Arch. Hydrobiol.
144
3
255-269
Stuttgart, February 1999
UV-absorbing mycosporine-like
compounds
planktonic and benthic organisms from a
high-mountain lake
Ruben-Sommaruga’
in
* and Ferran Garcia-Piche12
With 7 figures in the text
Abstract: We investigated the occurrence, concentration and composition of mycosporines (mycosporine-like amino acids, MAAs) in planktonic organisms and epilithic
cyanobacteria from a clear water high-mountain lake situated in the Central Alps,
Austria. Two bi-substituted MAAs were identified by HPLC in extracts made of 1996
plankton samples with 90 % aqueous methanol, i.e. asterina-330 (&= = 330 nm) and
shinorine (km = 334 run). Extracts with 20 % aqueous methanol for 2 h at 45 “C
revealed the additional presence of another MAA tentatively identified as palythine
(&.,, = 320nm) in the 1998 planktonic assemblage. In the upper 3 m of the water column the total concentration of MAAs decreased exponentially with depth, but the
maxima for both absolute and chlorophyll-a specific concentrations were. observed
close to the bottom at 8.5 m depth. This was explained by the accumulation of MAAs
in the copepod’cyclops abyssorum tatricus that stays in deep water, during daytime.
The copepodite III stage contained the 3 MAAs found in phytoplankton but also the
mono-substituted compound, mycosporine-glycine (mycosporine-gly: & = 310nm).
The concentration of MAAs in C. abyssorum tatricus was highest for shinorine
(1.45% of the dry weight) and lowest for mycosporine-gly (0.02 % of the dry weight).
Epilithic cyanobacteria had a more diverse MAA spectrum than plankton, and produced not only asterina-330 and shinorine but also palythinol (&, = 332 nm), mycosporine-gly and two unidentified compounds with &,,, = 330 and 340nm. The
composition and also the relative abundance of the cyanobacterial MAAs changed
with depth. Mycosporine-gly was found at the lakeshore where Gloeocapsa sp. dominates, but it was absent at 0.5 and 2.5 m depth dominated by Schizothrix sp. and Tolipothrix sp., respectively. We could not detect any MAAs in the cysts of the red snow
alga Chlamydomonas nivalis, which develops on top of the winter cover shortly be-
’ Authors’ addresses: University of Innsbruck, InstitutGof Zoology and Limnology,
Technikerstr. 25, A-6020 Innsbruck, Austria. E-mail: [email protected].
2 Max-Planck Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany.
* Author for correspondence.
0003-9136/99/0144-0255 $3.75
0 1999 E. Schweizerbart’sche
Verlagsbuchhandlung,
D-70176 Stuttgart
256
Ruben Sommaruga and Ferran Garcia-Pichel
fore ice-melt. These results expand to alpine lakes the range of ecosystems in which
these compounds may play a significant biological role.
Introduction
Alpine lakes experience high UV irradiances due to the natural increase of UV
radiation with elevation. In the Alps, for example, solar UV-B (290-320nm)
radiation was found to increase by about 20 % per 1,000 m of elevation
(BLUMTHALER et al. 1993). Therefore, high altitude lakes receive considerably
more UV-B radiation than lowland lakes. Additionally, lakes situated above
the tree line generally have a very high water transparency for UV due to their
low concentration of chromophoric or coloured dissolved organic matter
(MORRIS et al. 1995, SOMMARUGA & P~ENNER 1997). Consequently, highly
transparent lakes deserve special attention, particularly considering a scenario
of increasing UV-B fluxes causedby stratospheric ozone depletion.
Organisms living in such ecosystems, however, must have developed
adaptive mechanisms to cope with high-UV h-radiances.One possible strategy
is the synthesis of UV-absorbing compounds that may act as natural sunscreens. One group of compounds, most commonly known as mycosporines,
mycosporine-like amino acids or MAAs, is widely distributed in marine organisms (KARENTZ et al. 1991) and is also present in terrestrial microorganisms like fungi (FAVRE-BONVIN et al. 1987),cyanobacteria (GARCIA-PICHEL &
CASTENHOLZ 1993) and lichens (B~~DEL et al. 1997).However, almost nothing
is known about their occurrence, concentration and composition in freshwater
organisms.
MAAs are water-soluble, low molecular weight compounds having high
molar extinction coefficients and absorption maxima ranging from 310 to
360nm. Chemically, they consist of a cyclohexenone or cyclohexenimine substituted with amino compounds (amino, amine, amino acid, amino alcohol) in
carbon 1 (mono-substituted) or in carbons 1 and 3 (bi-substituted). Approximately 17 aminocyclohexenimine and 15 aminocyclohexenone derivatives
have been identified in different taxa of marine and terrestrial organisms
(KARENTZ et al. 1991, GARCIA-PICHEL & CASTENHOLZ 1993, BANDARANAYAKE et al. 1997,DUNLAP & SHICK 1998). Laboratory studies have demonstrated that MAAs can play a moderate sunscreenrole in cyanobacteria (GARCIA-PICHEL & CASTENHOLZ 1993) and metazoans (ADAMS & SCHICK 1996).
The frequently observed correlation between their concentration and solar exposure found in nature, suggeststhat they play an important role as UV sunscreens in general. In addition, MAAs may function as moderate antioxidants
(DUNLAP & YAMAMOTO 1995) and may be related to reproductive processes
(BANDARANAYAKE et al. 1997). A biosynthetic origin for mycosporines
Mycosporine-like compounds
257
through the early shikimic acid pathway has been suggestedfor fungi (FAVREBONVIN et al. 1987).Whereas this pathway is also found in algae and bacteria,
it is lacking in invertebrates and vertebrates (HERMAN 1983). Marine metazoans, however, were found to obtain MAAs either from their food or in
symbiotic association with unicellular autotrophs (SHICK et al. 1991, BANASZAK & TRENCH 1995, CARROL & SHICK 1996).
MAA composition can vary widely within large taxonomic groups, but it
seems to -be conserved within lower phylogenetic clades. Thus, for example
cyanobacteria, may contain cs many as 13 different MAAs (GARCIA-PICHEL et
al. 1993) but closely related clusters of a strain show considerable resemblance
in MAA complement (KARSTEN & GARCIA-PICHEL 1996, GARCIA-PICHEL et
al. 1998). In the marine dinoflagellate Prorocentrum micans, porphyra-334,
mycosporine-glycine, shinorine and asterina-330 were found (LESSER 1996),
whereas the red-tide dinoflagellate Lingulodinium polyedra, contained porphyra-334, mycosporine-glycine : valine, Galythine, palythinol and palythene
(VERNET & WHITEHEAD 1996). In another red-tide dinoflagellate Alexandrium
excavatum, porphyra-334, mycosporine-glycine, palythine, asterina-330, shinorine, palythenic acid and the isomeric mixtures of usujirene and palythene
were found (CARRETO et al. 1990).
Here we report for the first time the occurrence, concentration and composition of MAAs in planktonic organisms and benthic cyanobacteria of a
transparent high-mountain lake in the Alps.
Materials and methods
The study was carried out during summer1996in Gossenkiillesee,
situatedabovethe
tree line at 2,417m above sea level in the Central Alps, Tyrol, Austria (47” 13’ N,
11”Ol’E). The lake (area: 1.7ha, maximum depth: 9.9m) has a high UV transparency
with 10% of the surfaceW-B (nominal h: 305nm) and 33 % of the UV-A radiation
(nominalI.: 34Onm),asmeasuredwith a PWSOOunderwaterradiometer,reachingthe
bottom after ice break-up(SOMMARUGA& PSENNER1997).A field stationon the shore
facilitatedthe laboratorywork. Additional samplingwas carried out in the early 1998
summer.
For the analysisof MAAs in planktonsamples,2 to 3 liters of water were collected
aroundnoon at 0, 1.5, 3, 5 and 8.5m depthwith an opaquesamplerand immediately
filtered througha WhatmanGF/F filter. The filters were frozen at -20 “C until extraction within 1 week. Samplesof epilithic cyanobacteriawere collectedbetween0 and
2.5m depth.bySCUBA diving following the zonationdescribedin ROTT & PERNEGGER (1994).The only copepod present in this lake, CycZopS
abyssorumtat&us, was
collectedduringdaytimefrom 8.5m depthwith a 5 liter samplerandconcentratedon a
net sieve of 100pm mesh size. The sieve was washedseveraltimes with filtered
(co.45 pm) lake water to remove phytoplanktonand 160individualswere carefully
258
Ruben Sommaruga and Ferran Garcia-Pichel
picked-upandplacedon a wet WhatmanGF/F filter kept on an ice-pack.The biomass
of the copepoditeswas estimatedbasedon the relationshipbetweenlength and dry
weight calculatedfor this speciesby PRAPTOKARDIYO (1979).Cysts of the red snow
alga Chlumydomonas nivulis were collecteddirectly from the snow in late spring.
After melting at 4 “C, the samplewas filtered onto a WhatmanGF/F filter.
In most casesthe extractionof MAAs was donewith 90 % aqueousmethanolfor
24h in the dark at 4 “C. Occasionally,extractionswith 20% aqueousmethanolat 45 “C
for 2h were alsoperformed(GARCIA-PICHEL & CASTENHOLZ 1993).Thenthe samples
were b&fly sonicated(2 min) on an ice bath and the extractsclearedusing a 0.1pm
poresizeWhatmanAnodiscfilter. The extractswere scannedbetween250 and750nm
in a doublebeam Hitachi U-2000 spectrophotometerusing quartz cuvettesof 5 cm
pathlengthagainsta 90 or 20 % aqueousmethanolreference.Then the extractswere
evaporateduntil drynessunder vacuum using a SpeedVacconcentrator(Savant)at
45 “C. The sampleswere storedat -80 “C for further characterizationusinghigh performanceliquid chromatography(HPLC). For separationof MAAs, the concentrated
extracts were resuspendedin 100-200 pl of 20 % methanoland 20-100 pl aliquots
were injected in a Hypersil 5 urn pore size C is column for isocratic reverse-phase
HPLC analysis(GARCIA-PICHEL & CASTENHOLZ 1993).The mobile phasewas 25%
aqueousmethanolplus 0.2 % acetic acid. The MAAs in the eluatewere detectedby
onlineUV absorptionspectroscopy(220-450 nm, peakmeasurementwas carriedout
at 320nm). The MAAs were identified by co-chromatographywith authenticatedprimary standards(obtainedfrom Dr. DENEB KARENTZ and originally preparedby Dr.
WALTER DUNLAP) or with secondarystandardspreparedfrom cyanobacterialcultures.
The concentrationof MAAs was quantifiedby UV spectroscopyusing publishedmolar extinctioncoefficients(ITO & HIRATA 1977,TAKANO et al. 1978,CHIOCCARA et al.
1980).Since mycosporine-glycineis unstable,the molar extinction coefficient used
was that for the stablemethyl esterform. The extinction coefficient for asterina-330
has not beenreported,consequentlywe used a genericcoefficient of 1201g-’ cm-’
(GARCIA-PICHEL 1994).Concentrationsof the different MAAs were normalizedto the
concentrationof chlorophyll-a(Chl-a), which was analyzedin parallel samplesafter
the sameextractionprocedurebut using acetoneas solvent.The equationsof JEFFREY
& HUMPHREY (1975)were usedto calculatethe concentrationof pigments.
Particleabsorption(a,) measurementsof parallel plankton samples(zooplankton
excluded)were madeby the quantitativefilter techniquewith pathlengthcorrections
(MITCHELL 1990).WhatmanGF/F filters from a singlebatch were usedfor all measurements.The sampleswere kept at 4 “C in the dark until analysiswithin 24h. The absorbancewas measuredin the samespectrophotometer
describedabovewith a wetted
GF/F filter as a reference.A smoothingfunction (Savitsky-Golay)was appliedto the
scanobtained.Pigment-specificparticle absorption(a*,) was calculateddividing at,by
the concentrationof chlorophyll-aplus phaeopigment.
Results and discussion
Fig. 1 shows the characteristic absorbanceof MAAs in plankton samples ex,tracted with 90 % aqueous methanol. Two MAAs, shinorine (La = 334 nm)
Mycosporine-like compounds
--
:1
259
Om
1.z. :‘3*5,”
......... .. 5m
--8.5 m
300
400
500
Wavelength
600
700
(nm)
Fig. 1. Exampleof absorptionspectraof aqueousmethanol(90 %) extractsof plankton
for different depthsin the water column of Gossenkijllesee
on 22 July 1996.Samples
collectedat noonon a sunnyday.
and aster-ma-330(&,= = 330 nm) were found in 1996 plankton samples extracted with aqueousmethanol 90 %. From the surface to 3 m depth both absolute and chlorophyll-a specific concentrations of asterina-330 were higher than
that of shinorine, while at 5 and 8.5 m depth, concentrations of sbinorine were
higher (Fig. 2 b, c). Total concentrations of MAAs ranged from 21.6 to
139.4ngl-’ or from 0.015 to 0.053 ug ug-’ Chl-a (Fig. 2d). In the upper 3 m,
absolute concentrations of MAAs decreasedexponentially with depth, but this
pattern was reversed at 5 m depth and the highest total MAA concentration
was found at 8.5 m depth. Although the chlorophyll-a concentration was
highest at 8.5 m depth (Fig. 2 a), this pattern was also observed when the concentration of MAAs was normalized to the chlorophyll-a content.
The summer phytoplankton of this lake is dominated by nanoplanktonic
forms, mainly dinoflagellates such as Woloszynskiusp., Gymnodinium uberrimum, G. cnecoides, Gymnodinium sp., Amphidinium elenkinii, the chrysophytes Ochromonas sp. and Chromulina sp., and the diatoms Fragilaria deli-
260
Ruben Sommaruga and Ferran Garcia-Pichel
0
1
2
Asterina-330
Shinorine
Chlorophyll-a
(iJs i-7
(w w-’
3
0.00
ChW
0.02
bs id Chl-a)
0.04 f
0.00
0.02
Tot MAAs
W
0
0.00
id Chl-4
0.02 0.04 0
2-
4-
6
6-
8
0-
0
20
40
Shinorine
60
80
loo
(ng I”)
102030405080
Asterina-330
0
(ng I-‘)
40
00
Tot MAAs
120
(ng
160
I”)
Fig. 2. Depth distribution of chlorophyll-a (A), and absolute (0) and normalized (0)
concentrations of shinorine (B), asterina-330 (C) and total MAAs (D) in plankton
samples collected in the water column of Gossenkiillesee on 22 July 1996 at noon.
catissima, Cyclotella aff. gordonensis and C. distinguenda var. unipunctata
(NAUWERCK 1966, HALAC et al. 1997). A deep maximum of chlorophyll-a is
typical for transparent alpine lakes during summer (PECHLANER 1971). How-
ever, we expected to find a decreasein MAAs concentration with depth if they
were to play a role as sunscreens.In contrast to the pattern described above,
our particle absorption measurements from parallel samples, where only
phytoplankton was concentrated, indicated a higher pigment-specific particle
absorption (a*,) at 334nm at the surface and a decreasewith depth (Fig. 3).
This discrepancy prompted us to search for other possible sources of MAAs in
the plankton that may have masked the expected pattern.
The copepod Cyclops abyssorum tatricus characteristically found in deep
water layers during daytime (EPPACHER 1968) was a potential candidate to explain the maximum MAA concentration found at 8.5 m depth. Although at present, there is no evidence that invertebrates can synthesize MAAs de novo,
they can accumulate them through the diet (CARROLL & SHICK 1996). In addition, bacteria present in the gut may be an additional source of these compounds (ARAI et al. 1992). As expected, the methanolic extracts of C. abyssorum tatricus (copedodite stage III) clearly showed a very high absorption at
334nm that even exceeded the carotenoid peak at 474nm (Fig. 4). The very
low absorption at the red chlorophyll-a peak (665 nm) indicated that the occurrence of MAAs in this specieswas not associatedwith an occasional presence
of phytoplankton in their gut. Analysis of the extract by HPLC revealed the
Mycosporine-like compounds
261
0.00
300
400
600
500
Wavelength
700
(nm)
Fig. 3. Pigment-specificspectralabsorption(a*,,) of particles at different depthsin
Gossenktillesee
on 22 July 1996.
0.7
-+--
0.6
0.5
i!!
f
0.4
g
0.3
e
2
\
0.2
0.1
0.0
,.“.,.,.‘I.“‘.“‘,I’..“‘,‘.I”,“‘.*’1”.I
400
300
500
Wavelength
600
700
(nm)
Fig. 4. Methanolicextract (90 %) of the copepodCyclops ubyssowm tutricus showing
the peakof the carotenoidabsorptionmaximumat 474nm and of UV-absorbingcompoundsat 334nm. The inset shows the chemical structureof the mycosporine-like
aminoacidshinorine,the most abundantcompoundin this species.
262
Ruben Sommaruga and Ferran Garcia-Pichel
presence of shinorine, asterina-330, mycosporine-glycine (&.,, = 310nm) and
another MAA tentatively identified as palythine (&, = 320nm). The highest
MAA concentration was found for shinorine (0.046pg ind-i or 1.45 % of the
dry weight) followed by palythine (0.02Op.gind-’ or 0.63 % of the dry weight),
asterina-330 (0.008 pg ind-’ or 0.24 % of the dry weight) and mycosporine-glytine (0.0005 pg ind-’ or 0.02 % of the dry weight). To our knowledge, the work
of KARENTZ et al. (1991)is the only one that allows comparison of MAAs with
another’copepod species, Calanus propinquus. In this marine speciesthe peak
of absorption was found at 328 nm and the MAAs identified included palythine, porphyra-334, shin&-me, palythene and mycosporine-glycine; the last
being the most abundant. The distinct MAA composition between these species is likely to be a reflection of dietary differences. The total concentration of
MAAs in C. propinquus, however, was -16 times lower than in C. abyssorum
tatricus (0.00146 and 0.0234 kg p.g-’ dry weight, respectively).
An important difference between the lvlAA composition of the phytoplankton and C. abyssorum tatricus was the unique presence of mycosporine-glytine in the latter. As discussedabove, de novo synthesis of MAAs is not known
in metazoa, thus the presenceof mycosporine-glycine in this copepod may indicate a transformation from another MAA or another different source for this
compound like bacteria. Recently, DUNLAP & SHICK (1998) reported that shinorine can be converted into mycosporine-glycine by bacteria, and suggested
that this type of transformation may occur in the gut of a seaurchin species.The
hydrolytic conversion of some bi-substituted to mono-substituted MAAs occurs
also under mildly acidic conditions (PORTWICH & GARCIA-PICHEL unpubl.).
Although in Gossenkollesee, the adult stage of C. abyssorum tatricus is
never exposedto a maximal UV dose becauseit avoids shallow waters at midday, GLIWICZ (1986) reported its occurrence also in shallow ponds. The accumulation of these compounds by the female adults of C. abyssorum tatricus
may be an important adaptive strategy for the survival of the first life stages
that thrive close to the surface. In the Antarctic limpet Nacella concinna, concentrations of MAAs were.higher in ovary and eggs than in the gut, testis or in
the body tissue (KARENTZ et al. 1992). Furthermore, ADAMS & SHICK (1996)
demonstrated that MAAs play a photoprotective role in eggs of the sea urchin
Strongylocentrotus droebachiensis. These authors found an inverse relationship between the UV-induced cleavage delay of the embryos ‘and the MAA
concentration (mostly shinorine). Future studies should determine the concentration of MAAs in the different stagesof C. abyssorum tatricus and their photoprotective capacity in eggs of this species.
Another surprising result of our study was the low concentration of MAAs
found in the plankton samples (Fig. 2). For example, total MAAs concentration in natural phytoplankton assemblagesfrom Antarctica ranged from -0.36
to 1.67pg I.tg-’ Chl-a during a two-week exposure to UV radiation + photosyn-
Mycosporine-like compounds
400
*
500
Wavelength
263
600
(nm)
Fig. 5. Absorbancefrom extracts made with 90 % and 20 % aqueousmethanolin
parallelplanktonsamplescollectedfrom 8m depth(seetext for the methods).
thetically active radiation (VILLAFA~E et al. 1995). In our case, total MAA
concentrations in the water column were only between 0.015 to 0.053 ug ug-’
Chl-a. The low concentration was coincident with the relatively low absorption at the MAAs peak when compared, for example, with the maximum red
absorption of Chl-a at 665 nm (Fig. 1). However, the pigment-specific absorption of particles (a*,) indicated again an opposite pattern (Fig. 3). Moreover,
in several occasionsno distinct MAAs peak was observed with our routine extraction procedure (90 % aqueousmethanol). One possible explanation for this
discrepancy is the low extraction efficiency of MAAs in phytoplankton samples with 90 % aqueous methanol. Extractions in parallel with 20 and 90 %
aqueous methanol indicated that the efficiency of the latter was much lower
(Fig. 5). The HPLC analysis of the 20 % extract gave concentrations of MAAs
ranging between 0.30 and 0.69 pg pg-’ Chl-a. Our results are preliminary, but
we have serious grounds to caution against the use of high methanol concentrations for MAAs extraction in phytoplankton.
The methanolic extracts of the cysts of the red snow alga Chlumydomonas
nivalis had no peak in the UV range suggesting the absenceof MAAs, at least
in measurableamounts (data not shown). THOMAS & DUVAL (1995) also found
no peak or just a modest one in methanolic extracts of the same speciescollected from Sierra Nevada, USA. As suggestedby several authors, the extraordinary concentration of secondary carotenoids, in particular astaxanthin and its
esters, in snow algae plays an important role as photoprotective compounds
(BIDIGARJZ et al. 1993, KOBAYASHI et al. 1997,MILLER et al. 1998). Indeed the
264
Ruben Sommaruga and Ferran Garcia-Pichel
300
400
600
500
Wavelength
700
(nm)
Fig. 6. qpical spectraof aqueousmethanolicextracts(90%) from epilithic cyanobacteria collectedat different depthsin Gossenkiillesee.
sunscreenfactor provided by carotenoids in these cysts is among the highest
measuredfor microorganisms (GARCIA-PICHEL 1994).
Benthic cyanobacteria had more diverse MAAs than plankton, including
not only shinorine and asterina-330, but also palythinol (&, = 332nm), mycosporine-glycine (&, = 310nm), and two unidentified W-absorbing compounds with absorption maximum at 330 and 340 nm. A typical spectrum of
the methanolic extracts from benthic cyanobacteria collected at different
depths is shown in Fig. 6. The composition and relative concentration of
MAAs differed depending on the depth zones characterized by the dominant
cyanobacteria (Fig. 7). The samples obtained from the lakeshore in the zone
defined by ROTT & PERNEGGER (1994) as dominated by Gloeocupsa
sp. was
characterized by the absence of shinorine and the presence of mycosporineglycine, a compound not found in the other zones. The highest concentration
was found for an unknown compound with absorption maximum at 330nm
(Fig. 7 a). The samples collected at 0.5 and 2.5 m depth, characterized by the
Mycosporine-like compounds
B
265
60,
70-
c
60 50 40 30 20 -
Fig.7. Relativeconcentrationsof different MAAs in bentbiccyanobacteriacollectedat
the lakeshore(A), 0.5m depth(B) and2.5m depth(C) in Gossenk8llesee.
dominance of Schizothrix sp. and Tolipothrix sp., respectively, presented the
same qualitative composition with palythinol as the most abundant MAA (Fig.
7b,c).
The fact that mycosporine-glycine was only found in ,epilithic cyanobacteria exposed to high solar UV radiation is interesting because besides its absorption in the UV-B range (&, = 310nm), the shortest for any MAA, this
266
Ruben Sommaruga and Ferran Garcia-Pichel
compound has also an antioxidant function (DUNLAP & YAMAMOTO 1995) thus
providing protection against photooxidative stress.
In conclusion, the present study reports for the first time the presence and
characterization of MAAs in different organisms from a freshwater lake.
These results expand to alpine lakes the range of ecosystems in which these
compounds may play a significant biological role. Further investigations
should addressseveral open questions, for instance, which factors regulate the
synthesis of these substances,particularly the ability of different wavelengths
in the solar spectrum to induce MAAs synthesis, and their potential for UV attenuation in the water column (SOMMARUGA & PSENNER 1997).
Acknowledgements
We thank LEO F~~REDER and HARALD PEHO~R for SCUBA diving in Gossenkiillesee,
RAMONA APPLE for help with the HPLC analyses, BARBARA TARTAROTTI for separating the copepodites and estimating the dry weight, DENEB KARENTZ for supplying the
primary standards, and MALCOLM SHICK, ROLAND PSENNER and two anonymous reviewers for improving the clarity of a previous version of this manuscript. This work
was supported by a project from the Austrian Science Foundation (FWF, P 11856-BIO).
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Submitted: 15 July 1998; accepted: 13 October 1998.