A Dinoflagellate Amylax triacantha with Plastids of the Cryptophyte Origin: Phylogeny,Feeding Mechanism,and Growth and Grazing Responses

The gonyaulacalean dinoflagellates Amylax spp. were recently found to contain plastids of the cryptophyte origin, more specifically of Teleaulax amphioxeia. However, not only how the dinoflagellates get the plastids of the cryptophyte origin is unknown but also their ecophysiology, including growth and feeding responses as functions of both light and prey concentration, remain unknown. Here, we report the establishment of Amylax triacantha in culture, its feeding mechanism, and its growth rate using the ciliate prey Mesodinium rubrum (= Myrionecta rubra) in light and dark, and growth and grazing responses to prey concentration and light intensity. The strain established in culture in this study was assigned to A. triacantha, based on morphological characteristics (particularly, a prominent apical horn and three antapical spines) and nuclear SSU and LSU rDNA sequences. Amylax triacantha grew well in laboratory culture when supplied with the marine mixotrophic ciliate M. rubrum as prey, reaching densities of over 7.5 × 10³ cells/ml. Amylax triacantha captured its prey using a tow filament, and then ingested the whole prey by direct engulfment through the sulcus. The dinoflagellate was able to grow heterotrophically in the dark, but the growth rate was approximately two times lower than in the light. Although mixotrophic growth rates of A. triacantha increased sharply with mean prey concentrations, with maximum growth rate being 0.68/d, phototrophic growth (i.e. growth in the absence of prey) was ?0.08/d. The maximum ingestion rate was 2.54 ng C/Amylax/d (5.9 cells/Amylax/d). Growth rate also increased with increasing light intensity, but the effect was evident only when prey was supplied. Increased growth with increasing light intensity was accompanied by a corresponding increase in ingestion. In mixed cultures of two predators, A. triacantha and Dinophysis acuminata, with M. rubrum as prey, A. triacantha outgrew D. acuminata due to its approximately three times higher growth rate, suggesting that it can outcompete D. acuminata. Our results would help better understand the ecophysiology of dinoflagellates retaining foreign plastids.     

A novel type of kleptoplastidy in Dinophysis (Dinophyceae): presence of haptophyte-type plastid in Dinophysis mitra

Red-fluorescent, non-phycobilin-containing plastids were found in the heterotrophic dinoflagellate, Dinophysis mitra. Transmission electron microscopy showed that they contained a three-layer thylakoid, the absence of girdle lamella, and an embedded pyrenoid with thylakoid intrusions. These characteristics all coincide with haptophyte plastids. Phylogenetic analysis of the plastid small-subunit ribosomal DNA (SSU rDNA) revealed that the Dinophysis mitra sequences are distantly related to those of phycobilin-containing Dinophysis species and are positioned within a lineage of haptophytes belonging to Prymnesiophyceae. Because the plastid SSU rDNA sequences of Dinophysis mitra showed significant heterogeneity, despite being derived from a single species, it is highly likely that they were not established as plastids through an evolutionary process but are "kleptoplastids" (temporally stolen plastids) from multiple sources of haptophytes in the environment. We deduced that Dinophysis mitra takes up haptophytes myzocytotically and selectively retains the plastid with surrounding plastidal membranes, whereas other haptophyte cell components are degraded. This represents another type of kleptoplastidy in the Dinophysis species, which mostly harbor cryptophyte plastids, and is the first evidence of kleptoplastidy originating from haptophytes.     

DINOPHYSIS CAUDATA (DINOPHYCEAE) SEQUESTERS AND RETAINS PLASTIDS FROM THE MIXOTROPHIC CILIATE PREY MESODINIUM RUBRUM1

“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.     

PLASTID DYNAMICS DURING SURVIVAL OF DINOPHYSIS CAUDATA WITHOUT ITS CILIATE PREY1

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.     

The Effect of Starvation on Plastid Number and Photosynthetic Performance in the Kleptoplastidic Dinoflagellate Amylax triacantha

The dinoflagellate Amylax triacantha is known to retain plastids of cryptophyte origin by engulfing the mixotrophic ciliate Mesodinium rubrum, itself a consumer of cryptophytes. However, there is no information on the fate of the prey's organelles and the photosynthetic performance of the newly retained plastids in A. triacantha. In this study, we conducted a starvation experiment to observe the intracellular organization of the prey's organelles and temporal changes in the photosynthetic efficiency of acquired plastids in A. triacantha. The ultrastructural observations revealed that while the chloroplast‐mitochondria complexes and nucleus of cryptophyte were retained by A. triacantha, other ciliate organelles were digested in food vacuoles. Acquired plastids were retained in A. triacantha for about 1 mo and showed photosynthetic activities for about 18 d when measured by a pulse‐amplitude modulation fluorometer.