The Use of HPLC for the Characterization of Phytoplankton Pigments.

HPLC is still the technique of choice for the analysis and characterization of phytoplankton pigments. In this chapter we describe procedures for sample preparation and pigment extraction, and the use of octyl silica columns and pyridine-containing mobile phases to separate chlorophylls and carotenoids. The identification of pigments on the basis of their retention times and visible spectra, the preparation of pigment standards, and the quantitative analysis by either external or internal standard procedures are also described.

High cyst concentrations of the potentially toxic dinoflagellate Alexandrium tamarense species complex in Bedford Basin, Halifax, Nova Scotia, Canada.

We report a large cyst bed of the potentially toxic and bloom-forming dinoflagellate Alexandrium tamarense species complex in bottom sediments from the port of Halifax, Nova Scotia, Canada. The average cyst concentrations of that species ranged from 4033±2647 to 220872±148086 cysts g⁻¹ of dry sediments and the highest concentrations were found near ship terminals in Bedford Basin. Although this species is endemic to this region, our work strongly suggests that some of the cysts of A. tamarense species complex found in the port of Halifax were introduced through discharged ballast water and sediments.

Photosystem II photoinactivation, repair, and protection in marine centric diatoms.

Diatoms are important contributors to aquatic primary production, and can dominate phytoplankton communities under variable light regimes. We grew two marine diatoms, the small Thalassiosira pseudonana and the large Coscinodiscus radiatus, across a range of temperatures and treated them with a light challenge to understand their exploitation of variable light environments. In the smaller T. pseudonana, photosystem II (PSII) photoinactivation outran the clearance of PSII protein subunits, particularly in cells grown at sub- or supraoptimal temperatures. In turn the absorption cross section serving PSII photochemistry was down-regulated in T. pseudonana through induction of a sustained phase of nonphotochemical quenching that relaxed only slowly over 30 min of subsequent low-light incubation. In contrast, in the larger diatom C. radiatus, PSII subunit turnover was sufficient to counteract a lower intrinsic susceptibility to photoinactivation, and C. radiatus thus did not need to induce sustained nonphotochemical quenching under the high-light treatment. T. pseudonana thus incurs an opportunity cost of sustained photosynthetic down-regulation after the end of an upward light shift, whereas the larger C. radiatus can maintain a balanced PSII repair cycle under comparable conditions.

Relationship between propagule pressure and colonization pressure in invasion ecology: a test with ships' ballast.

Increasing empirical evidence indicates the number of released individuals (i.e. propagule pressure) and number of released species (i.e. colonization pressure) are key determinants of the number of species that successfully invade new habitats. In view of these relationships, and the possibility that ships transport whole communities of organisms, we collected 333 ballast water and sediment samples to investigate the relationship between propagule and colonization pressure for a variety of diverse taxonomic groups (diatoms, dinoflagellates and invertebrates). We also reviewed the scientific literature to compare the number of species transported by ships to those reported in nature. Here, we show that even though ships transport nearly entire local communities, a strong relationship between propagule and colonization pressure exists only for dinoflagellates. Our study provides evidence that colonization pressure of invertebrates and diatoms may fluctuate widely irrespective of propagule pressure. We suggest that the lack of correspondence is explained by reduced uptake of invertebrates into the transport vector and the sensitivity of invertebrates and diatoms to selective pressures during transportation. Selection during transportation is initially evident through decreases in propagule pressure, followed by decreased colonization pressure in the most sensitive taxa.

Cell size trade-offs govern light exploitation strategies in marine phytoplankton.

Marine phytoplankton show complex community structures and biogeographic distributions, the net results of physiological and ecological trade-offs of species responses to fluctuating, heterogeneous environments. We analysed photosynthesis, responses to variable light and macromolecular allocations across a size panel of marine centric diatoms. The diatoms have strong capacities to withstand and exploit fluctuating light, when compared with picophytoplankton. Within marine diatoms, small species show larger effective cross-sections for photochemistry, and fast metabolic repair of photosystem II after photoinactivation. In contrast, large diatoms show lower susceptibility to photoinactivation, and therefore incur lower costs to endure short-term exposures to high light, especially under conditions that limit metabolic rates. This size scaling of key photophysiological parameters thus helps explain the relative competitive advantages of larger versus smaller species under different environmental regimes, with implications for the function of the biogenic carbon pump. These results provide a mechanistic framework to explain and predict shifts in marine phytoplankton community size structure with changes in surface irradiance and mixed layer depth.

Photosystem II and pigment dynamics among ecotypes of the green alga Ostreococcus.

We investigated the photophysiological responses of three ecotypes of the picophytoplankter Ostreococcus and a larger prasinophyte Pyramimonas obovata to a sudden increase in light irradiance. The deepwater Ostreococcus sp. RCC809 showed very high susceptibility to primary photoinactivation, likely a consequence of high oxidative stress, which may relate to the recently noted plastid terminal oxidase activity in this strain. The three Ostreococcus ecotypes were all capable of deploying modulation of the photosystem II repair cycle in order to cope with the light increase, but the effective clearance of photoinactivated D1 protein appeared to be slower in the deepwater Ostreococcus sp. RCC809, suggesting that this step is rate limiting in the photosystem II repair cycle in this strain. Moreover, the deepwater Ostreococcus accumulated lutein and showed substantial use of the xanthophyll cycle under light stress, demonstrating its high sensitivity to light fluctuations. The sustained component of the nonphotochemical quenching of fluorescence correlated well with the xanthophyll deepoxidation activity. Comparisons with the larger prasinophyte P. obovata suggest that the photophysiology of Ostreococcus ecotypes requires high photosystem II repair rates to counter a high susceptibility to photoinactivation, consistent with low pigment package effects in their minute-sized cells.

GROWTH AND PHOTOPROTECTION IN THREE DINOFLAGELLATES (INCLUDING TWO STRAINS OF ALEXANDRIUM TAMARENSE) AND ONE DIATOM EXPOSED TO FOUR WEEKS OF NATURAL AND ENHANCED ULTRAVIOLET-B RADIATION(1).

Long-term growth response to natural solar radiation with enhanced ultraviolet-B (UVB) exposure was examined in two species of dinoflagellates [Alexandrium tamarense (M. Lebour) Balech, At, and Heterocapsa triquetra (Ehrenb.) F. Stein, Ht], including two strains of A. tamarense, one from Spain and another from UK, and one diatom species (Thalassiosira pseudonana Hasle et Heimdal). We examined whether variable photoprotection (mycosporine-like amino acids [MAAs] and xanthophyll-cycle pigments) affected photosynthetic performance, phytoplankton light absorption, and growth. Growth rate was significantly reduced under enhanced UVB for the UK strain of At and for Ht (both grew very little) as well as for the diatom (that maintained high growth rates), but there was no effect for the Spanish strain of At. MAA concentration was high in the dinoflagellates, but undetectable in the diatom, which instead used the xanthophyll cycle for photoprotection. The highest cell concentrations of MAAs and photoprotective pigments were observed in the UK strain of At, along with lowest growth rates and Fv /Fm , indicating high stress levels. In contrast, the Spanish strain showed progressive acclimation to the experimental conditions, with no significant difference in growth between treatments. Increase in total MAAs followed linearly the cumulative UVB of the preceding day, and both total and primary MAAs were maintained at higher constitutive levels in this strain. Acclimation to enhanced UVB in the diatom resulted in an increase in PSII activity and reduction in nonphotochemical quenching, indicating an increased resistance to photoinhibition after a few weeks. All four species showed increased phytoplankton light absorption under enhanced UVB. Large intrastrain differences suggest a need to consider more closely intraspecific variability in UV studies.

DIEL VARIATIONS IN OPTICAL PROPERTIES OF IMANTONIA ROTUNDA (HAPTOPHYCEAE) AND THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) EXPOSED TO DIFFERENT IRRADIANCE LEVELS(1).

Diel variations of cellular optical properties were examined for cultures of the haptophyte Imantonia rotunda N. Reynolds and the diatom Thalassiosira pseudonana (Hust.) Hasle et Heimdal grown under a 14:10 light:dark (L:D) cycle and transferred from 100 µmol photons · m(-2) · s(-1) to higher irradiances of 250 and 500 µmol photons · m(-2) · s(-1) . Cell volume and abundance, phytoplankton absorption coefficients, flow-cytometric light scattering and chl fluorescence, and pigment composition were measured every 2 h over a 24 h period. Results showed that cell division was more synchronous for I. rotunda than for T. pseudonana. Several variables exhibited diel variability with an amplitude >100%, notably mean cell volume for the haptophyte and photoprotective carotenoids for both species, while optical properties such as flow-cytometric scattering and chl a-specific phytoplankton absorption generally showed <50% diel variability. Increased irradiance induced changes in pigments (both species) and mean cell volume (for the diatom) and amplified diel variability for most variables. This increase in amplitude is larger for pigments (factor of 2 or more, notably for cellular photoprotective carotenoid content in I. rotunda and for photosynthetic pigments in T. pseudonana) than for optical properties (a factor of 1.5 for chl a-specific absorption, at 440 nm, in I. rotunda and a factor of 2 for the absorption cross-section and the chl a-specific scattering in T. pseudonana). Consequently, diel changes in optical properties and pigmentation associated with the L:D cycle and amplified by concurrent changes in irradiance likely contribute significantly to the variability in optical properties observed in biooptical field studies.

Photoacclimation to long-term ultraviolet radiation exposure of natural sub-Antarctic phytoplankton communities: Fixed surface incubations versus mixed mesocosms.

Solar UVB radiation (280-320 nm) is known to have detrimental effects on marine phytoplankton. Associated with the seasonal ozone hole in Antarctica, stratospheric ozone depletion occasionally influences the sub-Antarctic (Beagle Channel, Argentina) region, enhancing levels of UVB. The primary objective of this work was to study the effects of several (i.e. 6-10) days of exposure to UVB on the taxonomic composition and photosynthetic inhibition of local phytoplankton communities. For different light treatments, fixed-depth incubations placed in an outdoors water tank were compared with incubations in 1900 L mesocosms, where vertical mixing was present. Phytoplankton growth was inhibited by UV radiation (UVR) in fixed-depth experiments but not in the mixed mesocosms. Under fixed and mixed conditions alike, photosynthesis was significantly inhibited by UVB at the beginning of the experiment but no longer after several days of exposure, suggesting that cells had acclimated to radiation conditions. There was a change in species composition in response to UVR exposure in both experiments, which likely explained acclimation. In the community exposed to fixed conditions this change was from a phytoflagellate-dominated assemblage to a community with high relative abundance of diatoms after 6 days of exposure. UVA was responsible for most of the observed growth inhibition; however, the reduction in photosynthesis was produced by UVB. The reasons behind this variability in responses to UVR are associated with species-specific sensitivity and acclimation, and the previous light history of cells. In the community exposed in mesocosms, an assemblage codominated by phytoflagellates and diatoms was observed at the beginning of the experiments. After 10 days of exposure, green algae (Eutreptiella sp.) had increased, and phytoflagellates were the dominant group. The synthesis of mycosporine-like amino acids (MAAs), antioxidant enzymes and photosynthetic antenna pigments, in relation to repair and protection processes, may explain the reduced inhibition of both growth and photosynthesis that was observed in the phytoplankton community after several days of exposure. For environments such as the Beagle Channel seasonally exposed to the ozone hole, the results obtained from the fixed-depth experiments show that species can cope with UVR by means of MAA synthesis, while mixing would primarily promote a change in species composition and defense strategies.

UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities.

The reaction center of photosystem II is susceptible to photodamage. In particular the D1 protein located in the photosystem II core has a rapid, light-dependent turnover termed the photosystem II repair cycle that, under illumination, degrades and resynthesizes D1 protein to limit accumulation of photodamaged photosystem II. Most studies concerning the effects of UVB (280-320 nm) on this cycle have been on cyanobacteria or specific phytoplankton species rather than on natural communities of phytoplankton. During a 5-year multidisciplinary project on the effects of UV radiation (200-400 nm) on natural systems, the effects of UVB on the D1 protein of natural phytoplankton communities were assessed. This review provides an overview of photoinhibitory effects of light on cultured and natural phytoplankton, with an emphasis on the interrelation of UVB exposure, D1 protein degradation and the repair of photosystem II through D1 resynthesis. Although the UVB component of the solar spectrum contributes to the primary photoinactivation of photosystem II, we conclude that, in natural communities, inhibition of the rate of the photosystem II repair cycle is a more important influence of UVB on primary productivity. Indeed, exposing tropical and temperate phytoplankton communities to supplemented UVB had more inhibitory effect on D1 synthesis than on the D1 degradation process itself. However, the rate of net D1 damage was faster for the tropical communities, likely because of the effects of high ambient light and water temperature on mechanisms of protein degradation and synthesis.

Ultraviolet B-photoprotection efficiency of mesocosm-enclosed natural phytoplankton communities from different latitudes: Rimouski (Canada) and Ubatuba (Brazil).

Photoprotection against UV-B radiation (UVBR; 280-320 nm) was examined in natural phytoplankton communities from two coastal environments at different latitudes: temperate Rimouski (Canada) and tropical Ubatuba (Brazil). Mesocosm experiments were performed at these sites to examine the response of phytoplankton to increases in UVBR that corresponded to local depletions of 30% and 60% in atmospheric ozone levels (low and high UVBR treatments, respectively). A fluorescence method using a pulse amplitude modulation fluorometer (Xe-PAM, Walz, Germany) with selective UV filters was used to estimate photoprotection, and these results were compared with an index of mycosporine-like amino acid (MAA) concentrations determined using spectrophotometry of methanol extracts. The present study provided the first evidence, to our knowledge, of the suitability of this in vivo fluorescence method for the estimation of UV photoprotection efficiency in natural phytoplankton. No significant differences were found for most of the variables analyzed between the light treatments used at both sites, but differences were found between sites throughout the duration of the experiments. Vertical mixing, used to maintain cells in suspension, likely alleviated serious UVBR-induced damage during both experiments by reducing the length of time of exposure to the highest UVBR irradiances at the surface. In Rimouski, this was the main factor minimizing the effects of treatment, because optical properties of the coastal seawater rapidly attenuated UVBR throughout the water column of the ca 2 m deep mesocosms. In this location, synthesis of MAAs and photoprotective pigments likely contributed to the observed phototolerance of phytoplankton and, hence, to their growth; however, in a comparison of the UVBR treatments, these variables showed no differences. In Ubatuba, where nutrient concentrations were significantly lower than those in Rimouski, light attenuation was less than that in Rimouski and UVBR reached the bottom of the mesocosms. UVBR penetration and the forced vertical mixing of the cells, without the possibility of vertical migration below this photostress zone, resulted in photo-inhibition, because confinement in the mesocosms forced cells to remain constantly exposed to high levels of irradiance during the daytime. Hence, additional effects of UVBR were masked in this experiment, because cells were damaged too much and phytoplankton populations were rapidly declining. There was also an overall preservation of MAAs, in contrast with chlorophyll (Chl) degradation, in spite of the fact that this UV screening was not sufficient to counteract photo-inhibition, which suggests an important role for these molecules, either in the overall photoprotection strategy or in other physiological processes. Altogether, local water characteristics, namely attenuation, mixing, and nutrients concentration, can strongly modulate the photoprotection strategies used by natural phytoplankton populations in coastal environments.

UV effects on marine planktonic food webs: A synthesis of results from mesocosm studies.

UV irradiance has a broad range of effects on marine planktonic organisms. Direct and indirect effects on individual organisms have complex impacts on food-web structure and dynamics, with implications for carbon and nutrient cycling. Mesocosm experiments are well suited for the study of such complex interrelationships. Mesocosms offer the possibility to conduct well-controlled experiments with intact planktonic communities in physical, chemical and light conditions mimicking those of the natural environment. In allowing the manipulation of UV intensities and light spectral composition, the experimental mesocosm approach has proven to be especially useful in assessing the impacts at the community level. This review of mesocosm studies shows that, although a UV increase even well above natural intensities often has subtle effects on bulk biomass (carbon and chlorophyll), it can significantly impact the food-web structure because of different sensitivity to UV among planktonic organisms. Given the complexity of UV impacts, as evidenced by results of mesocosm studies, interactions between UV and changing environmental conditions (e.g. eutrophication and climate change) are likely to have significant effects on the function of marine ecosystems.

Ultraviolet-B radiation effects on the structure and function of lower trophic levels of the marine planktonic food web.

The impact of UV-B radiation (UVBR; 280-320 nm) on lower levels of a natural plankton assemblage (bacteria, phytoplankton and microzooplankton) from the St. Lawrence Estuary was studied during 9 days using several immersed outdoor mesocosms. Two exposure treatments were used in triplicate mesocosms: natural UVBR (N treatment, considered as the control treatment) and lamp-enhanced UVBR (H treatment, simulating 60% depletion of the ozone layer). A phytoplankton bloom developed after day 3, but no significant differences were found between treatments during the entire experiment for phytoplankton biomass (chlorophyll a and cell carbon) nor for phytoplankton cell abundances from flow cytometry and optical microscopy of three phytoplankton size classes (picoplankton, nanoplankton and microplankton). In contrast, bacterial abundances showed significantly higher values in the H treatment, attributed to a decrease in predation pressure due to a dramatic reduction in ciliate biomass (approximately 70-80%) in the H treatment relative to the N treatment. The most abundant ciliate species were Strombidinium sp., Prorodon ovum and Tintinnopsis sp.; all showed significantly lower abundances under the H treatment. P. ovum was the less-affected species (50% reduction in the H treatment compared with that of the N control), contrasting with approximately 90% for the other ones. Total specific phytoplanktonic and bacterial production were not affected by enhanced UVBR. However, both the ratio of primary to bacterial biomass and production decreased markedly under the H treatment. In contrast, the ratio of phytoplankton to bacterial plus ciliate carbon biomass showed an opposite trend than the previous results, with higher values in the H treatment at the end of the experiment. These results are explained by the changes in the ciliate biomass and suggest that UVBR can alter the structure of the lower levels of the planktonic community by selectively affecting key species. On the other hand, linearity between particulate organic carbon (POC) and estimated planktonic carbon was lost during the postbloom period in both treatments. On the basis of previous studies, our results can be attributed to the aggregation of carbon released by cells to the water column in the form of transparent exopolymer particles (TEPs) under nutrient limiting conditions. Unexpectedly, POC during such a period was higher in the H treatment than in controls. We hypothesize a decrease in the ingestion of TEPs by ciliates, in coincidence with increased DOC release by phytoplankton cells under enhanced UVBR. The consequences of such results for the carbon cycle in the ocean are discussed.

Effects of enhanced UV-B on pigment-based phytoplankton biomass and composition of mesocosm-enclosed natural marine communities from three latitudes.

A series of three outdoor mesocosm experiments was undertaken in Rimouski (Canada), Ubatuba (Brazil) and Ushuaia (southern Argentina) to examine the effects of lamp-enhanced UV-B (280-320 nm) on phytoplankton communities isolated from seawater at each site. Detailed pigment composition was used to identify these communities. Each experiment compared three replicated UV-B treatments, consisting of natural sunlight conditions (NUVB), low-level UV-B enhancement corresponding to local 30% ozone depletion (LUVB) and high-level enhancement corresponding to 60% ozone depletion (HUVB). Each mesocosm (ca 2 m deep) was mixed continuously (turnover time, ca 1.3 h) and samples were obtained daily over 7-10 days. In Rimouski a large diatom bloom occurred during the first week. Repeated-measures analysis of variance (RM-ANOVA), with time as the repeated factor, showed slight but statistically significant increases in the chlorophyll (Chl) a level with the HUVB treatment, which were especially obvious over the last 3 days of the experiment. A large decrease in grazers (ciliates) that was observed concurrently with this treatment is the most likely explanation for the increase in Chl a level. The lack of negative effect on algal biomass by enhanced UV-B is attributed to the mixing inside the mesocosms and to the relatively low UV-B penetration. In Ubatuba levels of most pigments decreased over time, particularly fucoxanthin, Chl c3 and alloxanthin. The RM-ANOVA showed no effect of the UV-B treatments, except for Chl c3, which had significantly lower concentrations under natural UVB conditions, indicating that enhanced UV-B directly or indirectly favored Chl c3 algae (likely prymnesiophytes). Although particulate organic carbon concentration was significantly larger during HUVB treatment than during the other treatments, Chl a was unaffected, suggesting that enhanced UV-B favored heterotrophs. Lack of algal growth during this experiment was attributed to low nutrient concentrations (which were the lowest of the three sites), high irradiances (which were the highest noon incident photosynthetically available radiation and UV of the three sites) and UV-B penetration down to the bottom of the mesocosms. In Ushuaia a small bloom took place over the first 5 days. The RM-ANOVA showed no overall effect of the UV-B treatments for any of the pigments examined but on the last 3 days of the experiment several green algae-type pigments, such as Chl b and siphonein, showed increased concentrations under the HUVB treatment. UV-B enhancement hence favored green algae, as seen from the stronger increase over time in the ratio of Chl b to Chl a associated with the HUVB treatment. UV-B enhancement also seemed to cause a slight decrease in physiological condition, because the relative concentration of chlorophyllide a and some pheophorbides that may be the product of dying algae increased during the HUVB treatments in Ubatuba and particularly in Ushuaia (where UV-B also penetrated to the bottom of mesocosms). For all three sites changes in phytoplankton biomass due to the UV-B treatments were minor, even though UV-B enhancement was important. This study indicates that effects of enhanced UV-B on the community structure of both phytoplankton and their grazers are potentially more important than effects on overall algal biomass.

INFLUENCE OF UV-B RADIATION ON NITROGEN UTILIZATION BY A NATURAL ASSEMBLAGE OF PHYTOPLANKTON.

A 7-day mesocosm experiment was conducted in July 1996 to investigate the effects of ambient UV-B radiation (UVBR) exclusion and two UVBR enhancements above ambient levels on NO3- , NH4+ and urea utilization in a natural plankton community (<240 µm) from the Lower St. Lawrence Estuary. The phytoplankton community was dominated by diatoms during the first 3 days and, afterward, by flagellates and dinoflagellates. The results of 4-h incubations just below the water surface show that, compared with ambient UVBR conditions, UVBR exclusion generally increased NO3- , NH4+ , and urea uptakes. During the last 4 days of the experiment, the percent increase in the specific uptake rate of urea under excluded UVBR conditions varied between 17% and 130% and was a linear function of the ambient UVBR dose removed. During the first 3 days, the phytoplankton community dominated by diatoms was able to withstand UVBR enhancements without any perceptible effect on nitrogen uptake. However, during the post-diatom bloom period, UVBR enhancements resulted in decreases in NO3- , NH4+ , and urea uptake compared with ambient UVBR conditions. The reduction of urea uptake under UVBR enhancements during the last 3 days varied between 23% and 64% and was linearly related to the enhanced UVBR dose. However, the different UVBR treatments did not affect the internal organic nitrogen composition (internal urea, free amino acids, and proteins) of the phytoplankton community experiencing vertical mixing in the mesocosms. The discrepancy between short-term uptake measurements at the surface and long-term effects in the mesocosms emphasizes the importance of vertical mixing on UVBR effects in natural ecosystems. This suggests that an increase in ambient UVBR would have a minimal effect on nitrogen utilization by natural phytoplankton assemblages if these are vertically mixed.