LETS SAVE OUR FIREFLIES FROM EXTINCTION

NATURE PRESERVATION

Lighting Up the Environment by a Living Organism Inspired and Fascinated by the Aesthetic Nature of this Living Organism

(Inquiry into innovative electrochemiluminescence of a Lightning Bugs-Lampyridae-Elateroid beetles-Elateroidea-Fireflies)

By: Jonathan Tetteh-Cole aka NORJGiX

FACTS ABOUT THE LIVING ORGANISM

BIOCHEMICAL EMISSION OF LIGHT BY THE LIVING ORGANISMS

Raphaël Dubois work on the discovery of Luciferase and Luciferin in the 19th century is highly acknowledged.

These living organisms were discovered due to the existence of Firefly. These living organisms are soft-bodied beetles commonly called fireflies, lightning bugs, or glowworms scientifically known as Firefly luciferin (s)-2-(6-Hydroxybenzo[d]thiazol-2yl)-4,5-dihydrothiazole-4-carboxylic acid. D-(-)-Luciferin for their conspicuous production of light, mainly during twilight to attract mates. Luciferin vary in chemical structure. The males glow to use their flash to attract females in the night. Each species has its own pattern of light flashing. In some places at some times, fireflies synchronize their flashing to yellow, green, or orange. Firefly larvae may glow living under water communicating to predators that they are not tasty and that they are not palatable. Relatively, they use their defensive steroids for protection against predators. As carnivorous, they enjoy eating snails and fireflies of other genera as well as feeding on nectar and pollen of plants. Fireflies produce their own light (bioluminescent). Nearly 100% of this efficient light which is chemically produced is emitted as light. However, it is important to note that the only way to get the chemical (Luciferase) is synthesized and stored in the cells of the firefly’s lantern organ.

Luciferase is an enzyme that produces the light in reaction with other compounds, while Luciferin facilitates and stimulates the light production. There are Luciferase in other bioluminescent animals. On the contrary, today synthetic Luciferase is being produced alongside harvesting the fireflies. These synthetic bioluminescence system is based on AkaLumine by modifying the luciferase gene which could be used in animal tissues. Notably, fireflies don’t migrate to other fields, they can disappear forever. Fireflies typically lives for approximately two months in the wild. Anecdotal evidence suggests that firefly populations may be on the decline. Fireflies live in fields, meadows, forests, and other natural habitats, and these areas are becoming fewer due to development. These insects live in a variety of warm environments, as well as in more temperate regions, and are a familiar sight on summer evenings. Fireflies love moisture and often live in humid regions of Asia and the Americas. In drier areas, they are found around wet or damp areas that retain moisture. Fireflies are found in temperate and tropical regions on every continent except Antarctica. They live throughout the United States in parks, meadows, gardens, and woodland edges. They are most commonly seen on summer evenings. Research shows that there are 2,400 firefly species in 144 genera (taxonomic category that ranks above species) described all over the world. The females of the Lamprigera firefly can grow to be the size of your palm. They are much larger than their male counterparts and lack wings. Two large light organs on their abdomen produce their characteristic glow. Research shows that in Africa, there are over 2,000 species which have been recorded, and one of them, the Luciola capensis can be found here in South Africa, mostly visible in spring and summer. They feed on other insects like snails, slugs, worms, and even other fireflies for their nourishment. Adult fireflies also eat nectar and pollen to survive. The fireflies’ species that are carnivorous spend all their life eating other fireflies. They are not edible. When predators attack, they start “reflex bleeding,” and produce drops of blood filled with nasty chemicals that are poisonous to lizards and birds. However, it is important to note that there are LUCs from other animals apart from firefly. Scientifically, this organism, the luciferase (Renilla-luciferin 2-monooxygenase) is closely related with a luciferinbinding protein as well as a green fluorescent protein (GFP). Additionally, luciferin produces light when it reacts with oxygen. All bioluminescent animals contain luciferin, but some (like tiny dinoflagellate plankton) produces their own, where others (like squid and some fish) absorbs bacteria that contain luciferin.

BIOLUMINESCENCE

Quantum rods and luciferase enzymes are nanomaterial and biomaterials respectively. When these two materials are combined correctly will produce bioluminescence – except, instead of coming from a biomaterial, such as a firefly enzyme, the light emanates from a nanomaterial, and is green, orange, red or near-infrared in color. A bioluminescent reporter assay consist of both a luciferase reporter enzyme and detection reagent that provides the enzyme substrate. When the reporter enzyme and detection reagent are combined, the light emitted is proportional to the reporter gene expression levels and is detected using a luminometer. Everyone knows how fireflies got their name, but many people don't know how the insects produce their signature glow. The glow is seen at the abdomen region of the fireflies. They emit light due to the chemical reaction of a protein that requires the substrates: luciferin with oxygen, calcium and ATP – the energy-carrying molecule of all cells resulting in production of glow whenever Luciferase is present. Males and females of the same species will flash signals back and forth as a way of communicating. This reaction is an oxidation reaction. The glow and light emission reaction is all known as bioluminescence. As highlighted, fireflies have dedicated light organs that are located under their abdomens. The insects take in oxygen and, inside special cells, combine it with a substance called luciferin to produce light with almost no heat. Firefly light is usually intermittent, and flashes in patterns that are unique to each species. Each blinking pattern is an optical signal that helps fireflies find potential mates. Scientists are not sure how the insects regulate this process to turn their lights on and off (1/0). Firefly light may also serve as a defense mechanism that flashes a clear warning of the insect's unappetizing taste. The fact that even larvae are luminescent lends support to this theory. Today there are Luciferase in other bioluminescent animals which serves as enzymes that produces light when they oxidize their substrate. Luciferase from other animals such as the sea pansy Renilla reniformis, the copepod Gaussia princeps, and the ostracod Cypridina noctiluca – are also used as reporters, but the gene from most common Luciferase is firefly.

HOW LUCIFERIN OF LUCIFERASE IS ADMINISTERED

As mentioned, Luciferin is a light-emitting compound, and Luciferase is an enzyme that accelerates the relation. When the two compounds interact with oxygen and some other compounds, energy is released as light. (Luciferin is typically applied intraperitoneally or intravenously) – in that you could inject 10MicroL of Luciferin stock solution per gram of body weight and wait 10-20 minutes before imaging for maximum luciferase signal plateau. Bioluminescence occurs when luciferase modifies the molecule luciferin which can emit bright yellow-green light. If necessary, luciferin solutions may be stored at four degrees Celsius or minus twenty degree Celsius for up to three weeks. However, prolonged storage at either temperature may results in degradation of signal. The gene that encodes luciferase enzyme is then introduced into cells from mammals or cancer cells and in the presence of luciferin these cells begin to glow which signifies or reports activities of the cell under experiment.

IMPORTANCE OF LUCIFERASE

Fireflies and Luciferase is the new insight (chemical Tool) that helps in biological reporting. Luciferase reporter gene assays have a number of important applications in biomedical science and cancer biology. Fireflies signal the effects of a changing climate. With a species so deeply connected to its habitat, its early arrival is a reminder of the importance of curbing climate change. Research shows that Luciferase (light-producing enzyme naturally found in insects such as fireflies and luminous marine and terrestrial microorganisms) is proven to be a useful chemical in food and forensic testing. Luciferase is used to detect levels of ATP which is the energy-carrying molecule of all cells in cells. Research shows that the luciferase-modified magnetic nanoparticles is a magnetic platform that can be utilized as a possible alternative for QD-based bio-imaging, and which also has potential for magnetic cellular manipulation and MRI applications and that the glow-in-the-dark protein called luciferase improves medical diagnostics.

A commonly used reporter gene is the Luciferase gene from the firefly Photinus pyralis. The gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and MG (++), yielding a fluorescent product that can be quantified by measuring the released light. The common reporter genes are: beta-galactosidase, luciferase, beta-lactamase, alkaline phosphatase and GFP (Green Fluorescent Protein). Luminescence, absorbance and fluorescence detection methods are used typically to measure expressed reporter gene protein. The main disadvantage of the reporter gene is the need of cofactors such as ATP and metal ions when compared to florescent reporters where a chemical substrate are necessary to facilitate the enzymatic reaction that leads to a quantifiable light signal. Relatively, the primary disadvantage of luciferase is the requirement for exogenous substrates and the 4-6 h delay from stimulus to response to allow transcription to occur. LUC-labeled-cells-acting as a reporter is a light emitting enzyme that helps in sending reports in a cell (e.g. level of growth of a tumor). It is important to note that the FLUC Reporter system or technology is a very sensitive reporter with high variety of applications. To utilize the reporter system to their full capacity, however, there is the need to study and understand the real characteristics of the phenomena. There are three (3) different Luciferase reporters: the NanoLuc Luciferase (Nluc, 19kDa), Renilla Luciferase (Rluc, 36kDa) and Firefly Luciferase (Fluc, 61kDa) which vary in size, brightness, and protein half-life. The popularity of native firefly luciferase as genetic reporter is due to the sensibility and convenience of the enzyme assay and tight coupling of protein synthesis with Enzyme activity.

The amount of light produced provides the quantitative measure of the effect of protein level measurement in the cell of a particular DNA or gene of a living organism using a luminometer. However, it is important to note that luminescence signal decays over the course of about 10 minutes of reaction time, although signal half-life may vary depending on luciferase expression levels. The light production resulting from the luciferase reaction leads to formation of suicidal adenyl-oxyluciferin at the enzyme surface. These can take place by conducting a Luciferase Assay Test Experiments with the help of a Reporter Gene Technology. As a results, cells can be sorted according to the level at which they illuminate. Luciferase Assay Test Experiments is commonly used as a tool to gene expression at the transcriptional level. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Research reveals that LUC can be used to study the nuclear delivery of oligonucleotides. A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene.

Luciferase-based assays are better than other reports because it is proven to be quick and offers a real-time measurement. Exceptionally high sensitivity than fluorescent reporters like the GFP (10-to1000 fold). The range of measurement is wide and dynamic.

THE DECLINE OF FIREFLIES

The bugs are endangered around due to habitat loss, toxic chemicals (which tend to linger in aquatic environments where fireflies start their lives) and light pollution. Researchers agree that protecting and enhancing firefly habitat is necessary to conserve their populations. However, it is important to note that effects of artificial light at night on fireflies has shown that light pollution can disrupt fireflies' courtship signals and even interfere with larval dispersal. Harvesting of Luciferase produced by fireflies themselves, habitat destruction, pesticides, including insecticides and herbicides also contributes to their decline in our biodiversity changes in terrestrial ecosystem characterized by climate change. Due to impact of climate change, it is observed that fireflies in some part of the world like United States of America lacks the ability to produce light.

Acknowledgements: www.sciencedirect.com www.nationalgeographic.com