Jason E. Adolf

Digital holographic microscopy reveals prey-induced changes in swimming behavior of predatory dinoflagellates

The shallow depth of field of conventional microscopy hampers analyses of 3D swimming behavior of fast dinoflagellates, whose motility influences macroassemblages of these cells into often-observed dense “blooms.” The present analysis of cinematic digital holographic microscopy data enables simultaneous tracking and characterization of swimming of thousands of cells within dense suspensions. We focus on Karlodinium veneficum and Pfiesteria piscicida, mixotrophic and heterotrophic dinoflagellates, respectively, and their preys. Nearest-neighbor distance analysis shows that predator and prey cells are randomly distributed relative to themselves, but, in mixed culture, each predator clusters around its respective prey. Both dinoflagellate species exhibit complex highly variable swimming behavior as characterized by radius and pitch of helical swimming trajectories and by translational and angular velocity. K. veneficum moves in both left- and right-hand helices, whereas P. piscicida swims only in right-hand helices. When presented with its prey (Storeatula major), the slower K. veneficum reduces its velocity, radius, and pitch but increases its angular velocity, changes that reduce its hydrodynamic signature while still scanning its environment as “a spinning antenna.” Conversely, the faster P. piscicida increases its speed, radius, and angular velocity but slightly reduces its pitch when exposed to prey (Rhodomonas sp.), suggesting the preferred predation tactics of an “active hunter.”

A dinoflagellate exploits toxins to immobilize prey prior to ingestion

Toxins produced by the harmful algal bloom (HAB) forming, mixotrophic dinoflagellate Karlodinium veneficum have long been associated with fish kills. To date, the perceived ecological role for toxins has been relief from grazing pressures. Here, we demonstrate that karlotoxins also serve as a predation instrument. Using high-speed holographic microscopy, we measure the swimming behavior of several toxic and nontoxic strains of K. veneficum and their prey, Storeatula major, within dense suspensions. The selected strains produce toxins with varying potency and dosages, including a nontoxic one. Results clearly show that mixing the prey with the predatory, toxic strains causes prey immobilization at rates that are consistent with the karlotoxins’ potency and dosage. Even prey cells that continue swimming slow down after exposure to toxic predators. The swimming characteristics of predators vary substantially in pure suspensions, as quantified by their velocity, radii of helical trajectories, and direction of helical rotation. When mixed with prey, all toxic strains that are involved in predation slow down. Furthermore, they substantially reduced their predominantly vertical migration, presumably to remain in the vicinity of their prey. Conversely, the nontoxic control strain does not alter its swimming and does not affect prey behavior. In separate experiments, we show that exposing prey to exogenous toxins also causes prey immobilization at rates consistent with potency. Clearly, the toxic predatory strains use karlotoxins as a means of stunning their prey, before ingesting it. These findings add a substantiated critical understanding for why some HAB species produce such complex toxin molecules.

Strain variation in Karlodinium veneficum (Dinophyceae): toxin profiles, pigments, and growth characteristics.

Karlodinium veneficum (D. Ballant.) J. Larsen strains, 16 from the U.S. Atlantic eastern seaboard and two from New Zealand (CAWD66 and CAWD83), were used to characterize toxin profiles during batch culture. All 18 strains were determined as the same species based on ITS sequence analyses, a positive signal in a chloroplast real‐time PCR assay and pigment composition. Five karlotoxin 1 (KmTx 1) containing strains were analyzed from the Chesapeake Bay, and 10 karlotoxin 2 (KmTx 2) strains were analyzed from Florida to North Carolina. One strain (MD5) from the Chesapeake Bay produced no detectable toxin. The two cultures from New Zealand contained both novel karlotoxins with lower masses and earlier elution times. Toxin type did not change during batch culture, although the KmTx phenotype did change in some strains under extensive (months) phototrophic growth in replete media. KmTx cell quota did not change during batch culture for most strains. The mass spectrum for every KmTx examined showed a pattern of multiple coeluting congeners within each HPLC peak, with masses typically differing by 16 amu. KmTx congeners tested showed nearly a 500‐fold range in specific hemolytic activity, with KmTx 1 (typically occurring at lower cellular levels) most hemolytic and CAWD66 toxin least hemolytic, while KmTx 2 and the CAWD83 toxin had similar intermediate specific activity. Despite morphological, genetic, and photopigment indicators consistent with species homogeneity among the 18 strains of K. veneficum, the high degree of toxin variability suggests different functional roles among strains that likely coexist in situ.

AUTOTROPHIC GROWTH AND PHOTOACCLIMATION IN KARLODINIUM MICRUM (DINOPHYCEAE) AND STOREATULA MAJOR (CRYPTOPHYCEAE)1

We compared autotrophic growth of the dinoflagellate Karlodinium micrum (Leadbeater et Dodge) and the cryptophyte Storeatula major (Butcher ex Hill) at a range of growth irradiances (Eg). Our goal was to determine the physiological bases for differences in growth–irradiance relationships between these species. Maximum autotrophic growth rates of K. micrum and S. major were 0.5 and 1.5 div.·d−1, respectively. Growth rates were positively correlated with C‐specific photosynthetic performance (PPC, g C·g C−1·h−1) (r2=0.72). Cultures were grouped as light‐limited (LL) and high‐light (HL) treatments to allow interspecific comparisons of physiological properties that underlie the growth–irradiance relationships. Interspecific differences in the C‐specific light absorption rate (EaC, mol photons·g C−1·h−1) were observed only among HL acclimated cultures, and the realized quantum yield of C fixation (φC(real.), mol C·mol photons−1) did not differ significantly between species in either LL or HL treatments. The proportion of fixed C that was incorporated into new biomass was lower in K. micrum than S. major at each Eg, reflecting lower growth efficiency in K. micrum. Photoacclimation to HL in K. micrum involved a significant loss of cellular photosynthetic capacity (Pmaxcell), whereas in S. major, Pmaxcell was significantly higher in HL acclimated cells. We conclude that growth rate differences between K. micrum and S. major under LL conditions relate primarily to cell metabolism processes (i.e. growth efficiency) and that reduced chloroplast function, reflected in PPC and photosynthesis–irradiance curve acclimation in K. micrum, is also important under HL conditions.

MODULATION OF POLYUNSATURATED FATTY ACIDS IN MIXOTROPHIC KARLODINIUM VENEFICUM (DINOPHYCEAE) AND ITS PREY, STOREATULA MAJOR (CRYPTOPHYCEAE) 1

We examined whether fatty acid (FA) composition changed when Karlodinium veneficum (D. Ballantine) J. Larsen (Dinophyceae) was grown phototrophically or mixotrophically on Storeatula major Butcher ex D. R. A. Hill (Cryptophyceae). We hypothesized that the FA composition of mixotrophic K. veneficum would not change relative to the FA composition of phototrophic K. veneficum. As in other phototrophic dinoflagellates, octadecapentaenoic acid (18:5n3) represented 9% to 20% of total FA in K. veneficum and was enriched within chloroplast‐associated galactolipid classes. The 18:5n3 content showed a highly significant positive correlation (r2 = 0.95) with chl a content and a highly significant negative correlation with growth rate (r2 = 0.88). A previously undescribed chloroplast galactolipid molecular species, digalactosyldiacylglycerol (DGDG; 18:5n3/18:5n3), was a dominant structural lipid in K. veneficum. Docosahexaenoic acid (22:6n3) represented 14% to 19% of total K. veneficum FA and was enriched within phospholipids. In the prey S. major, 18:5n3 was not present, but octadecatetraenoic acid (18:4n3) and α‐linolenic acid (18:3n3) represented approximately 50% of total FA and were enriched within chloroplast‐associated galactolipid classes. Eicosapentaenoic acid (20:5n3) and 22:6n3 represented approximately 18% of total FA in S. major and were enriched within phospholipids. The FA profile of mixotrophic K. veneficum, compared to phototrophic K. veneficum, showed elevated levels of 18:3n3, 18:4n3, and 20:5n3, and lower but persistent levels of 18:5n3. Production to ingestion (P:I) ratios >1 for major polyunsaturated fatty acids (PUFAs) indicated that direct assimilation from prey under balanced growth could not support rates of PUFA production in mixotrophic K. veneficum. These data suggest that the plastid plays a continuing and essential role in lipid metabolism during mixotrophic growth.