Cell cycle and cell mortality of Alexandrium minutum (Dinophyceae) under small-scale turbulence conditions

Decreased net population growth rates and cellular abundances have been observed in dinoflagellate species exposed to small-scale turbulence. Here, we investigated whether these effects were caused by alterations in the cell cycle and/or by cell mortality and, in turn, whether these two mechanisms depended on the duration of exposure to turbulence. The study was conducted on the toxic dinoflagellate Alexandrium minutum Halim, with the same experimental design and setup used in previous studies to allow direct comparison among results. A combination of microscopy and Coulter Counter measurements allowed us to detect cell mortality, based on the biovolume of broken cells and thecae. The turbulence applied during the exponential growth phase caused an immediate transitory arrest in the G2/M phase, but significant mortality did not occur. This finding suggests that high intensities of small-scale turbulence can alter the cell division, likely affecting the correct chromosome segregation during the dinomitosis. When shaking persisted for >4 d, mortality signals and presence of anomalously swollen cells appeared, hinting at the activation of mechanisms that induce programmed cell death. Our study suggests that the sensitivity of dinoflagellates to turbulence may drive these organisms to find the most favorable (calm) conditions to complete their division cycle.Postprin

Small-scale turbulence can reduce parasite infectivity to dinoflagellates

mall-scale turbulence and parasite infection are 2 important factors that govern the dynamics and fate of phytoplankton populations. We experimentally investigated the influence of turbulent mixing on the infectivity of the parasite Parvilucifera sinerae to dinoflagellates. Natural phytoplankton communities were collected during 3 stages of a bloom event in Arenys de Mar Har- bour (NW Mediterranean). The 15 to 60 μm size fraction was used as the inoculum and distributed into spherical flasks. Half of the recipients were exposed to turbulence while the rest were kept still. In the experiments, the dinoflagellate assemblage was mainly composed of Prorocentrum micans, Scrippsiella trochoidea and Alexandrium minutum. We observed a collapse of A. minutum and S. tro- choidea populations in the unshaken flasks, which coincided with an increase in parasite infectivity. After a short exposure to turbulence, the development of the dinoflagellate populations slowed down and stabilised as expected. In the shaken treatments, the infectivity was lower and the decay in the host cells numbers was delayed compared to the still treatments. The degree of interference of the turbulence with infectivity varied among the experiments, due to differences in cell abundances and possibly their physiological state. Results from a numerical model suggest that turbulence could lead to a 25 to 30% decrease in the maximum infection rate, which could be due to host population disper- sion and/or reduced host–parasite contact times. Turbulence may thus be effective in delaying the initial infection, but not in preventing it.Postprin

TURECOTOX and ECOALFACS Projects: Contributions of two GEOHAB Endorsed Projects

Berdalet, Elisa ... et. al.-- Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) Synthesis Open Science Meeting, 24-26 April 2013, Paris, FranceFrom 2009 to 2013, the TURECOTOX (CTM2006-13884-C02-00/MAR) and ECOALFACS (CTM2009- 09581) projects, endorsed by GEOHAB, focused on the interactions between small-scale turbulence and the biology of toxigenic dinoflagellates (toxin-producing HABs). Research included ecophysiological experiments and fieldwork in two contrasting areas: the Galician Rías Baixas (upwelling systems, Atlantic coast) and the microtidal estuary of Alfacs bay in the Ebro Delta (coastal embayment, stratified system, Mediterranean Sea). Laboratory experiments with cultures showed how small-scale turbulence can modulate different ecophysiological processes including growth rate, cell cycle patterns, asexual encystment, nucleic acids, toxin and DMSP cell quota and infection by parasites. However, the cellular mechanisms underlying the observed responses are still unknown. In the Rías, the population dynamics (division rate, viability, mortality) of Dinophysis spp. and their behavior (vertical migration, mixotrophy) were studied with the same spatio-temporal scale than the finescale hydrodynamical processes (water velocities, shear, vertical diffusion, turbulence). For the first time, data on the formation, maintenance and dissipation of thin layers of Pseudo-nitzschia spp. were obtained in this area. In Alfacs bay, several modeling approaches (3D hydrodynamic model combined with a Lagrangian particle-tracking module) validated by continuous records of physical and meteorological data have been implemented to understand how the complex circulation dynamics may facilitate water retention and thus phytoplankton biomass accumulation in the inner part of the bay. We hope that most undergoing efforts will improve the understanding of the link between physical dynamics and biological and ecological processes (growth, mortality, migration) of selected HAB taxa and/or functional groups in the bayPeer Reviewe

Effects of small-scale turbulence on dinoflagellate ecophysiology

Directora de tesis: Dra. Elisa Berdalet (Institut de Ciències del Mar, CSIC, Barcelona).Tesis defendida en Barcelona el 20 de Noviembre de 2009. Excelente cum laude por unanimidad. Los capítulos de la tesi 1, 4, 5 y 6 han sido publicados como artículos científicos en revistas del SCI entre los años 2007 y 2011Dinoflagellate proliferations seem to be often related to certain stability of the water column. Laboratory experiments have shown that small-scale turbulence can diminish the net growth rate and inhibit cell division, and simultaneously increase the cell size and the DNA and RNA content. The objective of this PhD Thesis was to explore direct effects of small-scale turbulence on physiological processes related to the cell and life cycles of dinoflagellates, and to investigate the underlying mechanisms in the observed responses. The gained knowledge should provide a better understanding of the development of their proliferations. The cell cycle of the dinoflagellate Alexandrium minutum was transiently arrested in the G2/M phase. It was explored whether such interference was due to an eventual alteration, due to turbulence, of the microtubular system implicated in the dinomitosis, whether ryanodine receptors were involved in the mechanotransduction pathway activated by this stimulus, and also whether a possible differential gene expression occurred under turbulence. Toxins and DMSP concentrations, the proportion of ecdysal cyst and the endoparasite Parvilucifera sinerae infection on dinoflagellates were also modulated by turbulence. Namely, infectivity decreased under shaking, and likely, asexual encystment did not constitute a preventive mechanism to infection. The suitability of some experimental systems for turbulence generation was also considerate. The orbital shaker is the most appropriate setup to carry out physiological studies on dinoflagellates, since the whole culture is exposed to turbulence, and cells cannot escape it. The present Thesis concludes that dinoflagellates prefer calm water column situations, since their physiology is sensitive to intense mixingSpanish projects: TURFI (REN2002-01591/MAR), TURDITOX (CTM2005-03547/MAR), TURECOTOX (CTM2006-13884-C02-00/MAR) VARITEC (CTM2004-04442-C02). G. Llaveria fue becaria FPU del MICINN (2004-2008

Modulation of dimethylsulfoniopropionate (DMSP) concentration in an Alexandrium minutum (Dinophyceae) culture by small-scale turbulence: A link to toxin production?

8 pages, 3 figuresSome marine dinoflagellates produce important amounts of dimethylsulfoniopropionate (DMSP), a common compatible solute, and its cleavage product dimethylsulfide (DMS), a climatically active trace gas. In the field, dinoflagellate proliferations appear to be favored by calm weather and water column stability; indeed, small-scale turbulence is a physical factor that directly affects ecophysiological aspects of this phytoplankton group, including toxin production. Here we report the effect of experimentally generated turbulence on DMSP production by a paralytic shellfish poisoning (PSP) toxin producing strain of Alexandrium minutum, a widespread bloomforming dinoflagellate species. With respect to still conditions, the populations exposed to turbulence grew at a slower growth rate and yielded low cell numbers turbulence. Concurrently, the cellular DMSP concentration increased by ca. 20% (from 0.22 ± 0.01 to 0.27 ± 0.03 fmol μm−3 on a cell volume basis) in the shaken cultures. DMSP was preferentially synthesized during the light period in both treatments. During the night, a slowdown of the division process caused DMSP accumulation in the cells exposed to shaking. The study suggests the existence of a tight link between the dynamics of DMSP concentration and other cell processes entrained by circadian rhythms in dinoflagellates. The observed effects of small-scale turbulence on the DMSP dynamics supports the suggested role of this compound as an overflow mechanism in metabolically unbalanced cells. Furthermore, considering all the effects on the physiology of A. minutum exposed to the same experimental setup, we propose a possible link between the DMSP and the PSP metabolismsThis work has been supported by the Spanish funded projects TURECOTOX (CTM2006-13884-C02-00/MAR) and ECOALFACS (CTM2009-09581), that are endorsed to the SCOR/IOC program GEOHAB (Golbal Ecology and Oceanography of Harmful Algal Blooms, www.geohab.info). G. Llaveria held an FPU fellowship from the Spanish Ministry of Science and Education. We appreciate the technical support kindly provided by Martí GalíPeer reviewe

Exploring Why Dinoflagellates Are So Sensitive To Small-Scale Turbulence.The Microtubule Assemblage Alteration Hypothesis

14th International Conference on Harmful Algae, 1-5 November 2010, Creta, GreeceSmall-scale turbulence has been shown to slow down the growth and/or some phases of the cell cycle of some dinoflagellate species, and it seems that turbulence or shear stress could alter the cell division processes. Here, we report our studies to investigate the unknown underlying mechanisms of the observed responses. We tested the hypothesis that turbulence could alter the correct assemblage of microtubules composing the mitotic spindle involved in the chromosome segregation during the dinomitosis. In shaking experiments performed on Akashiwo sanguinea, Prorocentrum micans and Oxyrrhis marina, microtubules were visualized through a combination of inmunocytochemistry (using anti-tubulin monoclonal antibodies) Confocal Fluorescent Laser Microscopy. Each species showed particular organizations of the nuclear, microtubular and other mitotic elements under still conditions. These mitotic organizations appeared unmodified by turbulence. Thus, our results did not provide evidences to support the microtubule hypothesis. Some alternative hypotheses also linked to the microtubular organization in the cell are suggestedPeer Reviewe

Particularities of dinomitosis pattern in different dinoflagellates

11th International Conference on Modern and Fossil Dinoflagellates (DINO11), 17-21 July 2017, Bordeaux, FranceDinoflagellates are primitive unicellular eukaryotes showing nuclear and mitotic peculiarities. Their chromosomes are visible and remain condensed at all stages of the cell cycle, they do not have true histones, the nuclear envelope persists during mitosis, whereas the mitotic spindle is usually cytoplasmic, making their division a complex process called dinomitosis. While there presently exist about 2000 species of dinoflagellates, only a few of them have been studied at the cellular level. Here, we show that dinomitosis differs greatly in several species, namely, Akashiwo sanguinea, Karenia brevis, Oxyrrhis marina and Prorocentrum micans. We suggest that dinoflagellates, which represent an heterogeneous group in various cellular, physiological and structural aspects, are also heterogeneous in terms of mitosis. Dinoflagellates must be regarded as an >experimental group> making an evolutive link between bacteria, fungi and higher eukaryotes in terms of cell division processes, that we call dinomitosesPeer Reviewe

Modulation of ecdysal cyst and toxin dynamics of two Alexandrium (Dinophyceae) species under small-scale turbulence

In some dinoflagellate species, physiological processes appear to be altered by exposure to certain turbulent conditions. Here we investigated how two levels of turbulent kinetic energy dissipation rates (ε = 0.4 and 27cm2s-3) affected the toxin and ecdysal cyst dynamics of two bloom forming species, Alexandrium minutum and A. catenella. The most striking responses were observed at the high s generated by an orbital shaker. In A. catenella, lower cellular toxin content was measured in cultures shaken for more than 4 days. The same trend was observed in A. minutum, although variability masked statistical significance. For the two species, inhibition of ecdysal cyst production occurred immediately and during the period of exposure of the cultures to stirring (4 or more days) at any time during their growth curve. Recovery of cyst abundances was always observed when turbulence stopped. When turbulence persisted for more than 4 days the net growth rate significantly decreased and the final biomass yield was lower than in the unshaken cultures. This study suggests that high levels of small-scale turbulence would contribute to the modulation of the harmful bloom dynamics through the interaction at the level of toxin and encystment processesThis work has been supported by the Spanish funded projects TURFI 25 (REN2002-01591/MAR) and TURDITOX (CTM2005-03547/MAR) and by the EU funded project SEED (GOCE-CT-2005-003875). L. Bolli held a “Leonardo da Vinci” grant within the StudEX program of Switzerland and Ò. Guadayol a predoctorate I3P fellowship from the CSIC. G. Llaveria holds an FPU grant of the Spanish Ministry of Science and Education (MSE). E. Garcés and F. Peters are sustained by the Spanish “Ramon y Cajal” contracts of the Spanish MSE and 30 K. van Lenning by the “Agència Catalana de l’Aigua” of the Catalan Autonomous GovernmentPeer Reviewe

Direct effects of small-scale turbulence on the physiology of red-tide involved dinoflagellates

Symposium GLOBEC-IMBER España celebrado del 28-30 marzo de 2007 en Valencia.-- 1 pageLaboratory experiments have shown that many dinoflagellate species display some degree of sensitivity to certain levels of small-scale turbulence (last review by Berdalet and Estrada 2005). The response is species-specific and highly dependent on the intensity and the experimental design and setup used. The reported effects (including alterations of morphology, population development -biomass yield and net growth rate-, mortality, migration pattern), point to a particular interference of turbulence with cell division and life cycle processes, although the involved mechanisms are still not known. In the field, dinoflagellate proliferations appear to be favoured by calm weather and water column stability (Margalef et al., 1979). Given the ecological and economic importance of such harmful events, our line of research is trying to unveil the effects of small-scale turbulence on the physiology of dinoflagellates, specially focussing on cyst production, toxin content, swimming and cell division patterns (Berdalet et al., in press). Here we will illustrate the main striking results of our experiments performed on widespread bloom forming species, such as, Alexandrium catenella and A. minutum,Prorocentrum micans and Akashiwo sanguinea. Turbulentce has been generated with an orbital shaker and the corresponding range of turbulent kinetic dissipation rates has been estimated by an acoustic Doppler velocimeter. Aware that any laboratory setup cannot mimic nature, we aim to set our physiological studies into an ecologic framework. Bibliography: Berdalet, E. and M. Estrada. 2005. “Effects of small-scale turbulence on the physiological functioning of marine algae” In Subba Rao, D. V., [Ed.] Algal cultures, analogues and applications. Science publishers, Enfield, NH, USA, pp. 459-500 Berdalet, E., F. Peters, V. L. Koumandou, C. Roldán, Ó. Guadayol, and M. Estrada. “Species-specific physiological reponse of dinoflagellates to quantified small-scale turbulence”, J. Phycol., in press. Margalef, R., M. Estrada, D. Blasco. 1979. “Functional morphology of organisms involved in red tides, as adapted to decaying turbulence. In Taylor, D. L. & Seliger H. H. [Ed.] Toxic dinoflagellate blooms. Elsevier North Holland, Inc., pp. 89-9