As a dinoflagellate bloom is a natural, environmental phenomenon, one has never been studied in the laboratory. The scales involved (tiny cells in a large body of water) make it impossible, and all aspects of a bloom (bacteria, viruses, parasites, grazers, competing phytoplankton, water movements, stratification, nutrients, rain, land runoff, season, temperature changes, pheromones, toxins, allelopathy etc. etc.) cannot be mimicked simultaneously. Different aspects are studied separately, and the laboratory environment necessitates keeping cultures in enclosures to enable quantitative studies on the factors of interest. The present study shows that the effect of confinement is probably much greater than previously known. Most dinoflagellate species cannot be grown into dense cultures; they stop growing at a cell density far from what is common for other cultivable phytoplankton. We used cages of plankton screen inserted in a larger volume of circulating medium to avoid confinement in a small volume of medium while still enabling the study of dense cultures. The experiment was inoculated with high densities of cells in the exponential vegetative growth stage, but exponential growth of cultures was not seen, nor lag phase or stationary phase. A high cell density simultaneously with access to fresh growth medium accelerated gamete formation rather than suppressing it. The results from the present experiment show two very important aspects of dinoflagellate growth:
1) Dinoflagellates could grow faster and into a denser culture with flow-cell culturing (confinement quickly restricts growth)
2) Dense cell accumulations triggered induction of the sexual phase without nutrient limitation, showing “quorum sensing” in dinoflagellates
Very high cell densities were reached with flow cell culturing, the highest measurement representing 342,000 cells · mL− 1. Thus, S. lachrymosa cells can grow very dense, but only when the medium is changed. The cells became much more numerous when they escaped outside the cage, which means that escaping cells grew faster than cells still inside the cage. Both cell density (closeness to other cells) and availability of nutrients/flow is apparently important. Cages with few escaping cells had fewer cells in total, even though the flow was fast and nutrients in the added medium not limiting. Was the medium the same or different outside the cage? The results indicate that it was probably different because of fast changes in nutrient uptake and/or waste disposal causing the environment to become different in the high cell density environment inside cages despite the fast flow of the medium. Cell-cell proximity (quorum sensing) was very important; it induced sexuality and inhibited continued vegetative growth. Because of the low Reynolds numbers, each cell is surrounded by a boundary layer of water (Breckels et al. 2010 and references therein) in which exudates have a higher concentration and nutrients are of lower levels than in the “free” water, and close proximity between cells has disadvantages at the same time as it is necessary for enabling gamete fusion. Carbon filters are used for cleaning STX from cyanobacteria in freshwater in Australia (e.g. Falconer et al. 1989; Orr et al. 2004). A preliminary test with Alexandrium catanella indicated that circulation through a filter with activated carbon could be favorable and we hypothesized it could remove inhibitory substances and enhance growth, but this was not confirmed the present experiment. No significant differences could be found between treatments with and without carbon filter.
Cultures did not reach stationary phase even though cell densities were very high, and we do not know where maximum cell density could be with the continued addition of nutrients (exchange of medium). The sexual phase was clearly initiated and the possibility of continued growth into even denser cultures remains unexplored. Experiments were also performed in the same way with A. catanella during methods development for the present experiment, but the high cell densities attained (up to 250,000 cells · mL− 1) caused fear of exposure to high toxin levels and were discontinued. A. catanella demonstrated so sharp and fast pattern formation that mixing of samples by hand stirring (with a glass rod or pipette tip) into an even distribution of cells was not possible. Cells, when pattern forming, used every water movement as an opportunity to assemble and formed patterns of accumulation. This behavior is probably very important in nature; the cells ‘use’ water movements for accumulation and transport. This behavior can, together with oceanographic factors such as stratification and movement of water masses, explain bloom formation. The pattern forming behavior observed macroscopically, occurred (in this experiment and all our previous, published and unpublished experiments) simultaneously with gamete formation as observed microscopically (described for S. lachrymosa in Smith and Persson 2005, and for A. fundyense in Persson et al. 2013 and Persson and Smith 2013).
The induction of gamete formation by bringing cells close to each other without nutrient limitation, shown here, suggests “quorum sensing” in dinoflagellates. The term quorum sensing is most often used to describe a form of cell-to-cell communication by which bacteria (by secreting signalling molecules) can regulate cell density- or growth-phase-dependent processes (e.g. Portugal 2013). When cells form patterns, a very high cell density is achieved within the pattern itself. Perhaps access to fresh medium at the same time as high cell numbers is something that occurs in nature but has never before been studied in the laboratory. We speculate that quorum sensing substances are formed rapidly and diffuse rapidly. Enclosed in a small container, these substances remain within the enclosure. The bloom-forming dinoflagellates we studied can swim and in nature stay in a gathered assembly of cells by swimming together, and substances produced diffuse away in the vast water mass. A high concentration of substances needed to attract co-specific gametes and deter predators and competitors requires continuous regeneration to counteract diffusion. This can lead to very high levels of such substances in a "batch culture", which in turn may explain the limitation in cell density (i.e., through auto-inhibition). Alternations between asexual growth and sexual reproduction are widespread in nature and very common in phytoplankton (Fryxell 1983). Induction of sexual behavior is often triggered by environmental cues when, or slightly before, environmental conditions deteriorate for the asexually dividing stages. In dinoflagellates, it is known that N-limitation alone can trigger gamete formation in the laboratory (e.g. Persson et al. 2008 and references therein). In nature, however, it is reasonable to assume that several signals together (light, temperature, stratification, nutrients from land run-off, etc.) make up a “seasonal cue” (Persson et al. 2008; Figueroa et al. 2011). Optimal timing of sexual reproduction is important in nature as sexual reproduction instead of asexual means major changes in metabolism and behavior that must be consistent with seasonal weather patterns that for dinoflagellates can be, for example, rain with land-runoff and stratification and/or water movements allowing accumulation and transport. Accumulating evidence points to “blooms”, e.g. densely accumulated cells, as consisting of sexual stages forming patterns of accumulation involving chemical attraction (Persson et al. 2013; Persson and Smith 2013; Wyatt and Zingone 2014; Brosnahan et al. 2015), and these cell accumulations being transported by hydrodynamic action into even denser patches or layers. It is well documented that high numbers of dinoflagellates can be found in thin layers during calm weather after rainfall with land runoff when water mass is density-stratified (e.g. Smayda 1997). Such water masses can subsequently be transported to the surface driven by shifts in meteorological forcing variables (Raine 2014). Surface blooms often form when wind causes upwelling and transport of water masses containing accumulated cells with currents into nearshore areas (Anderson et al. 2012, Ruiz-de la Torre et al. 2013, Lai and Yin 2014, McGillicuddy et al. 2014, Raine 2014). The cells in a bloom are thus accumulated by swimming behaviors and physical transport by larger forces in nature, and the cells in a sample from a bloom can originate from (have grown in) a much larger volume of water (and elsewhere) than the volume of the sample taken.
A comparison between cultures started with high or low cell numbers of Scrippsiella lachrymosa shows clearly that the environment (availability of nutrients and elimination of waste substances) is much more important than the starting number of cells. Also, in the field the same phenomenon occurs; the number of germinated cysts or starting cell number is far less important than growth conditions in the water (Martin et al. 2014). It is difficult to estimate the size of a background population that is thinly distributed in a large area and volume in the field. If conditions are favorable for growth, there is a possibility that growth can continue undiscovered until a seasonal signal induces sexuality and accumulation of cells, which are further concentrated by cell behavior and weather patterns as described above. A seemingly low-nutrient or low background population situation can thus still lead to dense dinoflagellate blooms, depending on local weather patterns and transport. Can growth rate in nature be vastly underestimated from lab experiments because of limitations set by culturing conditions? Our results show declines in growth (decreasing growth rate), even though the medium was exchanged and nutrients not limiting. When studying growth curves in published dinoflagellate experiments one can see that the exponential growth phase is very short. An extrapolation of the exponential phase imagining a period of optimum conditions answers our question. The growth rate in nature during optimum conditions can probably be much higher than previously thought. This is also suggested by Brosnahan et al. (2015) and Anderson et al. (2012).
Many have wished to mass culture dinoflagellates to enable extraction of toxins and other valuable substances in large scale. The small heterotrophic dinoflagellate Crypthecodinium cohnii is cultured in industrial scale for the extraction of docosahexaenoic acid (DHA), an omega-3 fatty acid, for infant formula (Mendes et al. 2009). The vast majority of dinoflagellates are, however, impossible to grow in large scale even though they are known to cause dense blooms. An understanding of the causes underlying dinoflagellate blooms in nature is necessary. Dinoflagellate blooms do not represent cells that have grown where they are found in large amounts. Underlying factors are, as discussed above, seasonal accumulation (possibly by sexual life stages) in water masses that are secondarily transported by upwelling and currents into the areas where blooms eventually are found.