2.7 Characterization of the underwater UV climate in lakes from the
Mt. Everest Region and assessment of DNA damage levels and
counteractive mechanisms (photorepair and photoprotection) in
planktonic organisms
University of Innsbruck, Institute of Zoology & Limnology, Laboratory of Aquatic
Photobiology and Plankton Ecology
Principal Investigator: Dr. Ruben Sommaruga
State of the art
Solar ultraviolet-B radiation (290-320 nm; UVB) has increased during the last 15 years
over many locations of the Earth because of the degradation of the stratospheric ozone
layer (Anderson et al., 1991). Beside Antarctica, where the increment is particularly
notorious, some studies have demonstrated that a moderate increase in UVB also
occurred in the northern hemisphere, for example, in northern America (Kerr & McElroy,
1993), and in the Swiss Alps (Blumthaler & Ambach, 1990). Because of the scientific and
public concern about the potential increase in UVB fluxes, the assessment of the
potential negative effects of solar UVB radiation on aquatic and terrestrial ecosystems
has received considerable attention during recent years. In addition, the problem of the
stratospheric ozone degradation has focused the attention of ecologists on several
biological and chemical processes in which solar UV radiation plays a significant role, as
well as on the different strategies and mechanisms that organisms have evolved to avoid
UV radiation, to protect themselves, and to repair potential damage.
Considering the ecological consequences of enhanced UVB radiation levels requires the
identification of potentially vulnerable aquatic ecosystems. Alpine lakes experience high
instantaneous UV fluxes due to the natural increase of solar UV radiation (UVR, 280-400
nm) with elevation. In the Alps, for example, solar UVBR increases by about 20% per
1000 m of elevation (Blumthaler et al., 1993). Consequently, high altitude mountain
lakes receive considerably more UVBR than lowland lakes. Lakes situated above the tree
line (except those influenced by glaciers) also have generally a very high water
transparency for UV due to their low concentration of chromophoric or colored dissolved
organic matter (Morris et al., 1995; Sommaruga & Psenner, 1997, Laurion et al. 2000).
Therefore, highly transparent lakes deserve special attention, particularly, considering a
scenario of increasing UVB fluxes caused by stratospheric ozone depletion.
Organisms living in such ecosystems, however, have developed adaptive mechanisms to
cope with high incident UVR. Nearly all groups of organisms have behaviors, habitat
selection or natural morphological features that mitigate the exposure of important
cellular targets to UV radiation. For example, it is known that in alpine lakes copepods
and flagellated phytoplankton, move away from the water surface at midday when
sunlight irradiances are strongest (Tilzer, 1973; Halac et al., 1999; Tartarotti et al.,
1999). Such an avoidance strategy may not be feasible in cases where the waterbody is
relatively shallow and the water is highly transparent to UVR. Strong vertical mixing of
the water column driven by wind-induced turbulence is an additional factor that may
lower the effectiveness of the avoidance strategy. Other organisms are protected by
living under stones or buried into the sediment. However, many species are either
continuously exposed to high solar UV intensities (e.g. benthic cyanobacteria at the
lakeshore), or intermittently, depending on the mixing conditions of the water column,
(e.g. planktonic species).
Ultraviolet radiation exerts negative effects either by direct damage to DNA, proteins,
pigments and lipids, or indirectly through the generation of reactive oxygen species
(Vincent & Roy, 1993). While UVB mainly causes damage by direct absorption, UVA (320400 nm) and also wavelengths in the visible range (400-700 nm, PAR) are more effective
in exerting indirect damage, although the final mechanism will depend on the
absorption characteristics of the chromophores. In any case, the synthesis or
accumulation of photoprotective compounds that intercept wavelengths in the UV range
(sunscreens) and dissipate the energy as heat without the formation of intermediate
radicals would be beneficial for organisms exposed to high UVR. Several photoprotective
compounds are known to absorb directly or indirectly the solar UV energy in aquatic
organisms
The occurrence of the yellow-brown pigment known as scytonemin is restricted to some
species of cyanobacteria (Nägeli, 1849; Garcia-Pichel & Castenholz, 1991) where it is
located in the extracellular polysaccharide sheath. Melanin is mainly found in the skin of
aquatic vertebrates and in the cuticle of cladocerans crustaceans (Hebert & Emery,
1990), while the putative protective compound sporopollenin has been detected in the
cell wall of some chlorophytes (Xiong et al., 1996). All these three photoprotective
compounds act as direct filters of UVR. On the other hand, the photoprotective action of
carotenoids is mainly related to their antioxidant properties (quenching of reactive
oxygen species, Bidigare et al., 1993). Primary or secondary carotenoids are found
mainly in phototrophic organisms but also in several planktonic crustaceans species
(Brehm, 1938; Hairston, 1976) and bacteria (Mathews & Sistrom, 1959).
The last group of photoprotective agents includes the UV-absorbing compounds known
as mycosporine-like amino acids (MAAs). These compounds are widely distributed
among marine organisms (Karentz et al., 1991; Dunlap & Shick, 1998). Information
about the occurrence of mycosporine-like compounds in freshwater organisms is still
scarce. These compounds have been reported recently for natural phytoplankton
assemblages (Sommaruga & Psenner, 1997; Sommaruga & Garcia-Pichel, 1999), benthic
cyanobacteria (Sommaruga & Garcia-Pichel, 1999), microalgae (Laurion et al. 2002),
mixotrophic ciliates (Sommaruga unpub.), and cyclopoid and calanoid copepods
(Sommaruga & Garcia-Pichel, 1999, Tartarotti et al. 2002).
Interestingly, these
compounds have not been found until now in Cladocera (including non-melanized
forms, Tartarotti et al. 2002).
The last line of defense against UVR exposure consists in repairing the damage after it
has occurred. By far, the most common type of DNA damage induced by UVC and UVB is
the formation of cyclobutyl pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone
photoproducts. These lesions block DNA replication and transcription leading to
mutagenic or lethal effects. Living organisms have evolved a specific mechanism for the
repair of pyrimidine dimers, called photoreactivation. This mechanism involves a single
enzyme (photolyase), which directly reverses the pyrimidine dimers into the two
pyrimidine nucleotides by using the energy of near ultraviolet and visible light
(Friedberg et al. 1995).
Photolyase activity is widely distributed in nature and
photoreactivation has been found in members of all kingdoms of life. Nevertheless, this
mechanism is absent in many species (Sancar 1994)
Recent investigations have shown that not all 3 main strategies to minimize negative
effects from UV radiation are feasible or work efficiently. For example, as discussed
above planktonic organisms from shallow and transparent lakes, will be still exposed to
significant UV fluxes in deeper layers. In fact, availability of depth refugia (1% UV
penetration depth:maximum depth is the main variable explaining the variability in
MAAs concentration in populations of the copepod Cyclops abyssorum (Tartarotti et al.
2002). Investigations in shallow and transparent water bodies in Antarctica, also suggest
that in cold lakes the main mechanism to repair DNA damage (i.e., photorepair) is
significantly reduced at low temperatures (Rocco et al. 2002). Thus, synthesis and
accumulation of photoprotective compounds might be the first line of defense in such
aquatic ecosystems. This type of strategy may be particularly efficient when visual
predators (e.g. fish) are absent as in natural remote lakes.
Rationale to investigate the high-altitude lakes in the Mt. Everest region
Although the “altitude effect” (i.e., the increase of UV radiation fluxes with altitude) is
variable for different mountain regions (Sommaruga 2001) and has not been established
for the Himalaya chain, assuming a conservative factor of 10% increase per 1000 m,
imply that organisms living in lakes from the Mt. Everest region experience extreme
high UV fluxes during the ice free season. In addition, preliminary measurements of
DOC concentration (Bertoni et al. 1998) in some of those lakes suggest that they are
very transparent to UV radiation. Finally, although phytoplankton species found in those
lakes seems to be cosmopolitan (Manca et al. 1998), several zooplankton species are
endemic of that region (Löffler 1968, Manca et al. 1994, 1998) and thus represent a
unique opportunity to understand their autoecology, as well as to assess the strategies
to minimize damage from UV exposure.
Objectives and hypotheses
The present proposal have as main objectives: 1) to evaluate the UV underwater climate
in the same lakes selected by the Pyramid Limno Group (PLG, directed by Dr. Lami), in
relation to their DOC content and absorption characteristics of the dissolved organic
matter (i.e., the two main factors controlling the attenuation of UV underwater), 2) to
assess the concentration and composition (only for MAAs) of photoprotective
compounds in planktonic organisms (phytoplankton as source of these compounds and
zooplankton) in the water column in some selected transparent lakes that offer different
depth refugia (Cadastre # 9, 10, 66) having different maximum depth (Tartari et al.
1998b), 3) to establish the natural levels of DNA damage in representative zooplankton
species of these 3 lakes, and 4) to test the efficiency of photorepair in those organisms.
The main hypothesis to test is whether photoprotection (carotenoids and MAAs)
represents the main strategy to minimize DNA damage in zooplankton from UVtransparent and cold lakes of the Himalaya region.
To my best knowledge, parallel assessment of the parameters indicated in objectives 2,
3, and 4 has never been done before for other lakes/organisms and thus will represent
the first study to assess natural DNA damage levels of zooplankton in relation to the
main strategies to minimize it. In particular, the planned measurements in lakes having
different maximum depth will give an excellent chance to test this hypothesis in lakes
having different depth refugia and for species having different types of photoprotective
compounds (e.g., melanized forms in Daphnia tibetana but not in D. longispina, (Manca
et al. 1994). In these three lakes, similar zooplankton community structure is found
(Tartari et al. 1998b).
Characterization of the underwater UV climate (objective #1) will not only provide a
basis to assess the degree of UV stress (i.e., UV penetration in relation to max depth) in
those lakes, but also will complement the activities of the PLG providing information
necessary for potential reconstruction of historical UV climate in those ecosystems.
Work plan, methodology, and timetable
Work plan
Field work will be done during September-October (post-monsoon period) together with
the PLG. Although these are not the months with the highest incident solar radiation,
they are characterized by less rain and clear sky conditions as indicated by the higher
clearness index of these months compared to the rainy season (Tartari et al. 1998a).
Measurements of UV penetration, natural fluorescence, DOC concentration and CDOM
absorption will be done in the same lakes selected by the PLG (see proposal by Dr.
Lami). Samples for Chlorophyll-a and MAAs in seston estimations will be collected in the
3 selected lakes at 3 discrete depths (surface, middle, and bottom) with a water sampler.
Zooplankton will be collected by vertical tows using a 55-µm-mesh plankton net.
Arctodiaptomus jurisvitchii and cladoceran species will be narcotized and counted and
sorted under a dissecting microscope. A given number of individuals will be stored in
triplicate at -20 °C and liquid N for MAA analysis, carotenoids, DNA damage, and
photolyase activity determinations (see below).
Materials and Methods
UV underwater measurements. Profiling of the water column will be performed with a
PUV-501 submersible radiometer from Biospherical Instrument (San Diego, CA). This
instrument measures the downwelling radiation at four nominal wavelengths (305, 320,
340 and 380 nm) with a full width half maximum waveband of  10 nm, and the depth
(cm resolution) by means of a pressure transducer. The instrument also measures PAR
(400-700 nm) and natural fluorescence. The attenuation coefficient (Kd) for UV
wavelengths and PAR will be calculated from the slope of the linear regression of the
natural logarithm of downwelling irradiance versus depth.
Dissolved organic carbon (DOC) concentration. Subsamples for DOC analysis will be
filtered on the same day through a previously combusted (2 h at 475°C) GF/F filter
(Whatman) placed on a stainless steel syringe holder (Poretics, 25 mm diameter) and
collected in combusted (same as above) brown glass vials with Teflon-faced screw caps
(Supelco). Samples will be preserved with HCl p.p.a and stored at 4 oC until further
analysis at Innsbruck (Laurion et al. 2000). Dissolved organic carbon will be measured
(direct method) with a high temperature catalytic oxidation method using a Shimadzu
TOC Analyzer Model 5000. The instrument is equipped with a Shimadzu platinizedquartz catalyst for high-sensitivity analysis. Blank analyses using Milli-Q water yield
usually DOC values of between 0.02 and 0.04 mg L -1. Controls will be done also with the
water available at the pyramid laboratory.
Absorption spectra of DOM. The filtrates (same as above) will be stored in the same type
of vials but will be only frozen. Absorption measurements between 250 and 750 nm will
be done in Innsbruck using a Hitachi U-2000 double beam spectrophotometer and 10cm quartz cuvettes. These measurements will be referenced against GF/F-filtered, Milli-Q
water.
MAAs analysis. For the analysis of MAAs in seston (metazooplankton will be prescreened with a net sieve of 85 µm mesh), 1-2 L of water (depending on cell density) will
be filtered at the Pyramid laboratory onto Whatman GF/F filters (25 mm diameter). A
known number of zooplankton individuals will be carefully picked-up and placed on
directly in a Eppendorf vial. The vials will be transported in a liquid nitrogen container
until their subsequent extraction previous lyophilization at Innsbruck. Extraction will be
done with aqueous-methanol (MeOH, HPLC grade) following Tartarotti & Sommaruga
2002). All extracts concentrated by freeze-drying will be resuspended in 100-200 µl of
20% methanol for analysis by high performance liquid chromatography (HPLC). For
separation of MAAs, 20-100 µl aliquots will be injected in a Phenomenex "Phenosphere"
C-8 column (4.6 mm ID x 250 mm) for isocratic reverse-phase HPLC analysis with a
mobile phase of 55% aqueous-MeOH plus 0.1% acetic acid. The MAAs in the eluate will
be detected by online diode array absorption spectroscopy. The MAAs will be identified
by co-chromatography and comparison of wavelength ratios with primary and secondary
standards (Sommaruga & Garcia-Pichel, 1999). Concentrations of the different MAAs will
be normalized to the chlorophyll-a in seston or to dry weight in the case of zooplankton
(see below).
Chlorophyll-a analysis. For chlorophyll-a (Chl-a) estimation, 1 to 2 liters will be filtered
onto Whatman GF/F filters (25 mm diameter) at low pressure (0.3-0.4 atm) and the
filters will kept frozen in liquid Nitrogen. In the laboratory, the filters will be extracted
overnight with 90% acetone and sonicated for 2 min with a tip sonicator (Bandelin Inst.)
on an ice bath. Next the extracts will be filtered through an Anodisc filter of 0.1-µm
pore-size (Whatman) and the absorbance measured in a double beam spectrophotometer
(Hitachi U-2000) against an acetone blank using cuvettes of 5 cm pathlength. The
equations of Jeffrey and Humphrey (1975) will be used to calculate the concentration of
pigments.
Zooplankton biomass. The biomass of the zooplankton in each vial will be estimated
knowing the abundance of the different species and the relationship between length and
dry weight calculated for the species/stages (copepods of the same stage will be sorted
out in the same vial) or if not available for Arctodiaptomus jurisovitchii, I will obtain an
approximation using the relationship for calanoid species of similar size. The biomass of
cladocerans will be calculated following Bottrell et al. (1976).
Total carotenoids in zooplankton. As for MAAs, zooplankton will be sorted out in vials,
transpoted in a liquid N container and freeze-dried at Innsmbruck. Carotenoids will be
extracted with absolute ethanol for 24 h (dark conditions, 20°C) or until to obtain a
complete extraction as checked by direct observation. The optical density of the extract
will be measured in the same spectrophotometer as above against an ethanol blank. The
carotenoid concentration will be calculated according to the following formula:
Carotenoid concentration (µg mg-1 dry weight)= (D* V* 104)/(E*W)
where D is the absorbance at peak, V is volume of ethanol (ml), E is the extinction
coefficient set at 2500 and W is the dry weight of the sample (mg) (Hairston, 1978).
Direct dosage of photolyase in zooplankton species. These measurements will be done
by Dr. Oscar Oppezzo at the Comisión Nacional de Energía Atómica, Departamento de
Radiobiología, Buenos Aires, Argentina. Shortly, the activity of DNA photolyase will be
assessed by measuring the light-dependent loss of thymine containing pyrimidine
dimers from UV-irradiated DNA. The method is described in detail in Rocco et al. (2002).
Determination of DNA damage. These analyses will be done by Dr. Anita Buma at the
University of Groningen, Biological Center, The Netherlands. The protocol used to
determine the concentration of T<>T dimers in DNA from zooplankton species will be a
modification of the method described by Vink et al. (1994). This modification has been
successfully applied to microzooplankton (heterotrophic flagellates and ciliates) and is
described in detail in Sommaruga & Buma (2000).