To survive, the marine dinoflagellate Dinophysis caudata Saville-Kent must feed on the plastidic ciliate Myrionecta rubra (=Mesodinium rubrum), itself a consumer of cryptophytes. Whether D. caudata has its own permanent chloroplasts or retains plastids from its ciliate prey, however, remains unresolved. Further, how long D. caudata plastids (or kleptoplastids) persist and remain photosynthetically active in the absence of prey remains unknown. We addressed those issues here, using the first established culture of D. caudata. Phylogenetic analyses of the plastid 16S rRNA and psbA gene sequences directly from the three organisms (D. caudata, M. rubra, and a cryptophyte) revealed that the sequences of both genes from the three organisms are almost identical to each other, supporting that the plastids of D. caudata are kleptoplastids. A 3-month starvation experiment revealed that D. caudata can remain photosynthetically active for ∼2 months when not supplied with prey. D. caudata cells starved for more than 2 months continued to keep the plastid 16S rRNA gene but lost the photosynthesis-related genes (i.e., psaA and psbA genes). When the prey was available again, however, D. caudata cells starved for more than 2 months were able to reacquire plastids and slowly resumed photosynthetic activity. Taken all together, the results indicate that the nature of the relationship between D. caudata and its plastids is not that of permanent cellular acquisitions. D. caudata is an intriguing protist that would represent an interesting evolutionary adaptation with regard to photosynthesis as well as help us to better understand plastid evolution in eukaryotes.

"Phototrophic"Dinophysis Ehrenberg species are well known to have chloroplasts of a cryptophyte origin, more specifically of the cryptophyte genus complex Teleaulax/Geminigera. Nonetheless, whether chloroplasts of "phototrophic"Dinophysis are permanent plastids or periodically derived kleptoplastids (stolen chloroplasts) has not been confirmed. Indeed, molecular sequence data and ultrastructural data lead to contradictory interpretations about the status of Dinophysis plastids. Here, we used established cultures of D. caudata strain DC-LOHABE01 and M. rubrum strain MR-MAL01 to address the status of Dinophysis plastids. Our approach was to experimentally generate D. caudata with "green" plastids and then follow the ingestion and fate of "reddish-brown" prey plastids using light microscopy, time-lapse videography, and single-cell TEM. Our results for D. caudata resolve the apparent discrepancy between morphological and molecular data by showing that plastids acquired when feeding on M. rubrum are structurally modified and retained as stellate compound chloroplasts characteristic of Dinophysis species.

The marine mixotrophic ciliate Mesodinium rubrum is known to acquire chloroplasts, mitochondria, nucleomorphs, and nucleus from its cryptophyte prey, particularly from species in the genera, Geminigera and Teleaulax. The sequestered prey nucleus and chloroplasts are considered to support photosynthesis of M. rubrum. In addition, recent studies have shown enlargement of the retained prey nucleus in starved M. rubrum and have inferred that enlargement results from the fusion of ingested prey nuclei. Thus far, however, little is known about the mechanism underlying the enlargement of the prey nucleus in M. rubrum. Here, we conducted starvation and refeeding studies to monitor the fate of prey nuclei acquired by M. rubrum when feeding on Teleaulax amphioxeia and to explore the influence of the retained prey nucleus on photosynthesis of M. rubrum. Results indicate that enlargement of the prey nucleus does not result from fusion of nuclei. Furthermore, the enlarged prey nucleus does not appear to divide during cell division of M. rubrum. The presence of a prey nucleus significantly affected photosynthetic performance of M. rubrum, while the number of retained chloroplasts had little influence on rate of carbon fixation. We interpret results within the context of a model that considers the dynamics of ingested prey nuclei during division of M. rubrum.

We verified an active uptake of kleptoplastids in the toxic and bloom-forming dinoflagellates of the genus Dinophysis from its preferred prey, the ciliate Myrionecta rubra, using a quantitative real-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 through the 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 at the same time, plastid DNA cell -1 showed a 3.5-fold decrease, and a 3.6-fold decrease in phycoerythrin fluorescence 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 population and that no new plastids were synthesised by the cells. When starved cells were re-fed by the ciliate M. rubra on Days 43 to 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.

In summer to autumn of 2008, a recently described thecate mixotrophic dinoflagellate, Fragilidium duplocampanaeforme Nézan et Chomérat, occurred in Masan Bay, Korea, where it frequently contained bright‐orange fluorescent inclusions. Using cultures of F. duplocampanaeforme isolated from Masan Bay, we investigated feeding, digestion, and prey specificity of this mixotroph. F. duplocampanaeforme fed exclusively on Dinophysis spp. when offered a variety of prey including dinoflagellates, a raphidophyte, a cryptophyte, a ciliate, and diatoms separately. In addition, F. duplocampanaeforme had allelopathic effects on other organisms, including cell immobilization/motility decrease (in Dinophysis acuminata, D. caudata, D. fortii, D. infundibulus, Gonyaulax polygramma, Heterocapsa triquetra, and Prorocentrum triestinum), breaking of cell chains (in Cochlodinium polykrikoides), cell death (in Prorocentrum minimum), and temporary cyst formation (in Scrippsiella trochoidea). F. duplocampanaeforme engulfed whole Dinophysis cells through the sulcus. About 1 h after ingestion, F. duplocampanaeforme became immobile and shed all thecal plates. The ecdysal cyst persisted for ∼7 h, during which the ingested prey was gradually digested. These observations suggest that F. duplocampanaeforme may play an important role in the Dinophysis population dynamics in the field.