1. Physiology of Bioluminescence in Fireflies
Coleopteran Lampyridae

2. Coleopteran Lampyridae
Coleopteran-Beetles
Lampyridae- fireflies and glow worms
Bioluminescent
Vary in flash patterns and colours
Fireflies also known as lightning bugs are of the Genus Coleopteran and the family Lampyridae
As we have learned in class, Coleopteran are the beetles and the family Lampyridae accounts for the fireflies and glow worms (which are their predatory larvae)
They are found in temperate and tropical environments
The richest faunas are found in South America and Asia
They are winged beetles with hard elytra. Some species are diurnal where most commonly known ones are nocturnal
These insects are bioluminescent meaning they intrinsicly produce light from a specialized organ located somewhere on their abdominal segments. These processes are curious because the light produced does not depend on high temperature also known as incandescence of the radiating substance. Nor does it depend on its prior absorption of light as in fluorescence. Bioluminescence depends instead on oxidation of a substrate.
While a lot of bioluminescent organisms simply glow for more or less uninterupted , long periods of time and others do so spontaneously or in response to a disturbance, fireflies regulate their bioluminescence under control of a pacemaker in the brain where they emit light in distinct flashes that are used in communication

3. Why do fireflies light up?
Mating
Sexual selection
Display indications of mate:
Quality
Sex
Species
Exact location
“critically timed signals”
Larvae-aposematic functions
Fireflies use bioluminescence as an adaptation for mating and courtship. In most species, the light displays are used in courtship to display certain attributes about the mate. The flash patterns are precise, rythmic and under central and peripheral control. In this timed and specialized dialogue, they are expressing their sex, species (as species differ in their patterns, colours, and light intensities) as well as their exact location. A short dialog between the male and female usually ensues and either the male departs or lands near the female and then mating may or may not occur.
In most species, the luminous displays serve to communicate something about the quality of the male (as the female is most often discriminating based on this courtship). In Photinus species of fireflies, the duration of the male flash provides the female with information about the quality of the nuptual gift it can give. Male Phtinus fireflies provide gifts to females in the form of spermatophores that the female ingests internally and gives her nutrients. Because these insects do not feed as adults, this gift is very important for female fedundity. There is a relation to body mass, lantern size and the amount of spermatophore and spermatophore size is highest the first time the male mates and decreases the more he mates. So the female only uses this as an indicator at the beginning of the season when this is the first time all the males are likely to be mating. The females choose a mate based flash rates and in the lab, even as flash rates exceed those possible in nature, they will always choose the highest flash rates
These photic signals are commonly termed “critically time signals” because the parameters of the male’s patterns show little variation and the species specific delay after the male’s last flash and the female’s response are precisely timed. Flash pattern duration is typically a few hundred milliseconds and the flash patterns vary within species.

4. Male to female signaling
1) Both males and females use bioluminescence (ex: Hotaria parvula)
2) Sedentary female produces light to attract male (ex: Microphitus)
3) Sedentary females use pheromones to attract males and when in close proximity, glow (ex: Pleotomus pallens)
4) Only larvae emit light,
rely solely on pheromones
(Lucidota atra)

5. Larvae Bioluminescence
Spend most of their lives in this stage (2-3 years vs. 10 days as an adult)
Same reactions
Differences: Izoenzymes, differences in location, morphology and physiology of light organs
Results: Different colours and behavioural displays
slide
The origin of photic organs actually precedes their use in sexual signalling and was originally used as a warning signal against predators indicating the larvae was indegestable. While firefly larvae do luminesce, they don’t do it for sexual purposes but ratherare thought to do it for aposematic purposes do display that fireflies possess distasteful steroids called lucibufagin in their hemolymph. When they tested this with mice and toads, they found that these predators could associates a bioluminescent glow with distasteful substances
Studies have shown that most larvae produce green light bioluminescence irrespective of what wavelength their adult form makes because it is the most conspicuous to many vertebrate and arthropod eyes and because they use this as a defence mechanism rather than in mating, they want to be the very visible

6. How do fireflies produce light?
Neural activity stimulates release of NT octopamine
Triggers the Lantern Organ in abdomen
Inside peroxisomes: luciferin- luciferase reaction
In very general terms, fireflies produce light through central and peripheral neural control stimulating the release of the neurotransmitter octopamine which triggers the light producing organ (the lantern organ) in the abdomen to emit light through the luciferin-luciferase reaction in organelles called peroxisomes.

7. Lantern organs 1) Photocyte 2)Trachea 3)Tracheole 4)Lantern nerve
5)tracheolar cell
6)Tracheal end
cell
Each lantern organs is a flat slab of tissue with a ventral and dorsal layers. The dorsal layer has large cells packed with granules. The ventral photogenic layers is the source of the light. The Lantern organ contains thousands of cylindrical units each consisting of several photocytes arranged radially around a central air conducting trachea. Within the photocytes there are organelles called peroxisomes which contain the enzymes that allow the light producing reaction to occur. The peripheral cytoplasm of these photocytes is densely packed with mitochondria which have been proposed to act as gatekeepers that control the access of required oxygen to light producing reactions that are more central in the peroxisomes.
The neurons releasing the neurotransmitters that inervate the lantern do not terminate directly on the photocytes themselves but rather synapse on the tracheolar cells surrounding the terminal branch poitns of the tracheal air supply. So, the activation of photocytes therefore require a signal to pass from the tracheolar cells to the peroxisomes which is a distance of about 17 micrometers. So how does the signal reach the peroxisomes within the mitochondrial covered photocytes?

8. The photocyte
As you can see the nerve is synapsing onto the tracheole rather than the photocyte itself and the signal must travel through the mitochondrial thick layer surrounding the photocyte to get to the peroxisomes so that the light reactions can occur

9. How does the signal reach the peroxisomes?
Several hypotheses:
1) The NOS model
2)The Osmotic control model
3)Hydrogen peroxide model
There are several proposed hypotheses as to how the signal reaches and triggers the light reaction in the peroxisomes. The first is the Nitrogen oxide model. This hypotheses suggests that one potential transimitter that can penetrate cell membranes and quickly cross such distances is the free radical gas nitric oxide. It has long been found that oxygen availibility is the immediate biochemmical trigger for light production and that the dark state represents repression of bioluminescence. and it is proposed that nitric oxide transiently inhibits mitochondrial respiration in photocytes and allow oxygen to permeate and reach the peroxisomes. The evidence for this is in testing where nitric oxide introduced into a chamber containing fireflies immediately induced biolluminescence, whereas when it was not present, they did not emit light. When a Nitric oxide scavenger was introduced into the system, the flashing activity was reduced and the fact that the enzyme responsible for producing nitric oxide is localized within the tracheal end cell, tracheolar cells, and the peripheral mitchondrial region of the photocytes. But this model needs furthur substantiation and has only been modeled in one species
The second hypotheses is the osmotic control hypotheses is the proposal that the control mechanism which mediates the flow of oxygen to the photocytes is likely to act by modulating the levels of fluid in the tracheoles. They suggest that the release of octopamine causes an increase in H+ concentration which causes swelling in the cytoplasmic proteins may cause the lantern tracheole to begin pumping out tissue fluid, allowing oxygen to overwhelm the lantern so that it can flow past the mitochondria to the peroxisomes. As oxygen supply to tissues in insects is mediated by changes in the tracheolar fluid levels which result from changes in the balance of osmotic pressure of the tracheolar fluid and intracellular middle, this seems like a feasible hypotheses
Thirdly, The Hydrogen peroxide model suggests the Nitric oxide model is the beginning steps of the cycle, but that oxygen concentration changes are not enough to initiate the bioluminescence reaction. It suggests that the Nitic oxide not only shuts down the mitochondria allowing oxygen to diffuse inThey suggest this because the light reaction happens so quickly and the sharp increase in hydrogen peroxide is another initiator of the bioluminescence reaction
But what is the light reaction?

10. What is the luciferin-luciferase or “light” reaction?
ATP + luciferase + luciferin
→Luciferase-luciferin-AMP + PPi
Luciferase-luciferin-AMP+O2
→ Luciferase+ oxyluciferin + CO2 + AMP + light
To produce light, fireflies employ a luciferin-luciferase reaction which produces light using oxygen. Firstly, luciferin (or the substrate) combines with luciferase (the enzyme) and ATP in the presence of Mg2+ to form an active intermediate that only needs oxygen to complete the photochemical reaction. When oxygen is combined, the active intermediate forms oxyluciferin which is in an excited state. As this excited state decays to its ground state, a photon of light is emited. Instead of glowing like some bio;luminescent organisms, fireflies can modulate their light to a greater or lesser extent and can actually flash by turning their lantern on and off.

11. Synchrony Certain fireflies exhibit synchrony Anticipatory mechanisms
Rate of one dictates the rate of another
VIDEO:
Certain fireflies, particularly those of southeast asia show synchronous flashing. In these species, synchrony was shown to use anticipatory mechanisms meaning that the flashes of one firefly could advance or delay the flashes of a second firefly either producing synchrony or maintaining it. As you can see in the video, they have pauses and then flash all at the same time.
They have actually been able to entrain fireflies to follow the synchrony of a pacer light and while it is said to be run by a timed pacemaker, when stimulated in synchrony by another source, it stops the timing cycle and restarts the pace-maker to the new cycle, being either faster or slower
This phenomena occurs but no one is actually sure why. A possible explanation is that in courtship signalling, there is a heightened response when two spatially seperated signals are emitted and only seperated by a short time frame.

12. Different colours in light
Different species of fireflies emit different wavelengths of light and therefore different colours
Reaction route universal
Light emissions span wide range of wavelengths from green (545 nm) to red (620 nm)
As I explained before, light arises from the oxidation of a substrate (luciferin) by an enzyme (luciferase) that in turn results in the formation of oxyluciferin which is in its excited state and as it decays to its ground state, a photon of light is emitted.
But there is an intriguing issue as to the chemical origin of the multicolor lights produced in this bioluminescence reaction despite the reaction route and substrate product structures being the same for all species. The emissions span a wide range of wavelengths from green (545 nm) to red (620 nm)

13. How can light emission vary in colour?
Oxyluciferin: keto form=red, enol form=yellow?
But 5,5-dimethyloxyluciferin can emit multicolour light despite being constrained to keto form!!
Twisted structure of the keto form can rotate to change colour
OR polarization of Oxiluciferin in microenvironment and the changes in polarity of the luciferase binding sites
Please bear with me as I try to explain the chemistry behind the varying light emission! One possibility that was proposed is that oxyluciferin(or the product of the luciferin-luciferase reaction that when going from an excited state to a ground state, emits light) can be deprotonated into a keto-form that can emit red light and that the formation of its enol form would emit yello light and that the concentration of each in the peroxisome dictated the colour of light emitted. However it was found that 5,5-dimethyloxyluciferin when constrained to a keto form, can emit multicolour lights and so this is not the case.
Another suggestion is that in its excited state, keto oxiluciferin has a twisted structure rather than a planar one and that the colour of the light emission could change upon rotation of the thiazolinone fragment.
A third hypotheses is that the colour of bioluminescence depends on the polarization of the oxyluciferin in its microenvironment and that the colour of light emission depends on the pH of the media in which it resides and can be varied by modifying certain binding site residues in the luciferase enzyme.

14. Firefly light receptors
3 visual receptors:
Near-UV
Blue
Green-yellow
Species specific bioluminescence matches visual receptors
Achromatic- transmit light without separating it into its constituent colours
Visual systems of fireflies have been shown to have 3 receptor types based on wavelength: Near UV, blue, and green yellow
For example, in species who restrict their flashing activity to twilight show a narrow visual spectral sensitivity that maxes out at yellow region of the spectrum, so that the spectrum of their bioluminescence or the light they are emitting matches their visual receptors.
However, the detection of the bioluminescent optical signals is achromatic (meaning they transmit light without seperating it into its constituent colours). Hence in sexual communication, fireflies do not use the colour of their bioluminescent optical signal as a parameter in decoding the signal, but rather the temporal patterning or timing, the duration and the threshold intesity of the male flash are the important parameters of the optical signaling used in male-female communication.

15. Increasing Bioluminescence?
Using genetic modification
10 fold increase in luminescence than the wild type
Modify luciferase by mutation
Recently, they have actually been able to increase the amount of bioluminescence produced by ten times more than the wild type by genetically modifying the luciferase enzyme

16. Recent uses of bioluminescence
Microbial detection in food and pharmaceuticals
Genetic marking

17. Look!

18. Questions?

19. Works cited
Aprille, J.R., Lagace, C.J., Napolitano, J., and Trimmer, B.A. (2004) Role of Nitric Oxide and Mitochondria in Control of Firefly Flash. Integr. Comp. Biol. 44:
Branham, M.A., Wenzel, J.W. (2005). The origin of photic behavior and the evolution of sexual communication in fireflies (Coleoptera: Lampyridae). Cladistics. 19:1-22
Copeland, J. and Moiseff, A. (1997) Effect of flash duration and flash shape on entrainment in Pteroptyx malaccae, a synchronic Southeat Asian firefly. Journal of Insect Physiology. 43:
Cratsley, C.K. (2004) Flash Signals, Nuptial Gifts and Female Preference in Photinus Fireflies. Interg. Comp. Biol. 44:
Day, J.C., Goodall, T.I., Bailey, M.J. (2009) The evolution of the adenylate-forming protein families in beetles: Multiple luciferase gene paralogues in fireflies and glow-worms. Molecular Phylogenetics and Evolution. 50:
De Cock, R. (2004) Larval and Adult Emission Spectra of Bioluminescence in Three European Firefly Species. Phytochemistry and Photobiology. 79:
Fujii, H., Noda, K, Asami, Y., Kuroda, A., Sakata, M., Tokida, A. (2007) Increase in bioluminescence intensity of firefly luciferase using genetic modification. Analytical Biochemistry. 366:

20. Works Cited cont’d
Ghiradella, H., Schmidt, J.T. (2004) Fireflies at One Hundred Plus: A New Look at Flash Control. Integr. Comp. Biol. 44:
Lall, A.B. and Worthy, K.M. (2000) Action spectra of the female’s response in the firefly Photinus pyralis (Coleoptera: Lampyridae): evidence for an achromatic detection of the bioluminescent optical signal. Journal of Insect Physiology. 46:
Michaelidis, C.I., Demary, K.C. and S.M. Lewis Male courtship signals and female signal assessment in Photinus greeni fireflies. Behavioral Ecology 17:
Moosman, P.R., Cratsley, C.K., Lehto, S.D., Thomas, H. (2009) Do courtship flashes of fireflies (Coleoptera: Lampyridae) serve as aposematic signals to insectivorous bats? Animal Behaviour. 78:
Orlova, G., Goddard, J.D. and L.Y. Brovko Theoretical Study of the Amazing Firefly Bioluminescence: The Formation and Structures of the Light Emitters. Journal of the American Chemical Society 125:
Oertel, D., Lindberg, K.A. and J.F. Chase. (1975).Ultrastructure of the Larval Firefly Light Organ as Related to Control of Light Emission. Cell and Tissue Research 164:
Timmins, G.S., Robb, F.J., Wilmot, C.M., Jackson, S.K. and Schwartz, H.M. (2001). Firefly Flashing is Controlled by Gating Oxygen to Light-Emitting Cells. Journal of Experimental Biology. 204:
Trimmer, B.A., Aprille, J.R., Duzinski, D.M., Lagace, C.J., Lewis, S.M., Mitchell, T., Qazi, S., and Zayas, R.M. (2001) Nitric Oxide and the Control of Firefly Flashing. Science. 292:
Viviani, V.R. (2001) Fireflies (Coleoptera:Lampyridae) from Southeastern Brazil: Habitats, Life History and Bioluminescence. Entomological Society of America. 94: