J. Dean Pakulski

An improved method for the hydrolysis and MBTH analysis of dissolved and particulate carbohydrates in seawater

Abstract An improved method for the bulk characterization of particulate and dissolved combined carbohydrates in seawater is presented. Seawater samples are dried under vacuum and the residue is hydrolyzed with 12 M H 2 SO 4 . Carbohydrates in the hydrolysate are subsequently quantified spectrophotometrically by MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) analysis. Sulfuric acid was more effective in hydrolyzing high-molecular-weight structural carbohydrates (cellulose, chitin, and alginic acid) than weak (0.09 N) hydrochloric acid. Sulfuric acid hydrolysis of dissolved and particulate samples yielded estimates of carbohydrates equal to or greater (two- to four-fold) than those resulting from dilute (0.09 N) HCl hydrolysis. In samples from the Gulf of Mexico, total dissolved carbohydrates determined after sulfuric acid hydrolysis varied from 20 to 56 μM C and from 10 to 28% of the dissolved organic carbon determined by high-temperature catalytic oxidation.

Sunlight-induced DNA Damage in Marine Micro-organisms Collected Along a Latitudinal Gradient from 70°N to 68°S

We examined ultraviolet radiation (UVR)‐induced DNA damage in marine micro‐organisms collected from surface seawater along a latitudinal transect in the Central Pacific Ocean from 70°N to 68°S. Samples were collected predawn and incubated under ambient UVR in transparent incubators at in situ temperatures until late afternoon at which time they were filtered into primarily bacterioplankton and eukaryotic fractions. Cyclobutane pyrimidine dimers (CPDs) and (6‐4) photoproducts [(6‐4)PDs] were quantified in DNA extracts using radioimmunoassays. UVB was lowest in the polar regions and highest near the equator and correlations between UVB and DNA damage were observed. The eukaryotic fraction showed significant CPDs across the entire transect; (6‐4)PDs were detected only in the tropics. The bacterial fraction showed no accumulation of (6‐4)PDs at any latitude, although residual (6‐4)PDs were observed. Bacterial cell volumes were greatest in the sub‐Arctic and northern temperate latitudes and lower in the tropics and southern hemisphere, a unique observation that parallels Bergmann’s rule. A strong negative correlation was observed between cell volume and CPDs. The environmental impact of solar UVR on marine micro‐organisms in the open ocean is complex and our results suggest that several factors such as DNA repair, cell size, temperature, salinity, nutrients and species composition are important in determining relative sensitivity.

Seasonal Fluctuation of DNA Photodamage in Marine Plankton Assemblages at Palmer Station, Antarctica ¶

Abstract Ultraviolet radiation–induced DNA damage frequencies were measured in DNA dosimeters and natural plankton communities during the austral spring at Palmer Station, Antarctica, during the 1999–2000 field season. We found that the fluence of solar ultraviolet-B radiation (UV-B) at the earths surface correlated with stratospheric ozone concentrations, with significant ozone depletion observed because of “ozone hole” conditions. To verify the interdependence of ozone depletion and DNA damage in natural microbial communities, seawater was collected daily or weekly from Arthur Harbor at Palmer Station, Antarctica, throughout “ozone season,” exposed to ambient sunlight between 0600 and 1800 h and fractionated using membrane filtration to separate phytoplankton and bacterioplankton populations. DNA from these fractions was isolated and DNA damage measured using radioimmunoassay. Under low-ozone conditions cyclobutane dimer concentrations in bacterioplankton and phytoplankton communities were maximal. DNA damage measured in dosimeters correlated closely with ozone concentrations and UV-B fluence. Our studies offer further support to the theory that stratospheric deozonation is detrimental to marine planktonic organisms in the Southern Ocean.

Effect of Stratospheric Ozone Depletion and Enhanced Ultraviolet Radiation on Marine Bacteria at Palmer Station, Antarctica in the Early Austral Spring

We investigated the interactions between ozone‐depleted air masses and subsequent changes in UVB on marine bacterial abundance and production at Palmer Station, Antarctica from September to November 1999. During periods of low total column ozone (TCO), bacterial cell concentrations declined by 57%. Photoinhibition of bacterial [3H]‐leucine (Leu) and [3H]‐thymidine (TdR) incorporation due to UVB was greatest during periods of low TCO in September and early October. During diel (∼28 h) exposure experiments, light treatment samples exhibited >75–100% inhibition of TdR incorporation by mid‐afternoon. Leu incorporation exhibited maximum inhibition (50–100%) at sunset and early evening hours. Leu and TdR incorporation in light treatment samples did not exhibit recovery during subsequent periods of darkness. Bacterial Leu and TdR incorporation rates were inversely related to Setlow Dose during a period of recovery from low TCO. These data further suggested a threshold exposure below which bacterial Leu and TdR incorporation recovered rapidly. Recovery of bacterial production after acute Setlow Dose exposures lagged recovery of TCO and was linearly related to TCO measured 2 days previously. This lag in recovery may have resulted from the energetically expensive repair of UVR‐induced DNA damage acquired during periods of low TCO.

The significance of the Dewar valence photoisomer as a UV radiation‐induced DNA photoproduct in marine microbial communities

Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.