Den 30 mars 2012 beviljade Länsstyrelsen i Kalmarbidrag till Kalmar kommun för det lokala vattenvårdsprojektet ”Uppföljning och restaurering av våtmarker i Kalmar kommun”. Projektet genomfördes under perioden 25 mars – 30 november i samarbete med miljöanalytiker Jonas Nilsson vid Havsmiljöinstitutets enhet vid Linnéuniversitetet och våtmarksexpert Geraldine Thiere. Delar av projektet genomfördes som ett examensarbete i miljövetenskap av Jessica Olofsson, Linnéuniversitetet. En bedömning visade att majoriteten av de inventerade våtmarkerna ur näringsretentionssynpunkt inte var placerade på optimala ställen. Flera hade till exempel en alldeles för liten andel åkermark i tillrinningsområdet. Andra var placerade för långt från recipienten. Även den lokala utformningenav respektive våtmark kunde i många fall varit bättre. Flera våtmarker var till exempel utformade så att vattenytans form ledde till låg hydraulisk effektivitet. Listan över negativ kritik i rapporten är visserligen lång, men den visar samtidigt att det finns stor utvecklingspotential vid anläggning av nya närsaltsfällor i Kalmar län.

We verified an active uptake of kleptoplastids in the toxic and bloom-forming dinoflagellatesof the genus Dinophysis from its preferred prey, the ciliate Myrionecta rubra, using a quantitativereal-time PCR technique. During a 65 d starvation/feeding experiment with Dinophysis caudata,changes in plastid 16S rRNA, plastid autofluorescence and plastid/nuclear DNA ratio throughthe cell cycle were followed with quantitative real-time PCR and flow cytometry. During starvation,the cultures initially showed a rapid growth and a 3.5-fold increase of number of cells ml–1, while atthe same time, plastid DNA cell–1 showed a 3.5-fold decrease, and a 3.6-fold decrease in phycoerythrinfluorescence cell–1. The decrease in plastid DNA cell–1 d–1 closely followed culture growth rate(Pearson correlation, r = 0.91), indicating that existing plastids were diluted within the growing populationand that no new plastids were synthesised by the cells. When starved cells were re-fed by theciliate M. rubra on Days 43 to 51 of the experiment, plastid DNA cell–1 increased 7-fold up to 14 00016S DNA copies per cell, thereby directly revealing the kleptoplastic behaviour. The implication isthat not only availability of the prey M. rubra itself, but also the supply of suitable kleptoplastidsmight be an important controlling factor for Dinophysis spp. bloom formation and decline.

This thesis deals with the question of whether planktonic protits of the genus Dinophysis have permanent plastids (=chloroplasts) or practice kleptoplasty, i.e. acquire plastids via predation on other microorganisms. Sequencing the plastid 16S rDNA of Dinophysis spp. collected from 4 different geographical regions unveiled two different plastid genotypes within this genera: one that was found at all locations investigated, identical to that of the free-living cryptophyte Teleaulax amphioxeia, and another found only in the Greenland Sea, closely related to that of the cryptophyte Geminigera cryophila. Both types were found within the species D. acuminata. These findings imply that the plastids in Dinophysis spp. were not inherited from a common ancestor, but acquired from feeding. By using flow cytometry in combination with an acidotrophic probe, it was shown that 71 % of the cells in a D. norvegica population in the aphotic zone of the Baltic Sea had food-vacuoles. Dinophysis used to be regarded as a primarily phototrophic organism, and this was a higher proportion of cells with food-vacuoles than reported earlier. To further study if Dinophysis needs constant refill of new plastids from the environment, a new method combining flow-cytometry and quantitative real-time PCR was developed to compare the levels of nuclear and plastid DNA in different phases of the cell-cycle. Results showed that plastid acquisition in Dinophysis was uncoupled with the cell-cycle, which is different than the pattern seen in microalgal species with permanent plastids. Furthermore, when quantitative real-time PCR combined with flow-cytometry was used to follow D. caudata cultures during a 65 days starvation/feeding experiment, the cells first went through a steady decrease in plastid DNA during starvation. In contrast, after feeding on the ciliate Myrionecta rubra, plastid DNA in starved cells increased 7-fold, thereby directly revealing the kleptoplastic behavior. The main conclusion from this thesis is that Dinophysis cells are actively taking up kleptoplastids from the ciliates on which they feed, and that kleptoplasty is an important key to understand Dinophysis ecology. Part of this thesis work has also been dedicated to the application and optimization of new methods, and it shows how quantitative real-time PCR, flow cytometry and molecular methods in different combinations can be used as powerful tools for the study of plankton ecology.

Several studies have proved that some Dinophysis species are capable of ingesting particulate organic matter besides of being photosynthetic, a form of nutrition termed mixotrophy. Phagotrophy may be an important aspect of the life history of the genus Dinophysis and the key to understand its ecology. We used modern techniques coupling flow cytometry and acidotropic probes to detect and score food vacuolated Dinophysis norvegica cells in natural samples. In addition, feeding experiments were conduced under controlled conditions to observe if D. norvegica would grow feeding on the cryptophyte Teleaulax amphioxeia. The results of the field observations showed a frequency of phagotrophy between 25 and 71% in a natural D. norvegica population from the Baltic Sea, which is higher than previous reports (1–20%). Although molecular methods have proved that the kleptoplastids of the D. norvegica from the Baltic Sea are from T. amphioxeia, the laboratory experiments showed that the presence of T. amphioxeia in the cultures did not enhance the survival rate of D. norvegica neither in phototrophic nor in heterotrophic conditions. We suggest that the D. norvegica Kleptoplats are obtained through a heterotrophic or mixotrophic protist, which have been feeding on cryptophytes, as it has recently been shown for Dinophysis acuminata. Our main conclusion is that D. norvegica, and probably all other species from the genus Dinophysis, is mainly phagotrophic and feeds on a larger prey than T. amphioxeia. Autotrophy through kleptoplastidy would be a secondary feature used as a complementary or short-term survival strategy. 

We verified an active uptake of kleptoplastids in the toxic and bloom forming dinoflagellates genus Dinophysis from its preferred prey, the ciliate Myrionecta rubra, using a quantitative real-time PCR technique. During a 65 days starvation/feeding experiment with Dinophysis caudata, changes in plastid 16S rRNA, plastid autofluorescence and plastid/nuclear DNA ratio through the cell-cycle was followed with quantitative real-time PCR and flow-cytometry. During starvation, the cultures initially showed a rapid growth and an 3.5-fold increase of cells ml^-1, when at the same time, plastid DNA cell^-1 showed a 3.5-fold decrease, and phycoerythrin fluorescence cell^-1 a 3.6-fold decrease. The decrease in plastid DNA cell^-1day^-1 closely followed culture growth rate (r = 0.91, Pearson correlation), indicating that existing plastids were diluted within the growing population, and that no new plastids were synthesised by the cells. When starved cells were re-fed by the ciliate M. rubra on day 43-51 of the experiment, plastid DNA cell^-1 increased 7-fold up to 14 000 16S DNA copies per cell, thereby directly revealing the kleptoplastic behaviour. The implication is that not only availability of the prey M. rubra itself, but also the supply of suitable kleptoplastids might be an important controlling factor for Dinophysis spp. bloom formation and decline.