This article reprinted from:
Goodwin, A. 2007. Use of Renilla bioluminescence to illustrate nervous function. Pages 217226, in Tested Studies for Laboratory Teaching, Volume 28 (M.A. O'Donnell, Editor).
Proceedings of the 28th Workshop/Conference of the Association for Biology
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Use of Renilla Bioluminescence to Illustrate
Nervous Function
Anne Goodwin
Department of Biology
Massachusetts College of Liberal Arts
375 Church Street
North Adams, MA 01247
[email protected]
Abstract: Renilla mulleri is a bioluminescent soft coral with a nervous system composed of a
simple nerve net. Bioluminescence in these animals occurs in a bright wave across the surface,
and is coordinated by nervous transmission. Groups of six students clustered around an animal
can easily see the light wave without the use of magnification. The bioluminescent wave can be
initiated by mechanical, electrical or chemical stimulation, and can be used to illustrate several
concepts related to nervous function, including threshold, refractory period and adaptation.
Students complete a homework assignment prior to the lab, in which they research mechanisms
and functions of bioluminescence in Renilla and in other organisms. In the lab, each group gives
a brief summary of the findings from the homework assignment; and examines the
bioluminescent response of Renilla to touch. Students then make hypotheses about effects of
varying the frequency and intensity of electrical stimulation, and about neurotransmitter and drug
effects. These hypotheses are then tested by altering settings of the Grass stimulator, and by
adding solutions of epinephrine, propranolol, and other chemicals. I used this lab in the context
of the nervous systems unit of a sophomore Animal Physiology class, but the lab would also
work well with more advanced classes, in which nervous control pathways could be better
defined, or with introductory courses, which might involve discussions of bioluminescence or
defense from predators. In this workshop, we will examine the morphology of Renilla
specimens and initiate bioluminescence using mechanical, electrical and chemical stimuli. We
will discuss applications of these experiments to various topics, and might also discuss other
biological functions of these animals, including water transport, locomotion, and feeding
responses.
Association for Biology Laboratory Education (ABLE) 2006 Proceedings, Vol. 28:217-226
218 ABLE 2006 Proceedings Vol. 28
Goodwin
Student Outline
Objectives:
To describe the cnidarian nervous system.
To explore the mechanisms & functions of bioluminescence.
To investigate effects of electrical and chemical stimulation on nervous function.
Introduction:
The sea pansy (Renilla spp.) is a soft coral, a cnidarian of the class Anthozoa. These animals
live on the ocean floor in shallow waters, and can often be found on the beaches of the southeast United
States at low tide. Renilla polyps have various forms, including the tentacled feeding polyps, called
autozooids, and the polyps that regulate water circulation, called siphonozooids (Figure 1). These
polyps share an umbrella-shaped colonial structure, the rachis, and a foot-like structure, the peduncle
(Figure 1). Both the rachis and the peduncle contain a water vascular system, muscular tissue, and
nervous tissue, and the coordinated movements of these structures allow Renilla to anchor into the sand
and move along the sea floor
autozooid
siphonozooid
peduncle
rachis
peduncle
rachis
peduncle
rachis
Figure 1. Anatomy of Renilla muelleri. Scale bars are approximately 1 cm in length.
Renilla, like other animals of phylum Cnidaria, has a nervous system in the form of a nerve net.
The nerve net coordinates colonial responses including contractions of the rachis and peduncle, polyp
movements, and bioluminescence. While the exact neurotransmitters used by the cnidarian nerve net are
poorly understood, some signaling chemicals are similar to those used by vertebrates.
Bioluminescence and nervous function
219
Bioluminescence is the generation of light via chemical reactions in living organisms.
Bioluminescent organisms include fireflies, some jellyfish, dinoflagellates that cause ‘sea sparkle,’ and
many deep-sea animals. The specialized bioluminescent cells of cnidarians, the photocytes, contain
membrane-bound vesicles called luminelles. When Ca++ ions move into the photocyte, as results from
nervous stimulation of these cells, a chemical reaction occurs in which a luciferin substrate reacts with
oxygen in the presence the enzyme luciferase to produce light.
Bioluminescence has a number of useful functions for the luminescent organism. The flashes of
light can be used to attract prey, hide from predators, distract predators, or attract mates. Renilla
bioluminescence occurs in response to mechanical disturbance of the colony. When the colony is
touched during the night phase, the nerve net sequentially activates the photocytes starting at the point of
contact, resulting in a wave of light passing over the colony. This light pattern may function to distract
predators – it has been reported that Renilla bioluminescence repels crabs, which may be predators of
the soft coral, and lowers the heart rate of these predators.
In this laboratory exercise we will test the effects of mechanical and chemical stimuli on nervous
function in Renilla. We will visualize the path of conduction through the nerve net by observing the
wave of luminescence over the colony surface.
Procedure:
Note that the room must be very dark while you are conducting the procedure. Be sure that you
are familiar with the equipment and protocol before the lights are turned out!
You will get two Renilla specimens in finger bowls. You may use red light to help to find your
materials, as this light will not cause the specimens to go into their day cycle (the bioluminescence
reaction only occurs at night!), but the light wave can be quite faint, so it is best to have your work area
as dark as possible.
Mechanical stimulation
1. To initiate bioluminescence, apply mechanical stimulation by gently poking the edge of the animal
with your finger. What do you observe?
Why does the wave of light only travel in one direction? (Hint: think about refractory periods)
2. Now, use mechanical stimulation to initiate the action potential on opposite sides of the animal.
Based on what you know about refractory periods, what do you expect to see?
What do you observe?
220 ABLE 2006 Proceedings Vol. 28
Goodwin
Electrical stimulation
For electrical stimulation, we will use a Grass stimulator and attached electrode (Figure 2). Be sure that
you are familiar with the controls before we turn out the lights!
Stimulator
Electrode
Figure 2. Grass stimulator.
Bioluminescence and nervous function
221
1. Note the location of the power switch and make sure that the controls on the Grass stimulator are
set appropriately:
Frequency: 2 pulses per second (note the multiplier switch below the frequency dial!)
Delay: minimum (note the multiplier switch below the frequency dial!)
Duration: 10 ms (note the multiplier switch below the frequency dial!)
Volts: 5 V (note the multiplier switch below the frequency dial!)
Stimulus: Regular.
To initiate the stimulus (don’t do this until you are ready to start the simulation of the animal!),
you will press the “mode” switch down toward the word “single.”
2. When you are ready, gently touch the electrode against the side of the colony, keeping your
fingers on the plastic arm of the electrode. DO NOT have your fingers in the water while an
electrical stimulus is applied! Apply several stimuli. What do you observe?
What is ‘threshold’ in the context of nervous function? Have we provided a threshold stimulus?
3. We will now increase the stimulus intensity. Increase the voltage gradually, not exceeding 50 V.
What do you expect will occur?
What do you observe?
4. Next, increase the frequency with which the stimuli are applied (temporal summation). To apply
multiple stimuli, you will press the “mode” lever up toward “repeat.” The frequency of pulses is
set by the “frequency” dial, which you have already set to 2 pulses per second.
Set the voltage to a level just below threshold. Start the pulses at 2 pps, then increase the
frequency by turning the “frequency” dial clockwise. Predict what will happen as the stimuli are
applied with greater frequency.
What do you observe?
222 ABLE 2006 Proceedings Vol. 28
Goodwin
Chemical stimulation
The two major vertebrate neurotransmitters used for communication between nerves and effector
tissues are acetylcholine and norepinephrine. Of these two neurotransmitters, only one has an effect on
Renilla nervous function. You will determine whether the Renilla nerve net responds to cholinergic or
adrenergic signals.
1.
Use a micropipettor to apply 50 μl of a neurotransmitter in the vicinity of the organism by
positioning the pipet tip at the bottom or side of the finger bowl and then moving the Renilla so
that it is just next to the pipet tip. Alternatively, you can hold the Renilla in your gloved hand
and use one of your fingers to maneuver the pipet tip near the surface of the colony. Release the
solution and observe the response.
Is the Renilla bioluminescence response mediated by cholinergic or adrenergic receptors?
Sources:
Anctil M. (1994) Monoamines and elementary behavior in a coelenterate. in Perspectives in
Comparative Endocrinology. Davey, Peter, Tobe, eds, p. 449-454. National Research Council of
Canada, Ottawa.
Awad EW and Anctil M. (1993) Identification of b-like aderenoceptors associated with bioluminescence
in the sea pansy Renilla koellikeri. J. Exp Biol 177:181-200.
Grober MS. (1990) Luminescent flash avoidance in the nocturnal crab Portunus xantusii. J. Exp Biol
148:415-426.
Wilson T and Hastings JW. (1998) Bioluminescence. Annu Rev Cell Dev Biol 14:197-230.
Parker GH. (1920). Activities of colonial animals; II. Neuromuscular movements and phosphorescence
in Renilla. J. Exp Biol. 31:475-515.
Thurman CL (2005) The chemical and neural basis for control of bioluminescence. in Laboratory
Manual for Physiology. Silverthorn, Johnson, Mills, eds, p. 821-829. Benjamin Cummings, San
Francisco.
Wilson T and Hastings JW. (1998). Bioluminescence. Annu. Rev. Cell Dev. Biol. 14:197-230.
Bioluminescence and nervous function
223
Instructor Notes
Student background:
This lab was incorporated into the nervous system unit of a sophomore level Animal Physiology
course. The students had already learned about nervous physiology and types of animal nervous system
in the lecture, and had done a simulation of nervous action potentials in the lab. All students in the class
had taken Introductory Biology, and most had also taken Zoology. This exercise could also be done as a
demonstration in an introductory biology class, or as part of a cnidarian lab exercise for Zoology.
Time required:
I conducted this lab in one three-hour time block. A shorter time block could be used,
particularly with a smaller class size or with the ability to darken the entire lab room. Since I had to
bring students into the dark room in groups, the students not conducting the experiments looked at
nervous system models and completed a case study assignment involving animal neurotoxins.
Renilla specimens:
I ordered the Renilla muelleri specimens from Gulf Marine Biological Labs at a cost of $9.20 per
specimen plus shipping. Most reports of Renilla function use Renilla kollikeri specimens from the
Pacific coast, but I was unable to find a supplier for this species. In fact the R. muelleri specimens
proved easier to use and maintain, as they thrive in room-temperature water, as opposed to the colder
temperature preferred by R. kollikeri.
Renilla bioluminescence only occurs when the specimens are in their night phase. The animals
must be dark-adapted for several hours before they will respond well to stimuli; I recommend having
them shipped to arrive the day of the lab and kept in the dark until the lab is completed. If you order a
few spare specimens, you and the students can examine morphology using dissecting scopes prior to the
experiments; otherwise, animals can be examined in the light after the bioluminescence studies are
completed. The animals are shipped cold, but do not put the finger bowls on ice to examine
bioluminescence – I found that this completely inhibited the response. The animals are not harmed in
the experiments described here, though surgical studies could be conducted to better analyze the path of
nervous conduction.
The animals can be maintained following the completion of the lab. I have ordered several
batches of animals, which have survived as long as 7 months in our aquarium, though 3 months is more
typical. The fluorescent lamps on the aquarium should be plugged into a timer to maintain a 12 hr
light/12 hr dark cycle. If you are conducting experiments during the day, be sure to keep the aquarium
in a dark room where the dark cycle can take place during working hours. I maintain the animals in an
established 55-gallon tank containing live rock and a few Aiptasia and brittle stars. The seawater is
room temperature, filtered and aerated; we replace half of the water with fresh artificial seawater (Instant
Ocean) once per month. Salinity is maintained at a specific gravity between 1.020 and 1.022. The
animals are fed 3 times per week with Cyclopeze, available in frozen blocks from aquarium stores or
online suppliers. We feed by mixing a small piece of the Cyclopeze with seawater and then pipetting
drops of this suspension directly onto the animals.
224 ABLE 2006 Proceedings Vol. 28
Goodwin
Darkened lab room:
This lab must be conducted in a dark room. I used a prep room adjacent to my lab room, and
therefore could only work with six students at a time; the students not actively conducting the
experiments looked at nervous system models and completed a case study exercise concerning
neurotoxins. It is critical that the students understand the procedure prior to turning out the lights –
although red lights may be used at the work stations, this light will not be sufficient to read the lab
handout or become familiar with the Grass stimulator. It takes awhile for everyone’s eyes to adjust to
the dark; during this initial time I had the lab groups discuss the answers to a homework assignment
focusing on Renilla and bioluminescence.
Lab setup
This might take about an hour. Each workstation will need:
1 desk lamp with a red light bulb, or flashlight with red filter paper
1 light-safe box (e.g. Styrofoam shipping box) containing:
at least 2 Renilla specimens in a finger bowl with seawater, or two finger bowls with one specimen
in each. Use the seawater that the specimens came in, if possible.
Extra seawater (1 liter or so); can be made from Instant Ocean powder
Grass stimulator and electrode
P200 micropipettor and pipet tips
100 μM solutions of epinephrine and acetylcholine, dissolved in seawater (add 1 mg/ml ascorbic
acid to epinephrine to prevent oxidation), clearly labeled so that the bottles can be distinguished in
dim light
1 dissecting scope and/or magnifying glass – to look at animals in the light
Protocol notes:
The Renilla may be inflated with water or deflated when you use them, and will likely be 2-5 cm in
diameter. An inflated animal might be 2 cm high, while a deflated animal will be almost perfectly
flat. An animal that started out inflated will likely deflate during the course of stimulation. Be sure
to provide at least two specimens per group, as some are more responsive than others.
In animals that are particularly sensitive, or are given a very strong stimulus a ‘frenzy response’ can
occur rather than a single wave of light. The frenzy response will either be a sequence of concentric
light circles or a spiraling pinwheel pattern of light that will continue uninterrupted for as long as
several minutes following the stimulus.
For mechanical stimulation, I found that a light squeeze with the fingers worked better than a light
poke with the glass rod.
Tape can be adhered over the lights on the Grass stimulator to darken the apparatus.
For chemical stimulation, Dr. Carl Thurman at the University of Iowa uses a syringe attached to fine
rubber tubing to apply chemicals to the top surface of the animal.
Bioluminescence and nervous function
225
Additional lab applications:
Other examples of bioluminescence can be displayed in addition to that of Renilla. Ward’s sells a
firefly bioluminescence kit, and an excellent protocol for dinoflagellate bioluminescence can be
found at www.lifesci.ucsb.edu/~biolum/organism/dinohome.html.
For more advanced courses, different concentrations, combinations & means of delivery for
neurotransmitters and drugs could be used. Other neurotransmitters expressed by Renilla include
melatonin, serotonin and antho-RF-amide, though in my preliminary study none of these chemicals
initiated bioluminescence.
The neural conduction pathways could be explored by disrupting the nerve net either surgically or
chemically using magnesium sulfate. Bioluminescence can then be observed following alteration of
conduction paths through the rachis, or connections between the rachis and the peduncle (references
below).
Rachidal peristalsis and feeding polyp retraction are also mediated by the nervous system; these
responses, too, could be examined (in the light!).
Renilla feeding polyps withdraw in a wave following exposure to certain chemicals (for example,
invertebrate food); this is likely mediated by the nerve net and could be explored further.
Renilla bioluminescence can be quantified using a luminometer (references below). Indeed, in dual
luciferase reporter systems for gene promoter activity, Renilla luciferase is used as a transfection
control.
Student feedback:
Students said that they enjoyed the lab, but aside from asking questions relating to the lab topics
on the lecture exam, I did not formally assess how well learning objectives were met. One student found
the topic so interesting that she has chosen to examine the contributions of nervous function and
circadian rhythm to the Renilla feeding response as her senior thesis project.
Lab source:
I first learned about Renilla bioluminescence from Dr. Carl Thurman’s lab writeup in the
Benjamin Cummings Laboratory Manual for Physiology. Dr. Thurman was an excellent source of
information and tips for conducting the lab.
Useful references:
Anctil, M. 1994. Monoamines and elementary behaviour in a coelenterate. In Perspectives in
Comparative Endocrinology. Davey KH, Peter RE, Tobe SS, editors. Ottawa: National
Research Council of Canada, p. 449-454.
Summary and images nervous-controlled Renilla functions, including bioluminescence.
Available online at: http://abonnes.collegebdeb.qc.ca/profs/ggermain/redaction/exemple2.html
Awad, E.W. and M. Anctil. 1993. Identification of beta-like adrenoreceptors associated with
bioluminescence in the sea pansy Renilla koellikeri. J. Exp Biol 177:181-200.
Germain, G and M. Anctil. 1996. Evidence for intercellular coupling and connexin-like protein in the
luminescent endoderm of Renilla koellikeri (Cnidaria, Anthozoa). Biol Bull 191:353-366
226 ABLE 2006 Proceedings Vol. 28
Goodwin
Use of luminometers to quantify Renilla bioluminescence.
Buck, J. 1973. Bioluminescent behavior in Renilla. I. Colonial responses. Biol Bull 144: 19-42.
Provides descriptions and diagrams of the various waveforms for bioluminescence.
Nicol, J.A.C. 1955. Nervous regulation of luminescence in the sea pansy Renilla koellikeri. Exp Biol
32:619-635.
Nicol, J.A.C. 1955. Observations on luminescence in Renilla (Pennatulacea). J Exp Biol 32:299-320.
Experimental analysis of facilitation, adaptation and summation in nervous control of Renilla
bioluminescence.
Parker, G.H. 1920. Activities of colonial animals. I. Circulation of water in Renilla. J Exp Biol. 31:343367.
Parker, G.H. 1920. Activities of colonial animals. II. Neuromuscular movements and phosphorescence
in Renilla. J Exp Biol. 31:475-515.
Great descriptions of Renilla structure and basic biology, as well as functions such as
bioluminescence, peristalsis and water circulation.
Diagrams of surgical patterns and use of magnesium sulfate to block nervous transmission.
Pieribone, V. and D.F. Gruber. 2005. A Glow in the Dark: The Revolutionary Science of
Biofluorescence. Belknap Press, Cambridge, MA.
Written for non-scientists.
Contains interesting historical anecdotes and scientific information about bioluminescence and
fluorescence.
Wilson, T. and J.W. Hastings. 1998. Bioluminescence. Annu Rev Cell Dev Biol. 14:197-230.
Excellent review of the chemistry of bioluminescence reactions in Renilla, fireflies,
dinoflagellates, bacteria, etc.
DVD: The Shape of Life series (NSF), Episode 2: Life on the Move – Cnidarians.
This whole series is a terrific way of introducing students to current research approaches with the
major phyla of the animal kingdom.
There is a 6-minute segment (at 24-30 minutes) in this episode describing the cnidarian nervous
system.
About the Author
Anne Goodwin received a B.A. in Biology from Albion College (Albion, MI) and a PhD in
Experimental Pathology from Harvard University, specializing in angiogenesis. She developed this lab
with great assistance from Carl Thurman (University of Northern Iowa) while teaching Animal
Physiology at Simmons College; this exercise is an adaptation of a protocol written by Dr. Thurman.
She will teach a variety of courses as an Assistant Professor of Biology at the Massachusetts College of
Liberal Arts (North Adams, MA) starting in July 2007, and will continue to conduct research on
cnidarians and tunicates.
©2007, Anne Goodwin
Use of Renilla bioluminescence
to illustrate nervous function.
ABLE 2006
Anne Goodwin
Simmons College
Workshop contents
„
„
„
Nervous function
Cnidarians & Renilla
Renilla bioluminescence
~ 1 cm
1
Workshop contents
„
„
„
Nervous function
Cnidarians & Renilla
Renilla bioluminescence
Nervous system basics
„
Nerve activity involves changes in
membrane potential
„
„
„
Differing ion concentrations on either
side of a membrane = voltage
When ions move across
membrane, voltage
changes
Nerve activity =
electrical activity
2
Nervous system basics
„
Ion channels open in response to
mechanical, chemical or electrical stimuli
© Prentice Hall
© Prentice Hall
© Brooks-Cole
Action potential propagation
3
Refractory periods
http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20102/Bi
o%20102%20lectures/Nervous%20System/neurons.htm
© Pearson
„
Refractory periods ensure unidirectional
impulse movement
Synaptic transmission
1.
2.
3.
Action potential reaches nerve end
Neurotransmitters are released
Neurotransmitters initiate action potential
on target cell
http://www.biologymad.com/NervousSystem/synapses.htm
4
Neurotransmitters
„
biogenic amines – vertebrate autonomic nervous
system, emotions; many invertebrate functions
(dopamine, norepinephrine, epinephrine,
serotonin, histamine)
„
acetylcholine – released at vertebrate
neuromuscular junctions, no activity in cnidarians
„
amino acids – in vertebrates & invertebrates
(GABA, glutamate)
„
peptides – vertebrate & invertebrate; endorphins,
antho-RF-amide, antho-RW-amide, etc.
Summation
„
„
„
Neurotransmitters
from multiple
neurons can act on
target cells
Neurotransmitters
may cause
stimulation or
inhibition
Multiple stimuli may
be needed to reach
threshold
http://www.biologymad.com/NervousSystem/synapses.htm
5
Facilitation
„
„
„
Closely spaced
impulses
Membrane does
not fully recover
from first
impulse
Resulting effect
is greater than
simple
summation
Intracellular recordings of facilitation of an EPSP.
Two identical stimuli, S1 and S2, were delivered to
the cortex about 6 msec apart; the resulting EPSP
is shown in A. When only S1 was delivered, the
EPSP was as shown in B. Notice the facilitation
(R2R1), not summation (R2=R1). (Porter R: J
Physiol (Lond) 207:733-745, 1970)
http://www.unmc.edu/Physiology/Mann/mann16.html
Model systems for nervous activity
„
Which organisms/model systems have
you used to illustrate nervous function
in laboratory exercises?
6
Workshop contents
„
„
„
Nervous function
Cnidarians & Renilla
Renilla bioluminescence
Cnidarians
„
„
„
Hydra, corals, anemones, jellyfish, etc.
Aquatic organisms with tissues, radial
symmetry
Have nervous system, water vascular
system, muscular activity
© Brooks-Cole
7
Cnidarian nervous function
„
Sensory cells, ganglion cells, effector
cells (muscles, glands, etc.)
Nerve nets
„
„
„
„
Neurons are non-myelinated
Conduction can be unidirectional or
bidirectional (nonpolar)
Neurotransmitters are
released at synapses
Multiple behaviors/
activities coordinated
by net
http://www.lifesci.ucsb.edu/~mcdougal/neurobehavior/modules_homework/jellies.html
8
Hydra nerve net
Koizumi (2002) Can J Zool 80:1678-89
Shimizu et al (2004) J Comp Physiol A 190:623-630
Cnidarian nerve net
DVD: Shape of Life, vol. 2: Cnidarians
9
Octocorallian corals
„
„
„
„
Have 8 tentacles
and 8 regions in
the gastrovascular
cavity
http://biology.kenyon.edu/courses/biol63/vocab.html
Grow in colonies
Have multiple polyp
types with different
functions
Soft or hard colony
structures
Renilla anatomy
„
„
„
Rachis – lobed, muscular structure
containing water vascular system
Peduncle – used for anchoring, locomotion
Polyps/zooids – have specialized functions
~ 1 cm
10
Renilla anatomy
„
„
Autozooids - feeding polyps
Siphonozooids - water flow polyps
Siphonozooid,
excurrent
Siphonozooid,
incurrent
Autozooid
http://www.usp.br/cbm/artigos/galer
ia/cnidaria/anthozoa/renilla.html
Spicules
Anctil et al. (2005) J Exp Biol 208:2005-2017
Renilla nervous system
Antho-RF-amidepositive neurons (a
subset of the total
neuronal population)
Pernet et al. (2004) J. Comp. Neurol 472:208-220
11
Renilla habitat
„
„
„
„
U.S.: Gulf, Atlantic & Pacific coasts
Sand or mud in intertidal region
Anchors into sand, expands & contracts
with tide
Can be found up to ~20 m depth
http://californiadiveboats.com/Truth/2005.05.28-30/
Renilla care
„
„
„
„
Will survive in aquarium for
several months, possibly up to
one year
Water: room temperature, wellaerated & filtered seawater
Food: ground seafood, “Cyclopeeze”, etc.
Light: 12 hr day/12 hr night
cycle
http://www.freezerbar.com/
12
Renilla nervous function
„
Nervous-regulated behaviors
„
„
„
„
Feeding responses
Peristalsis
Bioluminescence
Neurotransmitters:
„
„
„
„
Epinephrine (adrenaline)
Serotonin
Melatonin
Antho-RF-amide
Renilla peristalsis
Functions:
„ Shake off
sand
„ Move
„ Escape from
predators
http://abonnes.collegebdeb.qc.ca/profs/ggermain/red
action/exemple2.html
13
Nervous control of muscle activity
„
Peristalsis
Video clip
„
Polyp retraction
Video clip
Workshop contents
„
„
„
Nervous function
Cnidarians & Renilla
Renilla bioluminescence
14
Mechanisms of bioluminescence
„
„
Luciferin + oxygen + calcium +
luciferase = blue light (490 nm) + CO2
Blue light + green fluorescent protein =
green light (509 nm)
Bioluminescent cnidarians
utilize similar reaction
„
Fireflies, dinoflagellates &
bacteria use different
reactions
„
http://www.steve.gb.com/images/science/
aequoria.jpg
Stimulation of bioluminescence
„
Mechanical
„
„
Electrical
„
„
„
Touch to rachis or peduncle
Threshold 8-60 V
Facilitation required
Chemical
„
Epinephrine, not ACh or serotonin
15
Pattern of bioluminescence
Transmission
speed:
6.66-10.15
cm/sec (Buck, 1955)
http://abonnes.collegebdeb.qc.ca/profs/ggermain/redaction/exemple2.html
K. Patenaude
Renilla bioluminescent wave forms
“normal”
Refractory period
“volley”
“frenzy”
Buck (1973) Biol Bull 144:19-42
16
Facilitation & adaptation
„
„
Facilitation: repeated stimulation
increases intensity of response
Adaptation: continuous stimulation
eventually eliminates reponse
Nicol (1955) Exp Biol 32: 619-635
Summation
„
„
If pulses occur >1/sec, light never
completely returns to baseline
Subsequent flashes have greater intensity
(recruitment of more photocytes)
1 pulse/sec
3 pulses/sec
Nicol (1955) Exp Biol 32: 619-635
17
Stimulation notes
„
„
„
„
Renilla luminesce better if maintained in
non-stagnant water
Keep Renilla in dark at least 2 hr prior to
experiments
Mechanical stimulation gives a brighter
response than electrical stimulation
First electrical pulse not sufficient to
initiate response; facilitation required
Further studies
„
„
„
„
„
Renilla peristalsis
Surgical/chemical nerve blocks
Anemone tentacle contraction
Hydra digestive movements
Anemone nematocyst discharge
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Conduction pathway analysis
„
„
„
Pattern of
peristalsis can
be altered
Surgical
disruption
MgSO4
crystals
Parker (1920) J Exp Biol 343-67
Conduction pathway analysis
„
Surgical or chemical (MgSO4)
disruptions
Parker (1920) J Exp Biol 343-67
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More lab-friendly bioluminescence
„
Dinoflagellates
„
„
„
‘sea sparkle’
http://www.lifesci.ucsb.edu/
~biolum/organism/dinohome.
html
Fireflies
„
„
„
WARD’s kit for in vitro flash
Harvest & observe
http://iris.biosci.ohiostate.edu/projects/FFiles/biolum
.html
20