Effects
of Narrow Bandwidth Light Sources on Coral Host and Zooxanthellae
Pigments
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Lighting
is an extremely important consideration for reef aquarium hobbyists,
yet there are still many unanswered questions concerning artificial
light sources and their effects on captive corals. This article
will examine the results of experiments designed to investigate
the effects of light spectra on coral coloration, some of which
were unexpected.
Many
benthic invertebrates inhabiting coral reefs contain fluorescent
and reflective pigments, and it has long been speculated that
these are produced as a protective response to ‘strong
sunlight’ (suggested by Kawaguti in 1944 and re-examined
by Salih et al., 2000). Further, researchers have examined excitation
and emission spectra of fluorescent coral pigments (Mazel, 1995;
1997) and the instantaneous effects of altered spectral quality
on apparent coloration (Fux and Mazel, 1999, and unpublished).
However, very little information exists on influences of artificial
light sources (particularly those producing narrow bandwidths)
on coral host and zooxanthellae pigmentation.
Reef
aquarists often report drastic shifts in apparent and true coloration
of captive corals, especially when altering the spectral quality
of primary artificial light sources (especially metal halide
lamps). Some of these ‘changes’ are due simply to
the reflective nature of the coral host tissue and zooxanthellae.
Occasionally, reflected light cannot explain coloration shifts,
and intriguing reports state that certain coral colorations
can be maintained only under certain (usually ‘high’)
Kelvin lamps are sometimes heard.
A simple
experiment was devised to test the hypothesis that relatively
narrow spectral bandwidths can play a role in inducing colorful
coral pigments. Initial experiments involved a submersible lighting
system consisting of four LED lamps (blue, green, yellow and
red), which illuminated portions of genetically identical coral
fragments for 6 weeks. Replicate trials followed a few months
later with the same outcome. Results strongly suggest spectral
quality can have profound effects on host and algal pigmentation.
Procedure
– Round One
The
coral chosen for this experiment was the ‘Rose Coral’
(Pocillopora meandrina) – the common name for
this coral is well chosen, as coloration in some colonies is
red to hot pink. A small branch (approximately 13 cm long) was
obtained from an aquarium within the Natural Energy Laboratory
Hawaii (NELHA) complex in Kailua-Kona, Hawaii. The branch was
subdivided into 4 fragments, which were then glued to acrylic
pedestals.
The
branches were transferred to an open-system aquarium at NELHA’s
display area. This aquarium (122 cm X 46 cm X 46 cm, holding
approximately 257 liters) had been prepared in advance to meet
the requirements of this wave-washed P. meandrina specimen.
A Carlson Surge Device (CSD - approximately13 L capacity) situated
about 1 meter above the aquarium provided periodic wave surges
similar to those found in the coral’s natural environment.
Although the average water detention time within the aquarium
was only 30 minutes, adjustments of ‘warm’ and ‘cold’
water streams to the CSD were necessary to control temperature
within the tank. (The NELHA complex has available seawater pumped
from two depths – 25 and 615 meters, with temperatures
of ~27° and ~5°C, respectively.) Aquaculture and other
firms can dial in a temperature through simple feed water valve
adjustments. Minimum and maximum temperatures observed within
the aquarium during the experiments were 23.3° and 27.7°
C.
Natural
sunlight provided light to the aquarium, but was attenuated
to a maximum of ~10% intensity (200 – 215 µmol·m2·sec)
by two layers of shade cloth. This light intensity was well
below the maximum of ~1,100 µmol·m2·sec
experienced by most shallow-water corals in the area at noon.
Previous experiments suggested the intense pink coloration would
be lost below a ‘coloration threshold’ of about
200 - 250 µmol·m2·sec. All light measurements
were taken with a Li-Cor Model 189 quantum meter and 2 pi submersible
sensor. A sheet of Lexan clear glazing material attenuated ultraviolet
radiation to levels of ~ 1 µw·cm2 UV-A and <1
µw·cm2 UV-B (as measured with an Ultraviolet Products
radiometer and UV-A and UV-B sensors). An Ocean Optics USB-2000FL
spectrometer determined the effective wavelength cutoff of this
material was ~390 nm.
Five
Light-Emitting Diodes (LEDs) were used in these experiments.
Nichia America manufactures the Ultraviolet-A (UV-A) and blue
LEDs; the green, yellow and red LEDs were obtained at a local
Radio Shack. (See Figure 1. Spectral signature of the UV-A LED
is not shown. The maximum output of the red LED is dead on the
absorption peak of chlorophyll a.)
Figure
1.
Spectral Signatures of LEDs
Blue = Nichia Blue (peak emission @ 457 nm); Green
= Radio Shack Green LED (peak emission @ 564 nm);
Yellow = Radio Shack Yellow LED (peak emission @ 587
nm); Red = Radio Shack Red LED (peak emission @ 659
nm).
The
amplitudes in Figure 1 represent spectral quality and not absolute
intensity (since measurements were taken by positioning the
LEDs to deliver ~3,500 counts to the spectrometer’s sensor).
When measurements were made at a standardized distance (~6 mm)
from the tip of each LED with a Li-Cor quantum meter, the intensities
were:
Nichia
Blue LED = 400 µmol·m2·sec
Radio Shack Green LED = 15 µmol·m2·sec
Radio Shack Yellow LED = 50 µmol·m2·sec
Radio Shack Red LED = 323 µmol·m2·sec
Appropriate
resistors and wiring were housed within clear vinyl tubing,
and the LEDs were friction-fitted into the end of the tubing
and glued in place with silicon cement. U-shaped acrylic jigs,
each drilled to accept vinyl tubing and an individual LED were
attached to a black acrylic platform with nylon bolts and wing
nuts. PVC spacers under each jig allowed for vertical positioning
of the LEDs, while the nylon bolts acted as the pivot point
for horizontal movement (See Figure 2.)
Figure
2
Photograph of the experiment’s setup (specifically
that of the second set of experiments). Note how the
blue LED is positioned further away from the coral than
the red LED. This was done to standardize light intensity
falling upon the coral’s surface.
The
LEDs were pointed toward shaded areas of the coral fragments,
where maximum ambient light intensity (with the LEDs off) was
~20 µmol·m2·sec. A mechanical timer switched
the LEDs on and off and lamp photoperiod was incrementally increased
from an initial 2 hours to 12 hours over a 30-day period.
Results
– Round One
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The
reduced light level within the aquarium apparently caused loss
of pink coral coloration within 2 weeks after transfer, and
the animal turned to a ‘zooxanthellae brown’ color.
The fragment’s soft tissues in heavily shaded areas (those
not exposed to LED illumination) died and slowly receded.
On
Day 14 of the experiment, the yellow LED failed, due to a leak
in the silicon seal between the LED and vinyl tubing.
On
Day 22, loss of brown coloration in the area illuminated by
the red LED was noted (see Figure 3). Examination of this area
with Underwater Kinetics Light Canon dive light fitted with
NightSea excitation and barrier filters revealed no chlorophyll
red fluorescence, indicating the coral had ‘bleached.’
However, the coral tissue did not necrotize. (Note: Red fluorescence
of chlorophyll should not be confused with the reflective pink/red
pigments found in the coral host tissue. These are two entirely
different pigments!)
On
Day 28, coral fragments were again examined under the light
of the dive light and filters. The area illuminated by the blue
LED had strong chlorophyll red fluorescence. Normal, but not
elevated, chlorophyll fluorescence was noted on the fragment
illuminated by the green LED. The bleached area illuminated
by the red LED remained, as it did on Day 22, apparently free
of zooxanthellae and no chlorophyll fluorescence was noted.
On
Day 31, a spot of pink coloration (~5 mm in diameter) was noted
at the area illuminated by the blue LED (see Figure 4). Since
that day, the spot has intensified in coloration but not size.
Figure
#3
Figure
#4
On
the left, loss of zooxanthellae (within black circle)
apparently caused by red light on Day 22 of the experiment.
On the right: Day 31. Expression of the pink coloration
is noted (within the circle to the right of the bleached
area) in area illuminated by the blue LED.
These
results were briefly discussed during my 2002 MACNA presentation
in Fort Worth, Texas and this article was submitted to Advanced
Aquarist On-Line shortly thereafter. During the peer-review
process, it was suggested that the experiments be replicated;
the ‘new’ experiments should standardize the light
intensities falling upon the corals’ surfaces. I agreed,
but had no idea at the time that circumstances and time constraints
would delay initiation of the project for almost half a year.
(click
for a larger view)
Figure
5
A
close-up of the bleached and pink pigmented spots (circled).
Notice that these areas are recessed and were shaded
from direct sunlight during the experiment.
Procedure
- Round Two
It
was decided that the second round of the experiment would utilize
only the blue and red LEDs, plus an LED producing ultraviolet
radiation. Charlie Mazel of NightSea, Inc. was kind enough to
provide several LEDs producing UV-A with a peak output at 370
nm. This peak wavelength is very close to the UV-A output of
metal halide and other lamps containing the element mercury
– these lamps have a ‘spike’ at 365 nm. What
effect would UV-A radiation have on corals’ zooxanthellae
and pigmentation?
The
underwater cable and LED holder would have to be re-designed
to prevent leakage and failure (especially a concern since the
UV LEDs would be quite expensive to replace). The new underwater
LED system was built and tested, and passed with flying colors
(perhaps floating colors would be more appropriate).
Round
Two would also involved fragments from the Rose Coral (Pocillopora
meandrina). These fragments were collected (under a scientific
collection permit from the Hawaii Department of Aquatic Resources)
from a coral specimen damaged during a particularly strong early-Spring
southern swell. The coral fragments were glued to acrylic pedestals
and allowed to recover in the 257-liter aquarium at the Natural
Energy Laboratory. As in Round One, natural sunlight intensity
was attenuated with shade cloth and acrylic, and the coral fragments
faded from hot pink to brown in a couple of weeks’ time.
Blue
and red LED light intensities falling upon the coral fragments’
surfaces were standardized to 215 µmol·m2·sec.
The UV-A LED was positioned to deliver approximately 300 microwatts
per square centimeter (approximately 3X that measured in Walter
Bobe’s mainland aquarium containing magnificently colored
Acropora specimens). The photoperiod was set for 3 hours per
night, and the experiment began. Photoperiod was incrementally
increased every few days. There was some concern that the blue
and red lamp intensities would not be sufficient to induce either
color or bleaching. However, some lightening of the area illuminated
by the red LED was noted on Day 23 of the experiment, with the
photoperiod at 11 hours. No apparent changes were noted under
the UV and blue LEDs. No further visual changes were observed
through Day 50. A trip to the mainland prevented observations
between Days 51 through 70. On Day 71, it was found that the
coral areas under the blue LED had gained pink coloration, and
the area illuminated by the red LED had lost more zooxanthellae
– it had bleached (See Figures 6 and 7). No visible changes
were observed within the area irradiated by the UV LED.
Discussion
(click
for a larger view)
Figure
6
A
macro shot of the Pocillopora meandrina and
the area exposed to the blue LED. The pink pigmentation
is not as intense or pervasive as seen in the initial
experiments. Note: This is the same coral as in Figure
1 – a classic case of ‘Pocillopora
polymorphism’ in response to a change in environmental
conditions (perhaps water motion).
The
results of this experiment suggest that narrow bandwidths of
essentially ‘pure’ red and blue wavelengths have
profoundly different effects on zooxanthellae health and host
tissue pigmentation.
It
appears that red light induced bleaching in the two experiments.
It is also worthy to note that bleaching was noticed on Day
22 and Day 23 of the first and second set of experiments, respectively,
even when red LED lamp intensity differed by ~20%. What would
explain this? In the 1940’s, Emerson et al demonstrated
that monochromatic red light (at 680 nm) is about 36% more efficient
in the promotion of photosynthesis than monochromatic blue light
at 460 nm (reported in Hall and Rao, 1999). This is possibly
due to the direct absorption of red wavelengths by chlorophylls
and Pigment 680 (P-680) found in specialized chlorophyll molecules
within the reaction centers of Photosystem II. The relative
inefficiency of monochromatic blue light (as opposed to monochromatic
red light) to promote photosynthesis might be caused by the
less than perfect transfer of light energy collected by chlorophylls
a and c, as well as some accessory antennae pigments- the major
accessory carotenoid peridinin has been shown to transfer harvested
light energy with >85% efficiency (Schofield et al., 1996).
The energy collected by these pigments is channeled to the specialized
P-680 chlorophyll molecules, and the convoluted process of photosynthesis
is begun.
Red
light of different wavelengths has an ability to promote photosynthesis
in a phenomenon called the Emerson Enhancement Effect. Researchers
determined that the total amount of photosynthesis promoted
in the presence of a mixture of red (~650 nm) and far-red (>685
nm) light is greater than the sum of the amount of photosynthesis
observed during separate experiments with individual beams of
red and far-red light. These experiments’ results led
to the discovery of two different types of chlorophyll a –
one produces an oxidant and the other a reductant – and
the realization that there are two distinct photosystems (I
and II) acting in photosynthetic concert. In effect, far-red
light prevents a traffic jam of electrons from occurring on
the road connecting Photosystem II to Photosystem I (see Hall
and Rao, 1999). This is quite interesting since corals often
live in environments with depleted amounts of red light. To
overcome this, zooxanthellae of corals living in shaded areas
and deeper water alter the ratios of their photopigments and
become more efficient in absorption of wavelengths above 680
nm (Titlyanov et al, 1980).
(click
for a larger view)
Figure
7
Another
macro shot of P. meandrina. The area within
the circle is the general area illuminated by the red
LED. Note the reduced zooxanthellae within the coral
polyps – the coral has bleached, though not as
intensely as in the initial experiment where red light
intensity was greater.
In
order for photosynthesis to proceed smoothly, there must be
a balanced energy distribution between Photosystem I and Photosystem
II. Photosystem II produces an oxidant and PS I a reductant
– which is important in maintaining the electron flow
between the photosystems. This redox balance might be achieved
through control and/or alteration of photosynthetic pigment
content of zooxanthellae (Kinzie et al, 1984). In other words,
zooxanthellae can custom tailor (within limitations) their photopigments
to maximize use of available light energy. For instance, the
major accessory pigment peridinin (which harvests light in the
green portion of the spectrum, up to ~550 nm) transfers harvested
light energy to chlorophyll a, and hence the reaction centers
of PS II. On the other hand, the carotenoid beta-carotene transfers
its harvested energy to chlorophyll a and PS I. Increased absorption
of light energy above 680 nm is associated with aggregated forms
of chlorophyll a and PS I (Titlyanov et al., 1980).
Since
a water column rapidly attenuates red light, many, if not most,
corals found on natural reefs are exposed to only a fraction
of red light energy found at the water’s surface. Hence,
chromatic adaptations by corals are a reversal of ‘sun’
and ‘shade’ terrestrial plants: Corals found at
shallow depths (‘sun’ corals, if you will) are potentially
adapted to, among other wavelengths, moderate amounts of radiation
in the red portion of the spectrum, while deeper corals (‘shade’
corals) are likely adapted to an environment where green/blue
wavelengths predominate and red light is greatly reduced. It
is interesting to note that those pigments involved in photoprotective
dynamic photoinhibition (i.e. xanthophylls diadinoxanthin and
diatoxanthin) absorb blue wavelengths and not red wavelengths.
Hence, coral zooxanthellae do not possess an ability to rapidly
deal with red light and might bleach when suddenly exposed to
increased amounts of red radiation.
Kinzie
et al (1984, 1987) reported effects of different spectra (including
blue, white, green, blue-green and red) on two Hawaiian corals
(Pocillopora damicornis and Montipora verrucosa
- now M. capitata). The results of these experiments
suggest that red light promoted poor coral growth and zooxanthellae
growth/reproduction. Interpreted by some to mean that red light
is inefficient in the promotion of photosynthesis, it could
be that exactly the opposite is true – that bleaching
(either loss of algal cells or reduction in pigmentation) was
caused by an exposure to elevated levels of more photosynthetically
efficient red light. Consider that red light is attenuated by
~40% in the first meter of the clearest seawater – Type
1 Oceanic (Jerlov, 1976) – and much more in all other
optical classifications of seawater.
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The
results of Kinzie et al. experiments, and those in these procedures,
suggest that red light might play a role in regulating zooxanthellae
pigmentation and density.
What
are the possible effects on zooxanthellae of sudden exposure
to altered spectral quality? This question is not easily answered;
however, Iglesias-Prieto (1997) offers some interesting insights.
Although this paper discusses thermal effects on zooxanthellae
photosystems, some parallels are drawn between destruction of
photosynthetic ability by heat and photosynthetic photon flux
density. In essence, loss of re-oxidation capacity by Photosystem
II creates an ‘excitation pressure’ (through generation
of oxygen radicals) and can result in irreversible damage, possibly
resulting in bleaching or photosynthetic pigment loss. It is
possible that the red LED produced insufficient radiation to
drive PS I, resulting in destructive pressure on PS II.
With
that said, this discussion of bleaching and pigment loss will
end - the primary purpose of my experiments was to observe the
response of the coral host pigmentation to narrow bandwidth
light sources.
The reasons for corals’ production of a pink pigment under
blue light are not as easily explained - a theory could be advanced
that some corals (likely only those genetically predisposed
– see Takabayashi and Hoegh-Guldberg, 1995) react to blue
light by the manufacture of reflective/fluorescent pigments.
Could intensity of blue light be the environmental factor triggering
the production of pink/red pigment(s) to protect zooxanthellae
from the more photosynthetically efficient long-wave (red) visible
light? It is interesting to note the time scale of pink pigmentation
production under the differing blue light intensities used in
the two experiments. Expression of the pink pigment at a light
intensity of ~400 µmol·m2·sec was observed
on Day 31 of the first experiment. In the second experiment
with the red light intensity at 215 µmol·m2·sec,
the pink pigment was weakly expressed between Days 50 and 70.
These results suggest that the expression of the pink pigment
is a response to blue light intensity. The concentration of
the pigment (as judged visually) and the time frame required
for expression of the pigment seems also to be linked to the
intensity of blue light.
The
blue and red LEDs produce essentially no ultraviolet radiation,
strongly suggesting that UV played no part in either promotion
of the bleaching episodes or the expression of the pink pigment.
Near-infrared and infrared radiation (IR, which we perceive
as heat) production by all these LEDs is extremely low, hence
the energy transfer of near-IR and IR from water-cooled LEDs
makes any harmful effect of thermal stress unlikely.
The
UV-A LED produced no visual response on coral host coloration
and zooxanthellae. This result suggests that UV-A radiation
plays little or no part in inducing reflective/fluorescent pigments,
at least in the case of the pink/red pigment in these P.
meandrina specimens under the conditions of this experiment.
(Fluorescent coral pigmentation in low UV environments has been
previously reported – see Riddle and Amussen, 1998). Interestingly,
the coral did not bleach when irradiated with 300 microwatts
per square centimeter of UV-A energy (sunlight in Hawaii at
noon on a clear day delivers approximately 2,100 microwatts
of UV-A energy). Experiments are planned to examine the effects
of ultraviolet energy from artificial light sources on captive
corals. These experiments will measure the quantum yield of
zooxanthellae through use of a pulsed amplitude modulation (PAM)
fluorometer.
Lack
of apparent response to the output of the green LED is more
easily explained. First, the output intensity is very low at
only 15 µmol·m2·sec and, second, the peak
emission at 564 nm is just outside of the absorption bandwidth
normally associated with the antenna pigment peridinin. For
the second set of experiments an attempt to increase green light
intensity was made by bundling multiple green LEDs. This was
not successful – bundling did not significantly increase
light intensity - and insight on the effects of monochromatic
green light on coral host pigmentation remains elusive.
From
a practical standpoint for hobbyists, the results suggest that
narrow bandwidth blue light produced by the Nichia LED is sufficient
to not only maintain zooxanthellae health (at least short-term)
but can apparently promote colorful host tissue pigmentation,
if light intensity is high enough. Of more importance are perhaps
the observations of bleaching, and the realization of potential
effects of light quality from artificial light sources on captive
corals. The possibility certainly exists that red light content
of artificial light sources is the environmental trigger for
control of pigment content and/or zooxanthellae density within
captive corals.
Many
questions remain unanswered. What are the results on coral/zooxanthellae
pigments when red and blue LEDs are bundled and the coral is
subjected to a reasonably balanced spectrum? Are the results
of these experiments applicable to common aquaria lamps such
as fluorescent and metal halide lamps? Results of recent experiments
seem to confirm that broadband spectral qualities make little
difference to photoacclimated Fungia corals (within the context
of rates of photosynthesis and the conditions of the experiment
- Riddle, article in preparation). However, the effects of spectral
quality on corals with the ability to alter apparent coloration
through expression of reflective and fluorescent pigments seem
to be a different story.
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Acknowledgements
I
wish to thank Sara Peck of Sea Grant Hawaii for her unwavering
support, and to Charlie Mazel of NightSea, Inc. (www.nightsea.com)
for providing the Nichia LEDs and encouragement. Many thanks
also go to the Friends of NELHA for all those little things
that added up to a lot.
References
Fux,
E. and C. Mazel, Unpublished. An experimental method to separate
the fluorescence and reflectance components of the spectral
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spectra and predicting new spectra under different excitation
conditions. Applied Optics. 38, 3: 486-494.
Hall,
D. and K. Rao, 1999. Photosynthesis: Studies in Biology.
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R., 1997. Temperature dependant inactivation of photosystem
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