Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Interactions Between the Kleptoplastidic Dinoflagellate Shimiella gracilenta and Several Common Heterotrophic Protists

The newly described dinoflagellate, Shimiella gracilenta, is known to survive for approximately 1 month on the plastids of ingested prey cells during starvation, indicating kleptoplastidy. To understand the population dynamics of this dinoflagellate in marine planktonic food webs, its growth and mortality rate due to predation should be assessed. Thus, we investigated the feeding occurrence of eight common heterotrophic protists on S. gracilenta. We also determined the growth and ingestion rates of Oxyrrhis marina and the naked ciliate, Rimostrombidium sp. on S. gracilenta as a function of the prey concentration. The common heterotrophic dinoflagellates (HTDs) Gyrodinium dominans, O. marina, and Pfiesteria piscicida and a naked ciliate Rimostrombidium sp. were able to feed on S. gracilenta; whereas the HTDs Aduncodinium glandula, Gyrodinium jinhaense, Oblea rotunda, and Polykrikos kofoidii were not. Shimiella gracilenta supported positive growth of O. marina and Rimostrombidium sp. but did not support that of G. dominans and P. piscicida. With increasing prey concentrations, the growth and ingestion rates of O. marina and Rimostrombidium sp. on S. gracilenta increased and became saturated. The maximum growth rates of O. marina and Rimostrombidium sp. on S. gracilenta were 0.645 and 0.903 day−1, respectively. Furthermore, the maximum ingestion rates of O. marina and Rimostrombidium sp. on S. gracilenta were 0.11 ng C predator day−1 (1.6 cells predator−1 day−1) and 35 ng C predator day−1 (500 cells predator−1 day−1), respectively. The maximum ingestion rate of O. marina on S. gracilenta was lower than that on any other algal prey reported to date, although its maximum growth rate was moderate. In conclusion, S. gracilenta had only a few common heterotrophic protist predators but could support moderate growth rates of the predators. Thus, S. gracilenta may not be a common prey species for diverse heterotrophic protists but may be a suitable prey for a few heterotrophic protists.

Heterotrophic Dinoflagellate Growth and Grazing Rates Reduced by Microplastic Ingestion

Microplastics are ubiquitous contaminants in marine ecosystems worldwide, threatening fisheries production, food safety, and human health. Ingestion of microplastics by fish and large zooplankton has been documented, but there are few studies focusing on single-celled marine predators, including heterotrophic dinoflagellates. In laboratory experiments, the heterotrophic dinoflagellate species Oxyrrhis marina and Gyrodinium sp. readily ingested both algal prey and polystyrene microplastic spheres (2.5–4.5 μm), while Protoperidinium sp. did not ingest microplastics. Compared to algae-only fed dinoflagellates, those that ingested microplastics had growth rates reduced by 25–35% over the course of 5 days. Reduced growth resulted in a 30–50% reduction of secondary production as measured as predator biomass. Ingestion rates of algal prey were also reduced in the microplastic treatments. When given a mixture of microplastics and algal prey, O. marina displayed a higher selectivity for algal prey than Gyrodinium sp. Observations in the coastal ocean showed that phylogenetically diverse taxa ingested microplastic beads, and thus heterotrophic dinoflagellates could contribute to trophic transfer of microplastics to higher trophic levels. The results of this study may suggest that continued increase in microplastic pollution in the ocean could lead to reduced secondary production of heterotrophic protists due to microplastic ingestion, altering the flow of energy and matter in marine microbial food webs.

Taxonomy and Physiology of Oxyrrhis marina and Oxyrrhis maritima in Korean Waters

The genus Oxyrrhis is a heterotrophic dinoflagellate found in diverse marine environments. Oxyrrhis spp. have received attention owing to their ecological and industrial importance, high lipid contents, and docosahexaenoic acid formation. To the best of our knowledge, contrary to O. marina, ecophysiological characterization studies on O. maritima have not yet been reported. Therefore, we investigated the taxonomy and ecophysiology of four strains of O. marina from coastal waters and two strains of O. maritima from the littoral tidepool waters of Korea. Based on phylogenetic trees constructed using internal transcribed spacer ribosomal DNA (ITS rDNA) and SSU rDNA of dinoflagellates, the clade of all four O. marina strains was divergent from that of the two O. maritima strains. We measured the growth rates of both species at various water temperatures (10–36 °C), salinities (5–90), and light intensities (0–100 µE·m−2·s−1). The lowest (O. marina and O. maritima: 10 °C) and highest temperatures (O. marina: <35 °C, O. maritima: >35 °C) revealed that O. maritima has more tolerance to high salinity. This study provides a basis for understanding the ecophysiology of O. marina and O. maritima and their population dynamics in marine ecosystems.

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Influence of the Calcium Carbonate Shell of Coccolithophores on Ingestion and Growth of a Dinoflagellate Predator

Coccolithophores are an important group of ∼200 marine phytoplankton species which cover themselves with a calcium carbonate shell called “coccosphere.” Coccolithophores are ecologically and biogeochemically important but the reason why they calcify remains elusive. One key function may be that the coccosphere offers protection against microzooplankton predation, which is one of the main causes of phytoplankton death in the ocean. Here, we investigated the effect of the coccosphere on ingestion and growth of the heterotrophic dinoflagellate Oxyrrhis marina. Calcified and decalcified cells of the coccolithophore species Emiliania huxleyi, Pleurochrysis carterae, and Gephyrocapsa oceanica were offered separately to the predator as well as in an initial ∼1:1 mixture. The decrease of the prey concentrations and predator abundances were monitored over a period of 48–72 h. We found that O. marina did not actively select against calcified cells, but rather showed a size selective feeding behavior. Thus, the coccosphere does not provide a direct protection against grazing by O. marina. However, O. marina showed slower growth when calcified coccolithophores were fed. This could be due to reduced digestion rates of calcified cells and/or increased swimming efforts when ballasted with heavy calcium carbonate. Furthermore, we show that the coccosphere reduces the ingestion capacity simply by occupying much of the intracellular space of the predator. We speculate that the slower growth of the grazer when feeding on calcified cells is of limited benefit to the coccolithophore population because other co-occurring phytoplankton species within the community that do not invest energy in the formation of a calcite shell could also benefit from the reduced growth of the predators. Altogether, these new insights constitute a step forward in our understanding of the ecological relevance of calcification in coccolithophores.

Fat Tail in the Phytoplankton Movement Patterns and Swimming Behavior: New Insights into the Prey-Predator Interactions

Phytoplankton movement patterns and swimming behavior are important and basic topics in aquatic biology. Heavy tail distribution exists in diverse taxa and shows theoretical advantages in environments. The fat tails in the movement patterns and swimming behavior of phytoplankton in response to the food supply were studied. The log-normal distribution was used for fitting the probability density values of the movement data of Oxyrrhis marina. Results showed that obvious fat tails exist in the movement patterns of O. marina without and with positive stimulations of food supply. The algal cells tended to show a more chaotic and disorderly movement, with shorter and neat steps after adding the food source. At the same time, the randomness of turning rate, path curvature and swimming speed increased in O. marina cells with food supply. Generally, the responses of phytoplankton movement were stronger when supplied with direct prey cells rather than the cell-free filtrate. The scale-free random movements are considered to benefit the adaption of the entire phytoplankton population to varied environmental conditions. Inferentially, the movement pattern of O. marina should also have the characteristics of long-range dependence, local self-similarity and a system of fractional order.

Dimethyl sulfide acts as eat-me signal during microbial predator-prey interactions in the ocean

Phytoplankton are key components of the oceanic carbon and sulfur cycles 1. During bloom events, some species can emit massive amounts of the organosulfur volatile dimethyl sulfide (DMS) to the atmosphere, where it can modulate aerosol formation and affect climate. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic-level interactions. Yet its role in microbial predator-prey interactions remains elusive with contradicting evidence for its role in algal chemical defense and in grazer’s chemoattraction to prey cells. Here, we investigated the signaling role of DMS during zooplankton-algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme (Dimethylsulfoniopropionate lyase, DL) from the bloom-forming alga Emiliania huxleyi. We inhibited DL activity in live E. huxleyi cells by the novel DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate (Br-DMSP) , and overexpressed DL in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as anti-grazing chemical defense, and paradoxically enhanced grazing by the model microzooplankton Oxyrrhis marina and other micro- and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina’s growth. Overall, our results demonstrate that DMS-mediated herbivory may be ecologically important and prevalent during prey-predator dynamics in oceanic ecosystems. The role of DMS as an appetizing signal to grazers revealed here raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean.