Comparison of techniques used to count single-celled viable phytoplankton

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Journal of Applied Phycology 24 (2012): 751-758, doi:10.1007/s10811-011-9694-z.Four methods commonly used to count phytoplankton were evaluated based upon the precision of concentration estimates: Sedgewick Rafter and membrane filter direct counts, flow cytometry, and flow-based imaging cytometry (FlowCAM). Counting methods were all able to estimate the cell concentrations, categorize cells into size classes, and determine cell viability using fluorescent probes. These criteria are essential to determine whether discharged ballast water complies with international standards that limit the concentration of viable planktonic organisms based on size class. Samples containing unknown concentrations of live and UV-inactivated phytoflagellates (Tetraselmis impellucida) were formulated to have low concentrations (<100 ml-1) of viable phytoplankton. All count methods used chlorophyll a fluorescence to detect cells and SYTOX fluorescence to detect non-viable cells. With the exception of one sample, the methods generated live and non-viable cell counts that were significantly different from each other, although estimates were generally within 100% of the ensemble mean of all subsamples from all methods. Overall, percent coefficient of variation (CV) among sample replicates was lowest in membrane filtration sample replicates, and CVs for all four counting methods were usually lower than 30% (although instances of ~60% were observed). Since all four methods were generally appropriate for monitoring discharged ballast water, ancillary considerations (e.g., ease of analysis, sample processing rate, sample size, etc.) become critical factors for choosing the optimal phytoplankton counting method.This study was supported by the U.S. Coast Guard Research and Development Center under contract HSCG32-07- X-R00018. Partial research support to DMA and DMK was provided through NSF International Contract 03/06/394, and Environmental Protection Agency Grant RD-83382801-0

Iron and phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic

The role of iron in enhancing phytoplankton productivity in high nutrient, low chlorophyll oceanic regions was demonstrated first through iron-addition bioassay experiments1 and subsequently confirmed by large-scale iron fertilization experiments2. Iron supply has been hypothesized to limit nitrogen fixation and hence oceanic primary productivity on geological timescales3, providing an alternative to phosphorus as the ultimate limiting nutrient4. Oceanographic observations have been interpreted both to confirm and refute this hypothesis5, 6, but direct experimental evidence is lacking7. We conducted experiments to test this hypothesis during the Meteor 55 cruise to the tropical North Atlantic. This region is rich in diazotrophs8 and strongly impacted by Saharan dust input9. Here we show that community primary productivity was nitrogen-limited, and that nitrogen fixation was co-limited by iron and phosphorus. Saharan dust addition stimulated nitrogen fixation, presumably by supplying both iron and phosphorus10, 11. Our results support the hypothesis that aeolian mineral dust deposition promotes nitrogen fixation in the eastern tropical North Atlantic

High CO2 and Silicate Limitation Synergistically Increase the Toxicity of Pseudo-nitzschia fraudulenta

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si∶C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and ‘carbon fertilization’ of the coastal ocean

Influence of plankton community structure on the sinking velocity of marine aggregates

About 50 Gt of carbon is fixed photosynthetically by surface ocean phytoplankton communities every year. Part of this organic matter is reprocessed within the plankton community to form aggregates which eventually sink and export carbon into the deep ocean. The fraction of organic matter leaving the surface ocean is partly dependent on aggregate sinking velocity which accelerates with increasing aggregate size and density, where the latter is controlled by ballast load and aggregate porosity. In May 2011, we moored nine 25 m deep mesocosms in a Norwegian fjord to assess on a daily basis how plankton community structure affects material properties and sinking velocities of aggregates (Ø 80–400 µm) collected in the mesocosms' sediment traps. We noted that sinking velocity was not necessarily accelerated by opal ballast during diatom blooms, which could be due to relatively high porosity of these rather fresh aggregates. Furthermore, estimated aggregate porosity (Pestimated) decreased as the picoautotroph (0.2–2 µm) fraction of the phytoplankton biomass increased. Thus, picoautotroph-dominated communities may be indicative for food webs promoting a high degree of aggregate repackaging with potential for accelerated sinking. Blooms of the coccolithophore Emiliania huxleyi revealed that cell concentrations of ~1500 cells/mL accelerate sinking by about 35–40%, which we estimate (by one-dimensional modeling) to elevate organic matter transfer efficiency through the mesopelagic from 14 to 24%. Our results indicate that sinking velocities are influenced by the complex interplay between the availability of ballast minerals and aggregate packaging; both of which are controlled by plankton community structure

Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis

Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modes between autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutrition differ among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritional flexibility, although most species generally fall towards the heterotrophic end of the mixotrophy spectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growing mixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence of either heat-killed bacteria enriched with ^(15)N and ^(13)C, or unlabeled heat-killed bacteria and labeled inorganic substrates (^(13)C-bicarbonate and ^(15)N-ammonium). The alga exhibited high growth rates (up to 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotope analyses revealed that Ochromonas obtained 84–99% of its carbon and 88–95% of its nitrogen from consumed bacteria. The chrysophyte assimilated inorganic ^(13)C-carbon and ^(15)N-nitrogen when bacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient to support net population growth of the alga. Our use of nanoSIMS represents its first application towards the study of a mixotrophic alga, enabling a better understanding and quantitative assessment of carbon and nutrient acquisition by this species

Stable Isotope Evidence for Dietary Overlap between Alien and Native Gastropods in Coastal Lakes of Northern KwaZulu-Natal, South Africa

Tarebia granifera (Lamarck, 1822) is originally from South-East Asia, but has been introduced and become invasive in many tropical and subtropical parts of the world. In South Africa, T. granifera is rapidly invading an increasing number of coastal lakes and estuaries, often reaching very high population densities and dominating shallow water benthic invertebrate assemblages. An assessment of the feeding dynamics of T. granifera has raised questions about potential ecological impacts, specifically in terms of its dietary overlap with native gastropods.A stable isotope mixing model was used together with gut content analysis to estimate the diet of T. granifera and native gastropod populations in three different coastal lakes. Population density, available biomass of food and salinity were measured along transects placed over T. granifera patches. An index of isotopic (stable isotopes) dietary overlap (IDO, %) aided in interpreting interactions between gastropods. The diet of T. granifera was variable, including contributions from microphytobenthos, filamentous algae (Cladophora sp.), detritus and sedimentary organic matter. IDO was significant (>60%) between T. granifera and each of the following gastropods: Haminoea natalensis (Krauss, 1848), Bulinus natalensis (Küster, 1841) and Melanoides tuberculata (Müller, 1774). However, food did not appear to be limiting. Salinity influenced gastropod spatial overlap. Tarebia granifera may only displace native gastropods, such as Assiminea cf. ovata (Krauss, 1848), under salinity conditions below 20. Ecosystem-level impacts are also discussed.The generalist diet of T. granifera may certainly contribute to its successful establishment. However, although competition for resources may take place under certain salinity conditions and if food is limiting, there appear to be other mechanisms at work, through which T. granifera displaces native gastropods. Complementary stable isotope and gut content analysis can provide helpful ecological insights, contributing to monitoring efforts and guiding further invasive species research