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    Roxana Hernandez, Claudia Mengelt, Barbara B. Prézelin, Department of Ecology, Evolution and Marine
                              Biology University of California, Santa Barbara
                                        Due to the increased levels of organic and inorganic nitrogen from anthropogenic eutrophication, much effort has gone into studying possible
                                        effects on phytoplankton. This study intends to test whether the growth of Pseudo-nitzschia multiseries is enhanced by the addition of organic
                                        nutrients under various light conditions. Previous studies have shown that some phytoplankton species can use DON, and that P. multiseries might
                                        be included among this list (Palenik et al., 1990, Hillebrand and Sommer, 1996, Seitzinger et al., 1997 & 2002, Mengelt & Prézelin, 2002).
                                        Examining the heterotrophic abilities that this species has in the dark will further the knowledge on the link between eutrophication/runoff and bloom
                                        initiation via the extended survival of a “seed population”. This is especially important since P. multiseries is a marine planktonic diatom that
                                        produces the neurotoxin domoic acid. This neurotoxin is potentially deadly to humans and other marine mammals when consumed via
                                        contaminated shellfish and fish, such as anchovies (Mos, 2001). Consequently, P. multiseries can potentially cause large economical, social, and
                                        environmental damage. Therefore, this study will present results from organic and inorganic nitrogen addition experiments under various light/dark
                                        conditions to verify that growth is enhanced by these organic nutrient additions.

                                                                                                                                                     Definition Eutrophication:                                                                                                                                                               Hypothesized Model
   The intend of this project was to test whether Pseudo-
                                                                                                                                                                                                                                                                                                       Euphotic                                                                                   Photo-
   nitzschia multiseries is able to grow in the dark and                                                Point sources:
                                                                                                                                                                                                              Non - point sources:                                                                       Zone                                                                                     autotrophi
                                                                                                        Sewage                                                                                                                                                                                                                                                     aging                          c
   (various) light on organic nutrients. The experiment                                                 Industrial runoff
                                                                                                                                                                                                              Agricultural                                                                                                                               Bloom
                                                                                                                                                                                                              & urban runoff                                                                                                                                                           Senescence &
   examined the heterotrophic abilities in the dark and                                                                                                             Algal nutrient

   light. In order to determine the extend of the                                                                                                                    enrichment

   heterotrophic capacity P. multiseries, an indoor
                                                                                                                                                                    Algal biomass                                                                                                                      Aphotic
   growth experiment was conducted. The experiment                                                                                                                                                                                                                                                                                                                                                  heterotrophic
                                                                                                                                                                      increases                                                                                                                         Zone
   was run in an indoor system on inorganic media with                                                                                                                                                                                                                                                                                                                   Cells in the dark

   and without the presence of organic nutrients. This                                                                                                      Harmful Algal
                                                                                                                       Anoxia                                                             Decline in Seagrass                                                                                                                                                           (Adapted from Trainer et al, 1998)
   study intends to test the hypothesis that there will be                                                                                                     Blooms
                                                                                                                                                                                                                                                   Fig. 4: Conceptual Working Model - Pseudo-nitzschia blooms are
   a significant difference in growth in treatments with                                                                                                                                                                                           associated with moderate upwelling events (Trainer et al., 2000) and/or
   the addition of glutamate and glutamine. The results                                                                                                      Fish - Kills & Mortality                                Decline in                    runoff (Smith et al, 1990). If this is correct than why or how do P. multiseries
                                                                                                    Mortality in the                                        At Higher Trophic Levels                                fish habitat
   for P. multiseries are compared with a similar study by                                         Benthic Community                                                                                                                               survive in the dark. The question now becomes whether there is a resting
                                                                                                                                                                                                                                                   stage (seed like) or whether it has heterotrophic abilities. Aging of the
   Mengelt & Prézelin (2002). Repeating these                                                                                                                                                                                                      bloom will result in domoic acid production and most likely transparent
   experiments for other ecologically important algal                                             Fig. 3: Consequences of Algal Blooms due to anthropogenic N increase.                                                                            exopolymer particle (TEP) excretion, increasing the rate of aggregation
                                                                                                                                                                                                                                                   (Alldredge & Gotschalk, 1989; Bates et al., 1991).
   groups is crucial to make a comparison that allows to
   speculate on whether anthropogenic eutrophication
   could affect species composition in nature.
                                                                                                                                                                                                                               Previous research
                                                                                                                                                                                                                                                                                                                                                      Results - Dark Survival
                                                                                                                                                         Cellular Growth Rates
                                                                                                                 0.5                                                                                                                                                                                                                                  Dark Survival of Pseudonitzschia multiseries
                                                                                                                                           Cell growth - Control                                                                                                                                                                  35                                                                            100

                                                                                                                 0.4                       Cell growth + Organics                                                                                                                                                                                                                                               90
                                                                                                   Growth rate


                                                                                                                                                                                                                                                                                                           Cell Numbers (X10^4)


                                                                                                                                                                                                                                                                                                                                                                                                                      % viable cells
                                                                                                                 0.3                                                                                                                                                                                                                                                                                            60
                                                                                                                                                                                                                                                                                                                                  10                                                                            30
                                        Top Predators
                                                                                                                                                                                                               No Growth
                                                                                                                 0.1                                                                                                                                                                                                                                                                                            10
                                                                                                                                                                                                                                                                                                                                   0                                                                            0
                                                                                                                                                                                                                                                                                                                                           initial       1        2         3         4       5          6
                                                                                                                                                                                                                                                                                                                                                                        time in the dark (weeks)
                                                                                                                                           1         2         3              4           5            6
                                                                                                                                                     Number of weeks in the dark                                                                                                                                                                     Cell #s Control                   Cell #s With Organics
                                                                                                                                                                                                                                                                                                                                                     % viable Control                  % viable With Organics

                                                                                                Fig. 5: Growth rates in the light after 1 to 2 weeks in the dark. Cultures with
                                                                                                organics were higher compared to cultures without organics                                                                                    Fig. 6: Glutamate & Glutamine increased the cell numbers in the dark during the first week.

                                                                                                                                                                                                                               Results and Discussion
         NO3, NH4                                                          NO3, NH4,
                                                            HABs           Organic N
                                                                                                In our first experiment to determine dark heterotrophic, we did not observe any growth in the dark for P. multiseries
              Fig. 1: Mechanism of Bioaccumulation of Domoic acid
              through the food web by P. multiseries.                                           regardless of organic N concentration in the media (see figure 7). After only three days it was clear that bacterial growth
                                                                                                was compromising the experiment. Bacteria were observed (confirmed qualitatively under microscope) as medium
                                                                                                turned cloudy, which is an indication of high bacteria counts. As a result, the light experiment was aborted and only dark
                                                                                                treatment continued. There are many reasons why no growth was observed in this experiment. However, there are three
    Materials and Methods                                                                       main reasons why this could have occurred. One, contributing factor is bacteria contamination, which causes
     The set-up for the experiment will be conducted by                                         competition for the same organic nutrients between the bacteria and P. multiseries. Another is that in the previous
     preparing 10L of artificial sea water (ASW) and                                            experiment carried out in the Prézelin Lab utilized natural filter seawater, in contracts this experiment used artificial
     placing them in plastic bottles, which will then be                                        seawater. In order to definitively answer my question of heterotrophic growth, the experiment will have to be repeated
     refrigerated, see figure for detail set-up. A sample                                       using antibiotics to minimize bacterial contamination. A preliminary study was conducted to test whether antibiotics
     will be taken each day at the same time. Data                                              inhibit the growth of P. multiseries. This preliminary experiments revealed no algal growth inhibition by antibiotics (see
     analysis of growth and chl a of previous                                                   figure 8). The next step will be to repeat the first experiment by excluding bacteria by using the addition of antibiotics.
     experiments will be compared to indoor nutrient
     experiment. In order to determine the extend of the                                                                                             Pseudo-nitzschia multiseries cell concentration                                                                                                                              Pseudo-nitzschia multiseries Antibiotic Growth Experiment
                                                                                                                                           10                                                                                                                                              45.00
     heterotrophic capacity P. multiseries; an indoor                                                                                                                                                                   D-1C                                                               40.00
                                                                                                                                                                                                                                                                                                          With Antibiotics

     growth experiment will be conducted. The                                                                                               8
                                                                                                                                                                                                                        D-3C                                                               35.00
                                                                                                                                                                                                                                                           Cell Density [x10^4 cells/mL]

     experiment will run in an indoor system on                                                                                                                                                                                                                                            30.00
                                                                                                                         Cells/mL (x104)


     inorganic media with and without the presence of                                                                                                                                                                                                                                      25.00

     organic nutrients.                                                                                                                     4


                                                                                                                                            2                                                                                                                                              10.00

                                                                                                                                            11/18   11/19   11/20     11/21       11/22       11/23   11/24     11/25      11/26                                                            0.00
         Track                                                                                                                                                                                                                                                                                     0                                   2                   4             6                 8            10                  12
                     Cell #

         growth                                                                                                                                                                                                                                                                                                                                                  Day of Experiment
                                  while in log-phase
                                                                                                Fig. 7: Cell numbers in the dark over the course of the experiment for the                                                                           Fig.8: Cell numbers in the light over the course of the experiment
Grown in the light                                                                              inorganic culture (blue line D-1C), the mixed organic/inorganic culture (red                                                                         for the control culture (blue line), the antibiotic culture (red line)
                                                                                                line D-2C), and the organic culture (green line D-3C)

                              control                                                                  Dohler, G., 1997 Assimilation of Inorganic Nitrogen by Antarctic and Temperate Marine Phytoplankton Species under UV Stress, Russian Journal of Plant Physiology, Vol 44: 518-525
                              Light            (+ organic                                              Dohler, G., Drebes, G., Lohmann, M., 1997 Effects of UV-A and UV-B radiation on pigments, free amino acids and adenylate content of Dunaliella tertiolecta Butcher (Chlorophyta), Journal of Photochemistry and
                              treatment        Nutrients)                                              Photobiology B: Biology 40: 126-131
                                                              Daily Measurements:
                                                                                                       Kerfoot, John M., (2000) Growth and Photophysiological Responses of Dinoflagellate Heterocapasa Pygmaea to outdoor culturing in the presence and absence of solar ultra-violet radiation. M.S. thesis. University of
                              Dark                            • Growth (cell counts & chl a)           California, Santa Barbara.
                              control                         • Absorption Spectra                     Mengelt, C., Prézelin, B. B., 2002 Dark Survival and Subsequent Light Recovery for Pseudo-nitzschia multiseries. Poster, 10th HAB Conference, St. Petersburg, Florida.
                              Dark             (+ organic                                              Mos, L., 2001 Domoic acid: a fascinating marine toxin. Environmental Toxicology and Pharmacology, 9, 79-85
                              treatment        Nutrients)                                              Palenik, B., Morel, F. M. M., 1990 Amino acid utilization by marine phytoplankton: A novel mechanism, Limnology and Oceanography. 35(2), 260-269
                                                                                                       Parson, M.L., Q. Dorch, and R.E.Turner, 2002. Sedimentological evidence of an increase in Pseudo-nitzschia species (Bacillariophyceae) in Louisiana coastal waters: molecular probe field trials, genetic variability,
                                                                                                       and domoic acid analyses. J. Phycol., 35, 1368-1378
                                                                                                       Seitzinger, S. P., Sanders, R. W., 1997 Contribution of dissolved organic nitrogen from rivers to estuarine eutrophication, Marine Ecology Progress Series.                                        Vol. 159: 1-12
                                                                                                       Seitzinger, Sybil P., Sanders R. W., Styles Renee, 2002 Bioavailability of DON from natural and anthropogenic sources to estuarine plankton. Limnology and Oceanography. 47(2), 353-366
     Fig. 2: Experimental Set-up - Three different type of ASW medium will be made and of              Strickland, J. D. H., and T. R. Parson, 1972. Fluorometric determination of chlorophylls, In: A practical handbook of seawater analysis. Fisheries Research Board of Canada, Ottawa, 201-203
     each 3L will be made. The medium will be left acclimating in incubator temperature 2
     days before experiment. On the 1st day of the experiment, there will inoculation of
     75mL of cultures. The initial measurement will be taken at this point. Then, three vials                                                                                                                                      ACKNOWLEDMENTS
     of each ASW medium will be taken and placed in the light incubator and 3 vials in the                                                                                                        CAMP, National Science Foundation, #HRD-9624189, UC Marine Council, #02T CEQI 080094
     dark incubator.                                                                                                                                                                                                 Barbara B. Prézelin, Claudia Mengelt, Joseph Gorga
                                                                                                                                                                                                                                Steve Mckagan, Toni Mizerek