Monday, June 3, 2013

Lightening Bugs and Their Light.

Here come real stars to fill the upper skies,
And here on earth come emulating flies
That though they never equal stars in size
(And they were never really stars at heart),
Achieve at times a very star-like start.
Only, of course, they can't sustain the part.
-Robert Frost,  Fireflies in the Garden

As June begins and we dive head-first into summer, many people will be seeing a familiar nighttime site outside, the glowing of fireflies, also called "lightening bugs."  Lightening bugs are fascinating little creatures because of their seemingly magical ability to light up through a process called "bioluminescence" (bio means "life," while luminescence is the production of light).  Bioluminescence isn't something that we see often on land.  Most bioluminescent organisms live in the ocean, and their relative uniqueness on land makes them a beloved insect, particularly by children.  Fireflies are amazing, but as you can guess, there's no magic to what they do.  It's all science.  Read more about it after the jump...

In 1974, the firefly Photuris pennsylvanica, a common firefly species in the US and Canada, was named as the state insect of Pennsylvania, the home state of TheMadScienceBlog.  The state insect of Tennessee is also a firefly species, Photinus pyralis, also known as the "big dipper firefly" or "eastern firefly."  There are around 2,000 species of fireflies.  They are classified by taxonomists as the Lampyridae family, within order Coleoptera (the beatles) and the class Insecta (the insects).  Most species are nocturnal, and while some are carnivorous and eat other insects, others feed off of plant pollen or nectar.  Some don't eat at all, eating only during their larval stages and surviving as fireflies only long enough to mate and die.
At first thought, lighting up or glowing may seem like a major disadvantage to a small insect, because it would reveal their location to potential predators like bird or bats.  However, as most children who have encountered fireflies can tell you, in addition to the fireflies being very visible, they also are very easy to catch with your hand.  Fireflies aren't afraid, partly because there are few predators that try to eat them.  The reason is that they have evolved a natural protection by producing chemicals called steroid pyrones, specifically named lucibufagins, that are toxic to many animals and also smell and taste terrible.  When in danger, some types fireflies will reflexively secrete a sticky, terribly smelling and tasting sticky liquid that contains the lucibufagins.  This is has been called "reflexive bleeding."  The potential predator gets the foul liquid stuck to them, which is usually an effective deterrent.  However, in some animals, eating enough of the lucibufagins can cause cardic arrest, similar to the glycoside chemicals produced by poisonous toads.  Because of this, reptile and amphibian owners should never feed fireflies to their pets, just in case, though some species of frogs thrive on fireflies and eat so many of them that the frogs themselves sometimes glow.       
The light produced by fireflies was originally thought to serve as a warning to predators of their poisonous nature.  Many, but certainly not all, poisonous animals are very colorful, particularly certain frogs and snakes, which serves as a warning to potential predators.  Warning coloration is a type of aposematism (apo means "away," while semantism means "sign").  Aposematism is any type of defense mechanism that is based on sending a "warning signal" to a potential predator.  The purpose is to make the predator think that they will be harmed by eating or trying to eat the other animal because of some type of secondary defense.  In some animals, the secondary defense may be simply an unpleasant smell or taste, as in the case of a skunk.  In other animals, the secondary defense is an inherent poison that is more dangerous, as in the case of poisonous toads or brightly colored venomous snakes like the coral snake.       

However, we now know that most fireflies blink and flash primarily to signal to potential mates, not  to warn potential predators.  Female fireflies chose male mates based on their flashing patters, and when the female finds a male that she likes, she signals back to him to let him know to approach her.  Different types of fireflies have different flash patterns, and fireflies can distinguish whether another firefly they see is from the same or a different species.  Below is a graph from the National Park service showing the different flash patterns of the various firefly species in the park.

One exception to this, however, is seen with certain species of the female Photuris firefly, often called femme fatale fireflies, which blink to attract firefly males of other species to prey upon.  Photuris fireflies can mimic the blinking patterns of females of other species such as the Photinus fireflies. When the male comes in, attracted to the blinking, he thinks that he will get the chance to mate, but the Photuris firefly kills and eats him.  Not only does the Photuris get nutrition from this, but she also absorbs the male's defensive lucibufagin steroids and stores them to protect herself.
Firefly light production and its regulation has long fascinated humans.  While many other insects are considered pests, fireflies have the unusual ability to capture the human imagination because the light they produce seems almost magical.  Firefly light falls in the wavelengths of 510-670 nanometers, from the green to red range of the light spectrum.  Firefly light is a "cold light," because it doesn't contain any measurable infrared light (heat) or ultraviolet (UV) light.  Because almost none of the energy lost goes to heat, this light production process is very efficient.  This is in contrast to a standard incandescent filament light bulb, which produces a lot of heat (up to 80-90% of its energy output), and is one of the reasons they are being phased out for more-efficient CFL bulbs.  The high efficiency is very important for bioluminescence, because excess heat production could be lethal, and because efficiency lets the firefly produce more light from the calories and it consumes and energy it has stored.
Light production occurs within organs in the firefly abdomens called "lanterns," which contain layers of cells called "photocytes."  The photocytes are sandwiched between other layers of cells containing crystals of uric acid which reflect the light produced by the photocytes.  These layers are arranged in a circular rosette pattern around a central air tube, called a trachea, that is important for delivering oxygen to the organ.  We'll soon learn that oxygen plays a very important role in the light production. 

In 1885, the French scientist Raphael Dubois made some important observations while he was studying extracts he made from the abdomens of luminescent beetles.  He discovered that there was a substance that was consumed during the bioluminescent reaction, and called it "luciferin."  He also discovered that there was another component that catalyzed the reaction, which he called "luciferase."  The American scientist Newton Harvey also studied beetle luminescence several years later.  One of Harvey's important discoveries that oxygen is required for this reaction.  One of Harvey's PhD students, William McElroy, discovered in 1947 that the luminescence reaction also requires a molecule called ATP.  ATP stands for adenosine trisphosphate, which is a molecule made by cells to store energy.  ATP often requires a magnesium ion (Mg2+) to function, and that's the case here.  McElroy's work built on that of Dubois and Harvey and defined four main components of the luminescence reaction: 1) A substrate called luciferin, 2) an enzyme luciferase, 3) oxygen, and 4) ATP plus magnesium.  

The work of Dubois, Harvey, and McElroy laid the foundation for out understanding that fireflies make light through bioluminescence, which a form of chemiluminescence.  Chemiluminescence is the production of light from a chemical reaction that is exogenic (energy releasing).  While most exogenic chemical reactions result in excess energy being released as heat, chemiluminescent reactions result in excess energy being released as light. 
Luciferin has a complex chemical structure shown to the right, with carbon (C) atoms in green, hydrogen (H) in gray, oxygen (O) in red, sulfur (S) in yellow, and nitrogen (N) in blue.  In the presence of luciferase, luciferin can undergo a complex chemical reaction in which it first reacts with ATP to form adenylate luciferin, which is luciferin joined to AMP, which is ATP minus 2 phosphate groups.  This is followed by a reaction with oxygen to form a luciferase peroxide.  These first two steps are catalyzed by the luciferase enzyme.  What follows is a spontaneous decomposition of the luciferase peroxide into oxyluciferase into a more stable form.  The chemical reaction goes like this:

The actual reaction is very complicated with many intermediate steps, but the general point is that inside the photocyte cell, the luciferase enzyme catalyzes the reaction of luciferin with ATP and oxygen that changes the structure of the molecules and releases energy.  In the third step above, Luciferin peroxide decomposes to oxyluciferin, but the excess energy is still in the molecule, so we call this the "excited" state of oxyluciferin.  When the energy is spontaneously released, the oxyluciferin goes to its "ground" or "relaxed" state, and the energy comes out as a photon of light.   

An important mystery in early bioluminescence research was the relative amount of energy required to make light.  Scientists like McElroy knew ATP was involved, but they also knew that this couldn't easily explain where the energy for light production comes from.  Producing light requires a lot of energy.  It takes about 8 times the energy to produce a photon (the smallest unit of light) as the amount of energy you can get from breaking down an ATP molecule (50 kcal/mol vs 7 kcal/mol, respectively).  We now know that the key player in this reaction is the oxygen.  Luciferin reacts with oxygen to form a peroxide.  Peroxides contain a bond between 2 oxygens (-O-O-), and  they can be very unstable and very reactive, often releasing a lot of energy (up to 100 kcal/mol) when they do react to form a more stable molecule.  The energy for light comes from the peroxide formed when luciferin reacts with oxygen, and thus there can be no light generated without oxygen.  Fireflies actually use this this chemistry to their advantage to be able to control when they flash.    

Many scientific studies have now led to the belief that fireflies regulate flashing by controlling oxygen flow into the lantern organs and photocytes.  When cells at the end of the lantern organ trachea tubes open and allow oxygen into the tubes, the photocytes glow.  When they close and the oxygen is used up, the glowing stops because the reaction can't proceed.  Interestingly, many firefly larvae do not have the same regulatory cells at the end of the trachea tubes, which results in a steady constant glow instead of bright regulated flashing.  More recent studies have implicated a role for the production of another signaling molecule, nitric oxide, in further regulating this process, though many details of this remain to be worked out by researchers.  Despite the fact that scientists have been studying how fireflies light up for over a century, parts of the mystery still remain to be revealed.

However, in spite of the natural wonder that they inspire, firefly populations in the US are declining.   Researchers blame a combination of land development and light pollution.  Land development reduces the natural habitats that fireflies and their larvae need to grow and develop.  They particularly like marshy wetlands.  Light pollution is thought to disrupt their flashing signaling patterns and ability to find mates.  You can read more about fireflies and what you can do to help increase firefly populations here

© 2013 TheMadScienceBlog
Molecular structure of luciferin made with PyMOL for iPad.    

Sources and Further Reading
  • J.C. Day, L.C. Tisi, and M.J. Bailey.  "Evolution of beetle bioluminescence: the origin of beetle luciferin."  Luminescence, 2004.  19:8-20.  (subscription or pay-per-view only)
  • T. Eisner, D.F. Wiemer, L.W. Haynes, and J. Meinwald.  "Lucibufagins: Defensive steroids from the fireflies Photinus ignitus and P. marginellus (Coleoptera: Lampyridae)."  Proc Natl. Acad Sci. USA., 1978.  75:905-908.  Available here.
  • T. Eisner, M.A. Geotz, D.E. Hill, S.R. Smedley, and J. Meinwald.  "Firefly "femmes fatales" acquire defensive steroids (lucibufagins) from their firefly prey."  Proc. Natl. Acad. Sci. USA., 1997. 94:9723-9728.   Available here.
  • H. Fraga.  "Firefly luminescence: A historical perspective and recent developments." Photochem. Photobiol. Sci., 2008, 7:146–158.  (subscription or pay-per-view only)
  • J.W. Hastings.  "Bioluminescence."  Chapter 52 in Cell Physiology Sourcebook. Elsevier, 2012.  DOI: 10.1016/B978-0-12-387738-3.00052-4.
  • S.M. Lewis and C.K. Cratsley.  "Flash signal evolution, mate choice, and predation in fireflies."  Annu. Rev. Entomol., 2008.  53:293-321.  (subscription or pay-per-view only)
  • L. Pinto da Silva and J.C.G. Estaves da Silva.  "Firefly Chemiluminescence and Bioluminescence: Efficient Generation of Excited States."  Chem. Phys. Chem., 2012.  13:2257-2262.  (subscription or pay-per-view only) 
  • T. Wilson and J.W. Hastings.  "Bioluminescence."  Ann Rev. Cell Dev. Biol., 1998.  14:197-230.  (subscription or pay-per-view only) 
  • J.W. Hastings and T. Wilson.  "Bioluminescence and Chemiluminescence."  Photochemistry and Photobiology.  1976.  23:461-473.  Available here

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