Anita Buma

Monitoring ultraviolet-B-induced DNA damage in individual diatom cells by immunofluorescent thymine dimer detection

We developed a method to investigate the effect of ultraviolet‐B radiation (UVBR) on the formation of thy‐mine dimers in microalgal DNA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer‐specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance levels. Thy‐mine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR‐induced DNA damage on a cell‐to‐cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.

Dissolved aluminium in the Weddell-Scotia Confluence and effect of Al on the dissolution kinetics of biogenic silica

Abstract In the Scotia and Weddell Seas the concentration of dissolved Al was 1–1.5 nM in ice-free surface waters, up to 3 nM in ice-covered waters and about 2.6 nM in bottom waters. The solubility and the dissolution rate of diatomaceous silica, obtained from net samples and from incubations in the presence or absence of dissolved Al, were inversely related to solid-phase Al/Si ratios. At in situ temperatures, dissolution rates for Antarctic diatoms are higher than those for diatoms from temperate regions. Effects on distribution patterns of silicic acid in the Weddell Sea are discussed.

EFFECTS OF UV‐B‐INDUCED DNA DAMAGE AND PHOTOINHIBITION ON GROWTH OF TEMPERATE MARINE RED MACROPHYTES: HABITAT‐RELATED DIFFERENCES IN UV‐B TOLERANCE

The sensitivity to UV‐B radiation (UVBR: 280–315 nm) was tested for littoral (Palmaria palmata[L.] O. Kuntze, Chondrus crispus Stackhouse) and sublittoral (Phyllophora pseudoceranoides S. G. Gmelin, Rhodymenia pseudopalmata[Lamouroux] Silva, Phycodrys rubens[L.] Batt, Polyneura hilliae[Greville] Kylin) red macrophytes from Brittany, France. Algal fragments were subjected to daily repeated exposures of artificial UVBR that were realistic for springtime solar UVBR at the water surface in Brittany. Growth, DNA damage, photoinhibition, and UV‐absorbing compounds were monitored during 2 weeks of PAR + UV‐A radiation (UVAR) + UVBR, whereas PAR + UVAR and PAR treatments were used as controls. The littoral species showed a higher UV tolerance than the sublittoral species. After 2 weeks, growth of P. palmata and C. crispus was not significantly affected by UVBR, and DNA damage, measured as the number of cyclobutane‐pyrimidine dimers per 106 nucleotides, was negligible. Photoinhibition, determined as the decline in optimal quantum yield, was low and decreased during the course of the experiment, coinciding with the production of UV‐absorbing compounds in these species. In contrast, no UV‐absorbing compounds were induced in the sublittoral species. Growth rates of P. pseudoceranoides and R. pseudopalmata were reduced by 40% compared with the PAR treatment. Additionally, constant levels of DNA damage and pronounced photoinhibition were observed after the UVBR treatments. Growth was completely halted for Phycodrys rubens and Polyneura hilliae, whereas DNA damage accumulated in the course of the experiment. Because Phycodrys rubens and Polyneura hilliae showed the same degree of photoinhibition as the other sublittoral species, it appears that the accumulation of DNA damage may have been responsible for the complete inhibition of growth. The results suggest an important role of DNA repair pathways in determining the UV sensitivity in red macrophytes.

DEPTH DISTRIBUTIONS OF DNA DAMAGE IN ANTARCTIC MARINE PHYTO‐ AND BACTERIOPLANKTON EXPOSED TO SUMMERTIME UV RADIATION

During a survey from January to March 1998, the occurrence of UV‐B radiation (UVBR)‐ induced DNA damage in Antarctic marine phytoplankton and bacterioplankton was investigated. Sampling was done in Ryder Bay, off the British base Rothera Station, 67°S, 68°W (British Antarctic Survey). Samples were taken regularly during the survey period at fixed depths, after which DNA damage was measured in various plankton size fractions (>10, 2–10, and 0.2–2 μm). Incident solar radiation was measured using spectroradiometry, whereas attenuation of biologically effective UVBR was studied using a DNA dosimeter. A diatom bloom was found in the bay during the research period, judging from microscopic observations and HPLC analyses of taxon‐specific pigments. The high phytoplankton biomass likely caused strong attenuation of DNA effective UVBR (Kbd‐eff). Kbd‐eff values ranged from 0.83·m−1 at the peak of the bloom to 0.47·m−1 at the end of the season. UVBR‐mediated DNA damage, as measured by cyclobutane pyrimidine dimer (CPD) abundance, was detected in all plankton size fractions. Highest levels were found in the smallest size fraction, mainly consisting of heterotrophic bacteria. Clear CPD depth profiles were found during mid‐summer (January, beginning of February) with surface levels exceeding 100 CPDs per million nucleotides in the bacterioplankton fraction. At that time, melting of the continuously present shelf ice caused strong salinity gradients in the upper meters, thereby stimulating water column stabilization. At the end of February and beginning of March, this phenomenon was less pronounced or absent. At that time, DNA damage was homogeneously distributed over the first 10 m, ranging between 20 and 30 CPDs per million nucleotides for the smallest size fraction.

Effects of temperature on the photoreactivation of ultraviolet-B-induced DNA damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6‐4 photoproducts) induced by ultraviolet‐B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet‐A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6‐4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6‐4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6‐4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6‐4 photoproducts. Significant repair of both thymine dimers and 6‐4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6‐4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.

NUTRIENT LIMITATION AND HIGH IRRADIANCE ACCLIMATION REDUCE PAR AND UV-INDUCED VIABILITY LOSS IN THE ANTARCTIC DIATOM CHAETOCEROS BREVIS (BACILLARIOPHYCEAE)1

The effects of high PAR (400–700 nm), UVA (315–400 nm), and UVB (280–315 nm) radiation on viability and photosynthesis were investigated for Chaetoceros brevis Schütt. This Antarctic marine diatom was cultivated under low, medium, and high irradiance and nitrate, phosphate, silicate, and iron limitation before exposure to a simulated surface irradiance (SSI) treatment, with and without UVB radiation. Light‐harvesting and protective pigment composition and PSII parameters were determined before SSI exposure, whereas viability was measured by flow cytometry in combination with a viability stain after the treatment. Recovery of PSII efficiency was measured after 20 h in dim light in a separate experiment. In addition, low and high irradiance acclimated cells were exposed outdoors for 4 h to assess the effects of natural PAR, UVA, and UVB on viability. Low irradiance acclimated cells were particularly sensitive to photo induced viability loss, whereas no viability loss was found after acclimation to high irradiance. Furthermore, nutrient limitation reduced sensitivity to photo induced viability loss, relative to nutrient replete conditions. No additional viability loss was found after UVB exposure. Sunlight exposed cells showed no additional UVB effect on viability, whereas UVA and PAR significantly reduced the viability of low irradiance acclimated cells. Recovery of PSII function was nearly complete in cultures that survived the light treatments. Increased resistance to high irradiance coincided with an increased ratio between protective‐ and light‐harvesting pigments before the SSI treatment, demonstrating the importance of nonphotochemical quenching by diatoxanthin for survival of near‐surface irradiance. We conclude that a sudden transfer to high irradiance can be fatal for low irradiance acclimated C. brevis.

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet‐b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m−2 ·d−1 (BEDDNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.

Attenuation of biologically effective UV radiation in tropical atlantic waters measured with a biochemical DNA dosimeter

Abstract— A biochemical dosimeter was developed to study the attenuation of biologically effective UV radiation in marine tropical waters. Small quartz vials were used containing a solution of DNA molecules; the vials were incubated at discrete water depths. Subsequently, DNA damage was determined in these samples, using an antibody directed against thymine dimers followed by chemiluminescent detection. Measurements of DNA damage were compared with calculated biologically effective doses, as derived from spectroradiometer measurements. The biodosimeter was found to be a reliable and easy tool to determine levels of harmful UV radiation in marine waters. The highest attenuation coefficient (1.60 m‐l) measured with the biochemical dosimeter was found in eutrophic waters, at a coastal station off Curabcao, Netherlands Antilles. At the other stations attenuation coefficients ranged from 0.18 m‐1 in central Atlantic waters to 0.43 m‐1 close to the Curapcao coast line. Latter results indicate that biologically effective UV radiation may easily reach ecologically significant depths, e.g. coral reef communities.

Patterns of DNA damage and photoinhibition in temperate South-Atlantic picophytoplankton exposed to solar ultraviolet radiation

Natural marine phytoplankton assemblages from Bahía Bustamante (Chubut, Argentina, 45 degrees S, 66.5 degrees W), mainly consisting of cells in the picoplankton size range (0.2-2 microm), were exposed to various UVBR (280-315 nm) and UVAR (315-400 nm) regimes in order to follow wavelength-dependent patterns of cyclobutane pyrimidine dimer (CPD) induction and repair. Simultaneously, UVR induced photosynthetic inhibition was studied in radiocarbon incorporation experiments. Biological weighting functions (BWFs) for photoinhibition and for CPD induction, the latter measured in bare calf thymus DNA, differed in the UVAR region: carbon incorporation was reduced markedly due to UVAR, whereas no measurable UVAR effect was found on CPD formation. In contrast, BWFs for inhibition of photosynthesis and CPD accumulation were fairly similar in the UVBR region, especially above 300 nm. Incubation of phytoplankton under full solar radiation caused rapid CPD accumulation over the day, giving maximum damage levels exceeding 500 CPD MB(-1) at the end of the afternoon. A clear daily pattern of CPD accumulation was found, in keeping with the DNA effective dose measured by a DNA dosimeter. In contrast, UVBR induced photosynthetic inhibition was not dose related and remained nearly constant during the day. Screening of UVBR or UVR did not cause significant CPD removal, indicating that photoreactivation either by PAR or UVAR was of minor importance in these organisms. High CPD levels were found in situ early in the morning, which remained unaffected notwithstanding treatments favoring photorepair. These results imply that a proportion of cells had been killed by UVBR exposure prior to the treatments. Our data suggest that the limited potential for photoreactivation in picophytoplankton assemblages from the southern Atlantic Ocean causes high CPD accumulation as a result of UVBR exposure.

Ultraviolet-B-induced cyclobutane-pyrimidine dimer formation and repair in arctic marine macrophytes

The significance of ultraviolet‐B radiation (UVBR: 280–315 nm)–induced DNA damage as a stress factor for Arctic marine macrophytes was examined in the Kongsfjord (Spitsbergen, 78°55.5′N, 11°56.0′E) in summer. UVBR penetration in the water column was monitored as accumulation of cyclobutane‐pyrimidine dimers (CPD) in bare DNA. This showed that UVBR transparency of the fjord was variable, with 1% depths ranging between 4 and 8 m. In addition, induction and repair kinetics of CPD were studied in several subtidal macrophytes obtained from the Kongsfjord (5–15 m). Surface exposure experiments demonstrated CPD accumulation in Palmaria palmata, Devaleraea ramentacea, Phycodrys rubens, Coccotylus truncatus and Odonthalia dentata. In artificial light, field collected material of P. palmata, D. ramentacea, P. rubens and Laminaria saccharina showed efficient CPD repair, with only 10% of the artificially induced CPD remaining after 5 h. No significant differences in repair rate were observed among these species. CPD repair was slower or absent in O. dentata, C. truncatus and Monostroma arcticum, indicating that fast repair mechanisms such as photolyase were not continuously expressed in these species. CPD repair rates were not directly related to the vertical distribution of algae in the water column and to the reported UV sensitivity of the examined species. Dosimeter incubations showed that maximal exposure to DNA damaging wavelengths was low for all examined species. Furthermore, most species collected below the 1% depth for DNA damage displayed efficient CPD repair, suggesting that UVBR‐induced CPD currently impose a minor threat for mature stages of these species growing in the Kongsfjord, Spitsbergen.