BULLETIN OF MARINE SCIENCE, 67(2): 821–830, 2000
CORAL REEF PAPER
UV-ABSORBING COMPOUNDS IN THREE SPECIES OF
CARIBBEAN ZOOXANTHELLATE CORALS: DEPTH
DISTRIBUTION AND SPECTRAL RESPONSE
J. E. Corredor, A. W. Bruckner, F. Z. Muszynski,
R. A. Armstrong, R. García and J. M. Morell
ABSTRACT
We examined the ultraviolet (UV) absorption spectra (280–400 nm) of coral tissue
extracts and the concentration and composition of mycosporine-like amino acids (MAA)
in three common Caribbean reef-building corals using UV spectroscopy and high-pressure liquid chromatography. Two of the species examined Porites astreoides (green
morphotype) and Acropora cervicornis are widely distributed ranging from <1.0 to over
25 m depth, while the third, Mycetophyllia ferox, is a plating species usually confined to
the deeper parts of the reef. UV absorption spectra (280–400 nm) of coral tissue extracts
in seawater exhibited absorption maxima between 320 and 340 nm and protein-specific
absorption in general decreased with depth. A. cervicornis and P. astreoides exhibited
hypsochromic displacement in their UV-absorption spectra relative to M. ferox, denoting
greater capacity per mole unit MAA for absorption of the shorter, more energetic, and
thus more harmful radiation. MAA content of M. ferox was the lowest of the three species
examined, approximately one tenth that of P. astreoides across the depth range examined.
In all three species, mycosporine–glycine showed the least variation with depth while
content of other MAAs varied substantially.
Knowledge of the ability of corals and other marine organisms to chemically screen
their tissues from harmful ultraviolet (UV) light dates to the work of MacMunn (1903)
who reported the presence of “dark pigments that have the property of arresting the ultraviolet and violet rays of light”. With regard to the photoprotective role of these ‘pigments’, MacMunn noted that “they probably act as a screen, protecting the delicate organisms from the irritating effects of the rays of short wave-length”. Subsequently, Margalef
(1959) reported the presence of an unidentified compound with high absorption at low
wavelengths in reef building corals growing in shallow waters of southwest Puerto Rico;
again recognizing that such compounds might serve as a defense against the biological
effects of UV radiation. It was not, however, until the rediscovery of these compounds in
corals of the Great Barrier Reef by Shibata (1969) that modern work on their chemical
nature, taxonomic distribution and ecological significance came about. These compounds
are now recognized as ‘mycosporine-like amino acids’ (MAAs) a group of cyclohexenone/
amino acid-based water-soluble compounds believed to arise from the shikimic acid biosynthetic pathway. MAAs are abundant in benthic marine invertebrates exposed to high
fluxes of UV radiation, particularly in the reef building corals which are sessile and restricted to shallow depths within the euphotic zone. Dunlap and Shick (1998) have provided an extensive and up-to-date review of the biochemistry and environmental role of
MAAs in coral reef organisms.
Reef-building corals exhibit substantial variability in the concentration of these UVabsorbing compounds with depth. MAA content is usually greatest in shallow waters
presumably as a response to increased UV fluxes (Dunlap and Chalker, 1986; Dunlap et
al., 1986; Gleason and Wellington, 1993). The distribution of these photoprotective com821
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pounds within a single colony also responds to differences in the incident light flux with
higher concentrations on the upper part of the corallum and lower concentrations in the
shaded portions (MacMunn, 1903; Muszynski et al., 1998). MAA suites and total concentrations vary among species or even between two color morphs of a single species
(Gleason, 1993).
To assess the capacity for UV screening of the different coral species, we examined the
UV-absorption characteristics of coral extracts and the composition and concentration of
MAAs in three common Caribbean reef-building corals from a reef off La Parguera,
Puerto Rico. The species Porites astreoides (green morphotype), Acropora cervicornis
and Mycetophyllia ferox were selected based on their abundance and vertical distribution.
Two of the species examined are widely distributed from <1.0 to over 25 m depth, while
the third, M. ferox, is a plating species usually limited to the deeper parts of the reef
(Colin, 1978). In order to approximate the in vivo optical spectra of the coral MAAs, we
re-suspended dried extracts in filtered offshore seawater and recorded their absorption
spectra. These extracts were further characterized by high-pressure liquid chromatograhy.
We report significant differences in both the position and magnitude of absorption maxima
from different taxa as well as differences in MAA suite between species and within species along the depth gradient.
MATERIALS AND METHODS
SAMPLING PROCEDURE.—Coral samples were obtained at San Cristobal reef off La Parguera, on
the southwest coast of Puerto Rico. Sampling was performed on 12 April 1997 except for the
shallow (1.5 m) samples of A. cervicornis which were collected on 21 February 1997. Total MAA
concentration and composition were examined in coral colonies at three depths (1.5, 5 and 10 m).
At each depth, replicate coral tissue samples were taken from three colonies of each species, for a
total of 18 samples (nine colonies, two samples per colony). M. ferox was rare at 1.5 m depth. Only
one colony of this species was found oriented horizontally and exposed to direct incident light;
samples were taken from this colony as well as two colonies oriented vertically and shaded by reef
rock. We have previously demonstrated that MAA composition and content can vary up to five-fold
within an individual coral specimen according to the orientation of the surface sampled relative to
solar irradiation (Muszynski et al., 1998). To avoid such variability, we adopted the following sampling strategy: For all massive and plating species, samples were taken from the center of the horizontal upper surface. Two 3.5 cm2 cores were removed from three separate colonies of each coral
species using a pneumatic drill and a carbide-tipped hole saw. Each core was cut into two equal
portions with a high-speed rotary tool. For the coral A. cervicornis, one sample consisted of the top
10 cm of a vertically oriented branch approximately 15–20 cm in overall length; three vertical
branches were collected from each coral. Samples were transported to the laboratory in individual
containers submerged in seawater and shaded from direct sunlight.
SAMPLE EXTRACTION.—All samples were extracted sequentially in four 10 ml volumes of 20%
tetrahydrofuran in methanol (MeOH/THF) within 2 h of collection. Core sections were initially
extracted for 20 h at 4°C, followed by three additional 20 min extractions at room temperature.
Samples were then centrifuged at 5000 rpm for 5 min. The supernatant was decanted for MAA
analysis and the solid fraction was used for determination of total protein.
PROTEIN ANALYSIS.—Total protein was determined with a Bio-Rad DC protein assay kit using
bovine gamma globulin as a standard. The solid portion of each sample was heated in 3 ml 1N
NaOH for 30 min at 90°C, cooled to room temperature and neutralized with 3 ml 1N HCl prior to
reagent addition. Absorption was recorded at 750 nm using a Hewlett-Packard 8452-A diode array
spectrophotometer.
CORREDOR ET AL.: CARIBBEAN CORALS DEPTH UV SPECTRAL RESPONSE
823
WHOLE EXTRACT ABSORPTION SPECTRA.—Capacity for light absorption of coral species was assessed using the MeOH/THF extract and extracts re-dissolved in seawater. Aliquots of the extract
(5 ml) were evaporated to dryness in a rotary evaporator and re-dissolved in 5 ml filtered offshore
seawater to simulate the ionic strength of the intracellular fluids. The re-dissolved extracts were
passed through a C-8 Sep-Pak cartridge and MAAs retained in the cartridge were eluted with an
additional 5 ml seawater. The combined extracts were then scanned in a 1 cm quartz cell using a
HP8452 diode array spectrophotometer. Resulting data were normalized to protein content.
Seawater exhibits higher pH and ionic strength than intracellular fluids and both pH and ionic
strength can affect the absorption spectrum and magnitude of organic solutes (Görög, 1995). Consequently, the suitability of seawater for simulating the intracellular millieu was examined by varying the salinity and pH of representative extracts from P. astreoides re-dissolved in seawater over
ranges encompassing the physiological ranges.
MAA ANALYSIS.—MAAs were separated by reversed-phase isocratic HPLC using a LC-10AT
Shimadzu liquid chromatograph and a C-8 column (Upchurch α-Chrom) following a protocol modified from Dunlap and Chalker (1986). For each sample, 4 ml of supernatant was evaporated under
vacuum and re-suspended in 4 ml 0.1% aqueous acetic acid and 10% methanol. Non-polar photosynthetic pigments were removed with a maxi-clean C-18 SepPak solid-phase extraction cartridge
(900 mg), the filtrate was diluted appropriately (1:1 to 1:10), and sub-samples of 600 μL were
injected across a 200 μL injection loop at a flow rate of 0.2 ml.min −1. MAA elution was monitored
between 250 and 400 nm by diode array.
DATA HANDLING.—MAAs were identified by comparison of their absorption maxima and retention time to those of known compounds. Compounds were quantified as described previously
(Muszynski et al., 1998) using absorption coefficients for mycosporine-glycine e310 = 28,100 (Ito
and Hirata, 1977), for shinorine e334 = 44,668 (Tsujino et al., 1980), palythine e320 = 36,200 (Takano
et al., 1978), and e330 = 43,500 for asterina 330 and palythinol (Gleason, 1993).
UV absorption and MAA content were normalized to protein content (Chalker et al., 1983) for
statistical analysis. Between species differences, and depth variability within species were assessed
by analysis of variance. A two-factor ANOVA was used to determine if the total concentration of
UV absorbing compounds varied significantly among species and depths. For M. ferox from 1.5 m
a single factor ANOVA was used to examine for differences in MAA concentration between the
horizontally and vertically oriented, partially shaded colonies.
OPTICAL PROPERTIES.—Seawater absorption within the La Parguera reef complex was measured at
an adjacent reef (Laurel reef) on five occasions between December 1997 and January 1999. We
used a submersible Optronics OL-754 spectroradiometer with a 100 mm integrating sphere attached to a 11 m quartz fiber optic cable. Diffuse attenuation coefficients were calculated for each
2 nm band between 300 and 700 nm. Average daily sea-level UV irradiation for five channels of the
UVA/UVB region (305, 320, 340 and 380 nm) is routinely monitored at La Parguera using a
Biospherical Instruments GUV-511C radiometer. Estimates of daily UV irradiation at 320 nm were
obtained by averaging observations over the week of sampling.
RESULTS
UV absorption spectra (280–400 nm) of coral tissue extracts re-dissolved in seawater
exhibited UV-absorption maxima between 320 nm and 340 nm and absorption in general
decreased with depth (Fig. 1). Both the wavelength of maximum absorption and the total
absorption varied significantly within species. In shallow water (ca 1.5 m) P. astreoides
had the highest absorption (mean = 0.08 cm−1 mg protein−1; SD = 0.029) and A. cervicornis
had intermediate values (0.038 ± 0.007 cm−1 mg protein−1), while M. ferox exposed to
ambient light at 1.5 m demonstrated the lowest overall ability to absorb UV radiation
(0.0109 ± 0.0067 cm−1 mg protein−1).
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BULLETIN OF MARINE SCIENCE, VOL. 67, NO. 2, 2000
Figure 1. Absorption spectra of protein-normalized MAA tissue extracts (re-dissolved in seawater)
of A. cervicornis, M. ferox and P. astreoides from 1.5, 5 and 10 m depth. (n = 3 for all species and
depths except M. ferox at 1.5 m, n = 1).
CORREDOR ET AL.: CARIBBEAN CORALS DEPTH UV SPECTRAL RESPONSE
825
Table 1. Effect of varying seawater salinity and pH on the wavelength of maximum absorbance
of coral extracts (Porites astreoides). Methanol/tetraydrofuran extracts were evaporated to dryness
and re-dissolved in seawater. Sample salinity was progressively lowered by dilution with distilled
water. Sample pH was lowered by adding aliquots of a 10% HCl solution.
Salinity
(‰)
35
34
33
31
29
28
27
25
24
23
22
20
Wavelength
(nm)
329.8
330.0
329.8
329.8
329.2
329.4
329.4
329.6
329.6
329.4
329.6
329.6
pH
7.64
7.63
7.50
7.24
6.93
Wavelength
(nm)
330.1
330.3
329.7
329.9
329.4
Varying the ionic strength and pH of the seawater solutions of the P. astreoides extract
had no significant effect on the absorption spectrum (Table 1). In contrast, MAA extracts
for the most part exhibited substantial bathychromic absorption shifts in the MeOH/THF
solvent relative to those re-dissolved in seawater (Table 2). We conclude that seawater
adequately represents the intracellular milieu for assessment of wavelength-dependent
absorptive capacity.
HPLC revealed the presence of up to four distinct MAAs identified as mycosporineglycine, shinorine, palythine, and asterina 330 (Fig. 2). Asterina 330 was absent from A.
cervicornis and M. ferox. Shinorine and palythine were absent from P. astreoides.
Mycosporine-glycine was the prevalent MAA in P. astreoides. A. cervicornis contained
three MAAs at all depths; asterina 330 was absent from all samples. P. astreoides tissue
from 1.5 m contained the highest total quantity of MAAs (71 nmol mg protein−1). At the
same depth, A. cervicornis had an intermediate total MAA concentration (25 nmol mg
protein−1), while M. ferox had the lowest amount overall (10 nmol mg protein−1). All species examined exhibited a reduction of MAA content of approximately ten fold from 1.5
m to 10 m depth, with the largest decline observed in P. astreoides. Daily UV-doses at
Table 2. Position (nm) of maximum absorbance for MeOH/THF coral extracts and for extracts
re-dissolved in seawater.
1.5 m
5m
10 m
SW
Acropora cervicornis
Porites astreoides
Mycetophyllia ferox
322
326
332
324
no discernible peak
330
320
324
332
MeOH/THF
Acropora cervicornis
Porites astreoides
Mycetophyllia ferox
332
330
336
326
320
336
320
322
338
BULLETIN OF MARINE SCIENCE, VOL. 67, NO. 2, 2000
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Figure 2. Mean concentration of four MAAs identified in A. cervicornis, M. ferox and P. astreoides
tissue from 1.5, 5 and 10 m depth. (n = 3 for each species at each depth except M. ferox at 1.5 m, n
= 2).
depth were computed using the GUV data and the mean Kd320 discussed above (0.70 m−1).
A plot of individual MAA content as a function of the average daily UV dose (Fig. 3)
reveals little variation in mycosporine-glycine content, but larger variation in the content
of other MAAs.
Horizontal colonies of M. ferox exposed to ambient light at 1.5 m contained the same
three MAAs of A. cervicornis, however only trace quantities of shinorine were identified
in vertically-oriented, partially shaded M. ferox samples (mean = 1.06 nmol mg protein−1)
from the same depth. The quantity of MAAs was significantly different between 1.5 m
samples (t-test, P = 0.001), while no differences existed between vertical, shaded 1.5 m
samples and 10 m exposed colonies (t-test, P = 0.09).
Table 3. Two-way ANOVA of the effect of species and depth on the total concentration of UV
absorbing compounds. Species: Acropora cervicornis, Mycetophyllia ferox and Porites
astreoides; depths: 1.5, 5 and 10 m.
Source
Species
Depth
Interaction
Error
Total
DF
2
2
4
18
26
SS
0.004219
0.005005
0.002663
0.000348
0.012235
MS
0.002110
0.002502
0.000666
1.93Ε−05
F
109.3109
129.6580
P
<0.01
<0.01
CORREDOR ET AL.: CARIBBEAN CORALS DEPTH UV SPECTRAL RESPONSE
827
Figure 3. MAA distribution as a function of average calculated in situ daily UV dose at 320 nm.
The mean value (n = 5) for the diffuse attenuation coefficient (Kd320) of waters of Laurel
reef was 0.70 m−1. Kd320 varied from a minimum of 0.5 m−1 to a maximum of 1.05 m−1.
Average daily surface UV irradiance (280–400 nm) during the week of sampling was
0.41 J cm−2 d−1 for all samples except for the A. cervicornis collected at 1.5 m. The average daily UV dose at the time of sampling for this specimen was 0.35 J cm−2 d−1.
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DISCUSSION
Nineteen distinct MAAs have been identified in marine organisms (Dunlap and Shick,
1998). The corals we have characterized accumulate substantial quantities of only a limited number of these compounds. Our compound identifications are based on chromatographic elution sequence and UV absorption spectra and are not definitive given the lack
of authenticated standards. Compound quantification is also tentative as molar absorptivities may vary from the published values cited above due to solvent effects. On this
basis, mycosporine-glycine, shinorine and asterina 330 are the most abundant but palythine
is also widespread and, in some instances, may be the most abundant. Two-factor analysis
of variance comparing the total MAA concentration indicates that significant differences
exist both among species and depths (Table 2). Our results thus confirm previous observations (Dunlap et al., 1986; Gleason 1993; Shick et al., 1995) regarding the inverse
relationship between MAA concentration and depth over a varied range of species further
strengthening the argument for a UV-photoprotective role of these compounds. The values we report for the green color morph of P. astreoides MAA content are significantly
lower than those reported by Gleason (1993) for the reefs of St. Croix, U.S. Virgin Islands. Similarly, values for A. cervicornis are approximately three-fold lower than those
reported for A. microphthalma; a congeneric Pacific species (Shick et al., 1995). While
these discrepancies may be due to methodological differences in compound extraction
and protein quantification, they may indeed reflect variations of physiological nature.
Dunlap et al. (1986), for example, report a tendency for seasonal changes in total MAA
content of A. formosa. Differences reported in the nature of the specific MAAs are only
tentative as we lacked authenticated standards.
Species of the genus Mycetophyllia are usually limited to deeper waters in Caribbean
reefs (Colin, 1978). Accordingly, we tested the hypothesis that M. ferox might exhibit
decreased UV photoprotection. MAA content of M. ferox was the lowest of the species
examined being approximately one tenth of that of P. astreoides across the depth range
examined. While we have demonstrated that M. ferox indeed contains MAAs and that the
content varies with depth, its capacity for accumulation of these compounds appears to be
limited.
Previous studies of reef coral photoadaptation have demonstrated significant photosynthetic response (Zvalinskii et al., 1980) and adjustment of photosynthetic pigment
content to total irradiance in the photosynthetically active range (Wethey and Porter, 1976).
We here demonstrate that three widespread Caribbean species exhibit this capacity to
varying degrees with reference to the content of UV-absorbing compounds. Our results
further suggest that reef corals may be able to adjust their MAA content not only in terms
of increasing their total UV absorption capacity but also in adjusting UV-absorbing compound suites to spectral quality of UV irradiation. Inhibition of diverse physiological
functions in both plants and animals (Caldwell, 1977; Häder et al., 1995), including photosynthesis by corals (Lesser and Lewis, 1996) becomes apparent at about 320 nm and
increases notably at lower wavelengths. Seawater selectively absorbs shorter wavelengths
in such a manner that radiation at 320 nm can be attenuated 50 to 100 fold over a span of
only 14.5 m (Baker and Smith, 1981). In order to overcome the deleterious effects of the
shorter wavelength radiation close to the surface, photochromatic adaptation might be
expected to cause shifts in the absorptivity of photoprotective compounds in this direction. Our results support this contention between species. A. cervicornis, and P. astreoides
CORREDOR ET AL.: CARIBBEAN CORALS DEPTH UV SPECTRAL RESPONSE
829
exhibited hypsochromic displacement in their UV-absorption spectra relative to M. ferox,
denoting greater capacity for absorption of the shorter, more energetic, and thus more
harmful radiation. Individual species do not, however exhibit substantial wavelength
maximum shifts with depth. As expected, those species with a wide depth range exhibited
a large range of variation in MAA composition. In contrast, M. ferox, a stenobathic species, exhibits the lowest capacity for photoadaptation with restricted MAA suite composition.
ACKNOWLEDGMENTS
This work was supported by NASA grant #NCCW 0088. We acknowledge comments by M.
Shick and an anonymous reviewer that contributed substantially to the quality of the manuscript.
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DATE SUBMITTED: February 25, 1999.
DATE ACCEPTED: May 12, 2000.
ADDRESS: Department of Marine Sciences, P. O. Box 908, Lajas, Puerto Rico 00667-0908.