.// Since then, GFP and other members of the Green Fluorescent Protein family were described in a multitude of other species of various independent lineages as a part of their bioluminescent capabilities. GFP-like proteins are capable of catalyzing the entire chromophore synthesis pathway, and all share a similar “beta can” structure. [17] A wide variety of biotechnology applications have been developed based on GFP from the jellyfish //Aequorea victoria,// from the development of GFP-like fluorescent polymers that can be tracked using cellular imaging after assembling itself into aggregates [18] to a cellular marker that can then be used to identify and further react with other reagents. [19]

GFP, while it is one of the most widely used, is not the only biological light emitters used in scientific research. There are many other types of bioluminescent applications in scientific research. For instance, several different types of bioluminescent and chemiluminescent compounds are used as labels in immunoassays. For instance, firefly luciferase, the same compound used in nature by fireflies, can be used to analyze the methotrexate that may be present in a sample; similarly, luminol can be used to analyze Immunoglobulin G. In addition to this, chemiluminescent and bioluminescent reactions may be used in order to detect other immunoassay labels (and therefore enzymes) through the light emitted by the reaction. [20]

Bioluminescence is also useful in areas besides scientific research, the analysis of certain compounds in food, for instance. Even in this field, there are too many applications and reactions to innumerate in this paper, but some examples will be useful to get an idea of the versatility of light emission in food analysis. [21]

One simple example of the application of bioluminescence and chemiluminescence reactions in food analysis is the determination of alcohols in food and beverages. This analytical method involves a test alcohol being treated with alcohol oxidase; after the reaction occurs, the amount of hydrogen peroxide formed can be measured with a chemiluminescence assay (using luminol). The quantity of alcohol in the food or beverage can therefore be determined in this manner, and similar reactions (that create hydrogen peroxide) can be used to measure the quantities of other compounds in food products, such as sugars. [21]

Another way luminescent reactions are used in food analysis is in the detection of irradiated foodstuffs. Both luminol and lucigenin solutions can be used for this application as an indicator for food exposure to gamma radiation, although the effectiveness of these types of analyses is greatly dependent on particle size, protein content, and storage time of the food. [21]

__ Conclusion: __

In summary, bioluminescence is an interesting and important topic of study. Most bioluminescent mechanisms involve the reaction of a luciferin molecule with its corresponding luciferase enzyme. These two structures vary across species, and this type of reaction is used by a whole host of completely different species of organisms for a variety of purposes in all environments. It very likely evolved upwards of thirty separate times across extremely different lineages of organisms. Indeed, some organisms within the same family can have very different structures for the luciferin and luciferase involved in the reaction, although some may have similar types of these compounds that may even be able to cross react. The study of the chemical reactions involved has lead scientists to use similar mechanisms to advance scientific research in multiple fields and therefore benefit humans.

__ References: __

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