Daniel J. Franklin

EVOLUTION OF AN ARTIFICIAL SEAWATER MEDIUM: IMPROVEMENTS IN ENRICHED SEAWATER, ARTIFICIAL WATER OVER THE LAST TWO DECADES

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro‐ and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species (Apedinella spinifera), growth rate and final yield was no different between the media but in one case (Emiliania huxleyi) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species.

What is the role and nature of programmed cell death in phytoplankton ecology

Cell death is a fundamental process in all metazoan organisms. In contrast, the ecological role of cell death in phytoplankton has been sorely neglected: the causes and biochemistry of cell death, and the quantitative significance of cell death in the ecology of phytoplankton populations and in broader biogeochemical cycles, are not well understood. Metazoan cell death is much better described, due to its accepted roles in the regulation of multicellular life. In metazoan cells, an influential paradigm suggests that there are two morphological outcomes of cell death, one caused by an ‘active’ pathway within the cell (so-called ‘programmed cell death’; PCD), and the other from a ‘passive’ externally-driven process (necrosis). Here, we examine the development of this paradigm, and associated concepts, in plant, animal, and microbial life, and discuss the role of cell death amongst the diverse taxa of the phytoplankton. Several recent studies suggest PCD operates in cyanobacteria, chlorophytes, and dinoflagellates. A better understanding of phytoplankton cell death will potentially provide insight into bloom development, intercellular signalling and population regulation. Understanding the role of PCD in phytoplankton life-history will likely come through examination of metabolic differentiation within phytoplankton populations, of which at present there are only isolated reports. Although bacterial metabolic differentiation (e.g. in the formation of biofilms) is well accepted, metabolic differentiation and group selection amongst microalgae are poorly understood, and are ideas which merit greater research effort. If a process similar to metazoan PCD is widespread amongst unicellular algae, then a rethinking of the ecological relationships between and within phytoplankton populations will be necessary. We highlight the semantic difficulties present in this relatively new field of study and make recommendations for future study.

Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness

The study of cell death in higher plants and animals has revealed the existence of an active (‘programmed’) process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5–20% of the population) were found in late–phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.

Protection of cells from salinity stress by extracellular polymeric substances in diatom biofilms

Diatom biofilms are abundant in the marine environment. It is assumed (but untested) that extracellular polymeric substances (EPS), produced by diatoms, enable cells to cope with fluctuating salinity. To determine the protective role of EPS, Cylindrotheca closterium was grown in xanthan gum at salinities of 35, 50, 70 and 90 ppt. A xanthan matrix significantly increased cell viability (determined by SYTOX-Green), growth rate and population density by up to 300, 2,300 and 200%, respectively. Diatoms grown in 0.75% w/v xanthan, subjected to acute salinity shock treatments (at salinities 17.5, 50, 70 and 90 ppt) maintained photosynthetic capacity, Fq′/Fm′, within 4% of pre-shock values, whereas Fq′/Fm′ in cells grown without xanthan declined by up to 64% with hypersaline shock. Biofilms that developed in xanthan at standard salinity helped cells to maintain function during salinity shock. These results provide evidence of the benefits of living in an EPS matrix for biofilm diatoms.

Exploiting eco-physiological niche to facilitate the separation of the freshwater cyanobacteria Microcystis sp. and Synechococcus sp.

In a novel approach to separate the co-occurring freshwater cyanobacteria Microcystis and Synechoccous, published ecological characteristics are used to manipulate temperature and nutrient concentrations to successfully establish a unialgal Microcystis strain. The simple protocol has implications for future cyanobacterial culturing approaches and the establishment of new cyanobacteria strains.

Occurrence of chlorophyll allomers during virus‐induced mortality and population decline in the ubiquitous picoeukaryote Ostreococcus tauri

During viral infection and growth limitation of the picoeukaryote Ostreococcus tauri, we examined the relationship between membrane permeability, oxidative stress and chlorophyll allomers (oxidation products). Chlorophyll allomers were measured in batch-cultures of O. tauri in parallel with maximum quantum efficiency of photosystem II photochemistry (Fv /Fm ), carotenoids, and reactive oxygen species and membrane permeability using fluorescent probes (CM-H2 DCFDA and SYTOX-Green). Viral infection led to mass cell lysis of the O. tauri cells within 48 h. The concentration of the allomer hydroxychlorophyll a peaked with a 16-fold increase (relative to chlorophyll-a) just after the major lysis event. In contrast, cell death due to growth limitation resulted in a twofold increase in allomer production, relative to chl-a. Two allomers were detected solely in association with O. tauri debris after viral lysis, and unlike other allomers were not observed before viral lysis, or during cell death due to growth limitation. Conversely, the component chl-aP276 was found in the highest concentrations relative to chl-a, in exponentially growing O. tauri. The components described have potential as indicators of mode of phytoplankton mortality, and of population growth.