Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.     

Feeding by Phototrophic Red-Tide Dinoflagellates on the Ubiquitous Marine Diatom Skeletonema costatum

ABSTRACT We investigated feeding by phototrophic red‐tide dinoflagellates on the ubiquitous diatom Skeletonema costatum to explore whether dinoflagellates are able to feed on S. costatum, inside the protoplasm of target dinoflagellate cells observed under compound microscope, confocal microscope, epifluorescence microscope, and transmission electron microscope (TEM) after adding living and fluorescently labeled S. costatum (FLSc). To explore effects of dinoflagellate predator size on ingestion rates of S. costatum, we measured ingestion rates of seven dinoflagellates at a single prey concentration. In addition, we measured ingestion rates of the common phototrophic dinoflagellates Prorocentrum micans and Gonyaulax polygramma on S. costatum as a function of prey concentration. We calculated grazing coefficients by combining field data on abundances of P. micans and G. polygramma on co‐occurring S. costatum with laboratory data on ingestion rates obtained in the present study. All phototrophic dinoflagellate predators tested (i.e. Akashiwo sanguinea, Amphidinium carterae, Alexandrium catenella, Alexandrium tamarense, Cochlodinium polykrikoides, G. polygramma, Gymnodinium catenatum, Gymnodinium impudicum, Heterocapsa rotundata, Heterocapsa triquetra, Lingulodinium polyedrum, Prorocentrum donghaiense, P. micans, Prorocentrum minimum, Prorocentrum triestinum, and Scrippsiella trochoidea) were able to ingest S. costatum. When mean prey concentrations were 170–260 ng C/ml (i.e. 6,500–10,000 cells/ml), the ingestion rates of G. polygramma, H. rotundata, H. triquetra, L. polyedrum, P. donghaiense, P. micans, and P. triestinum on S. costatum (0.007–0.081 ng C/dinoflagellate/d [0.2–3.0 cells/dinoflagellate/d]) were positively correlated with predator size. With increasing mean prey concentration of ca 1–3,440 ng C/ml (40–132,200 cells/ml), the ingestion rates of P. micans and G. polygramma on S. costatum continuously increased. At the given prey concentrations, the maximum ingestion rates of P. micans and G. polygramma on S. costatum (0.344–0.345 ng C/grazer/d; 13 cells/grazer/d) were almost the same. The maximum clearance rates of P. micans and G. polygramma on S. costatum were 0.165 and 0.020 μl/grazer/h, respectively. The calculated grazing coefficients of P. micans and G. polygramma on co‐occurring S. costatum were up to 0.100 and 0.222 h, respectively (i.e. up to 10% and 20% of S. costatum populations were removed by P. micans and G. polygramma populations in 1 h, respectively). Our results suggest that P. micans and G. polygramma sometimes have a considerable grazing impact on populations of S. costatum.     

Feeding by the newly described, nematocyst-bearing heterotrophic dinoflagellate Gyrodiniellum shiwhaense

We explored the feeding ecology of the newly described, nematocyst-bearing heterotrophic dinoflagellate Gyrodiniellum shiwhaense (GenBank accession number=FR720082). Using several different types of microscopes and high-resolution video-microscopy, we investigated feeding behavior and types of prey species that G. shiwhaense feeds upon. Additionally, we measured its growth and ingestion rates on its optimal algal prey, the cryptophyte Teleaulax sp. and the dinoflagellate Amphidinium carterae, as a function of prey concentration. These rates were measured for other edible prey at single prey concentrations at which the growth and ingestion rates of G. shiwhaense were saturated. After anchoring the prey with a tow filament, G. shiwhaense fed using a peduncle, ingesting small algal species with equivalent spherical diameters (ESDs) of <13 μm. However, it did not feed on larger algal species that had ESDs≥13 μm or the small diatom Skeletonema costatum. The specific growth rates for G. shiwhaense feeding upon Teleaulax sp. and A. carterae increased rapidly with increasing mean prey concentration before saturating at concentrations of ca. 180-430 ng C/ml. The maximum specific growth rate of G. shiwhaense on Teleaulax sp. and A. carterae were 1.05 and 0.82/d, respectively. However, Heterosigma akashiwo did not support positive growth of G. shiwhaense. The maximum ingestion rates of G. shiwhaense on Teleaulax sp. and A. carterae were 0.35 and 0.54 ng C/grazer/d, respectively. The calculated grazing coefficients attributable to G. shiwhaense on co-occurring cryptophytes and Amphidinium spp. were 0.01-1.87/d and 0.08-2.60/d, respectively. Our results suggest that G. shiwhaense can have a considerable grazing impact on algal populations.     

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology,Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.     

Differential interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and common heterotrophic protists and copepods: Killer or prey

To investigate interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and different heterotrophic protist and copepod species, feeding by common heterotrophic dinoflagellates (Oxyrrhis marina and Gyrodinium dominans), naked ciliates (Strobilidium sp. approximately 35 μm in cell length and Strombidinopsis sp. approximately 100 μm in cell length), and calanoid copepods Acartia spp. (A. hongi and A. omorii) on P. shiwhaense was explored. In addition, the feeding activities of P. shiwhaense on these heterotrophic protists were investigated. Furthermore, the growth and ingestion rates of O. marina, G. dominans, Strobilidium sp., Strombidinopsis sp., and Acartia spp. as a function of P. shiwhaense concentration were measured. O. marina, G. dominans, and Strombidinopsis sp. were able to feed on P. shiwhaense, but Strobilidium sp. was not. However, the growth rates of O. marina, G. dominans, Strobilidium sp., and Strombidinopsis sp. feeding on P. shiwhaense were very low or negative at almost all concentrations of P. shiwhaense. P. shiwhaense frequently fed on O. marina and Strobilidium sp., but did not feed on Strombidinopsis sp. and G. dominans. G. dominans cells swelled and became dead when incubated with filtrate from the experimental bottles (G. dominans + P. shiwhaense) that had been incubated for one day. The ingestion rates of O. marina, G. dominans, and Strobilidium sp. on P. shiwhaense were almost zero at all P. shiwhaense concentrations, while those of Strombidinopsis sp. increased with prey concentration. The maximum ingestion rate of Strombidinopsis sp. on P. shiwhaense was 5.3 ng C predator⁻¹d⁻¹ (41 cells predator⁻¹d⁻¹), which was much lower than ingestion rates reported in the literature for other mixotrophic dinoflagellate prey species. With increasing prey concentrations, the ingestion rates of Acartia spp. on P. shiwhaense increased up to 930 ng C ml⁻¹ (7180 cells ml⁻¹) at the highest prey concentration. The highest ingestion rate of Acartia spp. on P. shiwhaense was 4240 ng C predator⁻¹d⁻¹ (32,610 cells predator⁻¹d⁻¹), which is comparable to ingestion rates from previous studies on other dinoflagellate prey species calculated at similar prey concentrations. Thus, P. shiwhaense might play diverse ecological roles in marine planktonic communities by having an advantage over competing phytoplankton in anti-predation against potential protistan grazers.     

Mixotrophy in the sand-dwelling dinoflagellate Thecadinium kofoidii

Thecadinium kofoidii is a marine sand-dwelling dinoflagellate that sometimes forms dense blooms. This species was previously thought to be an exclusively autotrophic dinoflagellate, and its mixotrophic ability has not been explored yet. By investigating its ecophysiology, its trophic mode should be revealed. We explored the mixotrophic ability of T. kofoidii by examining its protoplasm under light and transmission electron microscopes with diverse algal prey species. Furthermore, the feeding mechanism of T. kofoidii and prey species on which it feeds were investigated. In addition, the growth and ingestion rates of T. kofoidii as a function of prey concentration were determined when feeding on the benthic cryptophyte Rhodomonas salina. Thecadinium kofoidii was able to feed on R. salina and the dinoflagellate Symbiodinium voratum, which had equivalent spherical diameters (ESDs) ≤ 10.1?µm, while it did not feed on the benthic dinoflagellates Levanderina fissa, Prorocentrum concavum or Ostreopsis cf. ovata, which had ESDs ≥ 15?µm. Thecadinium kofoidii fed on the edible prey cells using the peduncle. The maximum ingestion rate of T. kofoidii on R. salina was 1.3 cells predator^?1 d^?1. However, feeding on R. salina did not significantly increase the growth rate of T. kofoidii. The low ingestion rate of T. kofoidii on R. salina may have partially resulted in the lack of significant increase in its growth rate due to mixotrophy. The present study discovered predator–prey relationships between T. kofoidii and R. salina and S. voratum, which may change our view of the energy flow and carbon cycling in marine benthic food webs.     

Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum

Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms and red tides in estuarine ecosystems. While known to be mixotrophic, previous attempts to document feeding on algal prey have yielded low grazing rates. In this study, growth and ingestion rates of P. minimum were measured as a function of nitrogen (‐N) and phosphorous (‐P) starvation. A P. minimum isolate from Chesapeake Bay was found to ingest cryptophyte prey when in stationary phase and when starved of N or P. Prorocentrum minimum ingested two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte species. In all cases ‐P treatments resulted in the highest grazing. Ingestion rates of ‐P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by ‐N cells saturated at 1 prey per predator per day. In the presence of prey, ‐P treated cells reached a maximum mixotrophic growth rate (μ_max) of 0.5 d^?1, while ‐N cells had a μ_max of 0.18 d^?1. Calculations of ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotrophy can be an important source of all three elements. While P. minimum is a proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.     

Mixotrophy in the phototrophic harmful alga Cochlodinium polykrikoides (Dinophycean): prey species, the effects of prey concentration, and grazing impact

We first reported here that the harmful alga Cochlodinium polykrikoides, which had been previously known as an autotrophic dinoflagellate, was a mixotrophic species. We investigated the kinds of prey species and the effects of the prey concentration on the growth and ingestion rates of C. polykrikoides when feeding on an unidentified cryptophyte species (Equivalent Spherical Diameter, ESD = 5.6 microm). We also calculated grazing coefficients by combining field data on abundances of C. polykrikoides and co-occurring cryptophytes with laboratory data on ingestion rates obtained in the present study. Cocholdinium polykrikoides fed on prey cells by engulfing the prey through the sulcus. Among the phytoplankton prey offered, C. polykrikoides ingested small phytoplankton species that had ESD's < or = 11 microm (e.g. the prymnesiophyte Isochrysis galbana, an unidentified cryptophyte, the cryptophyte Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellate Amphidinium carterae). It did not feed on larger phytoplankton species that had ESD's > or = 12 microm (e.g. the dinoflagellates Heterocapsa triquetra, Prorocentrum minimum, Scrippsiella sp., Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum). Specific growth rates of C. polykrikoides on a cryptophyte increased with increasing mean prey concentration, with saturation at a mean prey concentration of approximately 270 ng C ml(-1) (i.e. 15,900 cells ml(-1)). The maximum specific growth rate (mixotrophic growth) of C. polykrikoides on a cryptophyte was 0.324 d(-1), under a 14:10 h light-dark cycle of 50 microE m(-2) s(-1), while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.166 d(-1). Maximum ingestion and clearance rates of C. polykrikoides on a cryptophyte were 0.16 ng C grazer(-1)d(-1) (9.4 cells grazer(-1)d(-1)) and 0.33 microl grazer(-1)h(-1), respectively. Calculated grazing coefficients by C. polykrikoides on cryptophytes were 0.001-0.745 h(-1) (i.e. 0.1-53% of cryptophyte populations were removed by a C. polykrikoides population in 1 h). The results of the present study suggest that C. polykrikoides sometimes has a considerable grazing impact on populations of cryptophytes.     

Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates

Takayama spp. are phototrophic dinoflagellates belonging to the family Kareniaceae and have caused fish kills in several countries. Understanding their trophic mode and interactions with co-occurring phytoplankton species are critical steps in comprehending their ecological roles in marine ecosystems, bloom dynamics, and dinoflagellate evolution. To investigate the trophic mode and interactions of Takayama spp., the ability of Takayama helix to feed on diverse algal species was examined, and the mechanisms of prey ingestion were determined. Furthermore, growth and ingestion rates of T. helix feeding on the dinoflagellates Alexandrium lusitanicum and Alexandrium tamarense, which are two optimal prey items, were determined as a function of prey concentration. T. helix ingested large dinoflagellates ≥15 μm in size, except for the dinoflagellates Karenia mikimotoi, Akashiwo sanguinea, and Prorocentrum micans (i.e., it fed on Alexandrium minutum, A. lusitanicum, A. tamarense, A. pacificum, A. insuetum, Cochlodinium polykrikoides, Coolia canariensis, Coolia malayensis, Gambierdiscus caribaeus, Gymnodinium aureolum, Gymnodinium catenatum, Gymnodinium instriatum, Heterocapsa triquetra, Lingulodinium polyedrum, and Scrippsiella trochoidea). All these edible prey items are dinoflagellates that have diverse eco-physiology such as toxic and non-toxic, single and chain forming, and planktonic and benthic forms. However, T. helix did not feed on small flagellates and dinoflagellates <13 μm in size (i.e., the prymnesiophyte Isochrysis galbana; the cryptophytes Teleaulax sp., Storeatula major, and Rhodomonas salina; the raphidophyte Heterosigma akashiwo; the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum minimum; or the small diatom Skeletonema costatum). T. helix ingested Heterocapsa triquetra by direct engulfment, but sucked materials from the rest of the edible prey species through the intercingular region of the sulcus. With increasing mean prey concentration, the specific growth rates of T. helix on A. lusitanicum and A. tamarense increased continuously before saturating at prey concentrations of 336–620 ng C mL⁻¹. The maximum specific growth rates (mixotrophic growth) of T. helix on A. lusitanicum and A. tamarense were 0.272 and 0.268 d⁻¹, respectively, at 20 °C under a 14:10 h light/dark cycle of 20 μE m⁻² s⁻¹ illumination, while its growth rates (phototrophic growth) under the same light conditions without added prey were 0.152 and 0.094 d⁻¹, respectively. The maximum ingestion rates of T. helix on A. lusitanicum and A. tamarense were 1.23 and 0.48 ng C predator⁻¹d⁻¹, respectively. The results of the present study suggest that T. helix is a mixotrophic dinoflagellate that is able to feed on a diverse range of toxic species and, thus, its mixotrophic ability should be considered when studying red tide dynamics, food webs, and dinoflagellate evolution.     

The Newly Described Heterotrophic Dinoflagellate Gyrodinium moestrupii,an Effective Protistan Grazer of Toxic Dinoflagellates

Few protistan grazers feed on toxic dinoflagellates, and low grazing pressure on toxic dinoflagellates allows these dinoflagellates to form red‐tide patches. We explored the feeding ecology of the newly described heterotrophic dinoflagellate Gyrodinium moestrupii when it fed on toxic strains of Alexandrium minutum, Alexandrium tamarense, and Karenia brevis and on nontoxic strains of A. tamarense, Prorocentrum minimum, and Scrippsiella trochoidea. Specific growth rates of G. moestrupii feeding on each of these dinoflagellates either increased continuously or became saturated with increasing mean prey concentration. The maximum specific growth rate of G. moestrupii feeding on toxic A. minutum (1.60/d) was higher than that when feeding on nontoxic S. trochoidea (1.50/d) or P. minimum (1.07/d). In addition, the maximum growth rate of G. moestrupii feeding on the toxic strain of A. tamarense (0.68/d) was similar to that when feeding on the nontoxic strain of A. tamarense (0.71/d). Furthermore, the maximum ingestion rate of G. moestrupii on A. minutum (2.6 ng C/grazer/d) was comparable to that of S. trochoidea (3.0 ng C/grazer/d). Additionally, the maximum ingestion rate of G. moestrupii on the toxic strain of A. tamarense (2.1 ng C/grazer/d) was higher than that when feeding on the nontoxic strain of A. tamarense (1.3 ng C/grazer/d). Thus, feeding by G. moestrupii is not suppressed by toxic dinoflagellate prey, suggesting that it is an effective protistan grazer of toxic dinoflagellates.     

GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) FEEDING ON ALGAE

The growth and grazing characteristics of Poterioochromonas malhamensis (Pringsheim) Peterfi (= Ochromonas malhamensis Pringsheim) (ca. 8 μm) feeding on phytoplankton, including the cyanobacteria Synechococcus sp. (ca. 2 μm) and Microcystis viridis (A. Brown) Lemmermann (ca. 6 μm) and the green alga Chlorella pyrenoidosa Chick (ca. 13 μm), were investigated in laboratory experiments involving the following treatments: (1) light without added algal prey (autotrophy), (2) light with added algal prey (mixotrophy), and (3) dark with added algal prey (phagotrophy). There were significantly higher cell numbers under mixotrophic and phagotrophic growth than under autotrophic growth. With phytoplankton as food, growth rates under both mixotrophy and phagotrophy were about two or three times higher than those under autotrophy, indicating that the algal diets were readily able to support the population growth of P. malhamensis. There were no significant differences in growth rate between mixotrophic and phagotrophic cultures during exponential growth. The ingestion rate of P. malhamensis with algal prey was also similar under both continuous light and dark. Poterioochromonas malhamensis ingested on average 0.27 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous light and 0.25 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous dark during exponential growth. The results showed that light had no effect on the growth and ingestion rates of P. malhamensis for phagotrophy during exponential growth. However, phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days. Populations of P. malhamensis showed no increase when prey was added again after 4 days in continuous darkness, indicating that light is necessary for sustained phagotrophic growth of P. malhamensis. The study suggests that P. malhamensis, which has strong tolerance for light, is light dependent for phagotrophy.     

Ultrastructure and Systematics of Two New Species of Dinoflagellate,Paragymnodinium Asymmetricum sp. nov. and Paragymnodinium Inerme sp. nov. (Gymnodiniales,Dinophyceae)1

The genus Paragymnodinium currently includes two species, P. shiwhaense and P. stigmaticum, that are characterized by mixotrophic nutrition and the possession of nematocysts. In this study, two new dinoflagellates belonging to this genus were described based on observations using LM, SEM, and TEM together with a molecular analysis. Cells of P. asymmetricum sp. nov., isolated from Nha Trang Beach, Vietnam, were 7.9–12.6 μm long and 4.7–9.0 μm wide. The species showed no evidence of feeding behavior and was able to sustain itself phototrophically. Paragymnodinium asymmetricum shared many features with P. shiwhaense, including presence of nematocysts, absence of an eyespot, and a planktonic lifestyle, but was clearly distinguished by the asymmetric shape of the hyposome, possession of a single chloroplast, and its nutritional mode. Cells of P. inerme sp. nov., isolated from Jogashima, Kanagawa Pref, Japan, were 15.3–23.7 μm long and 10.9–19.6 μm wide. This species also showed no evidence of feeding behavior. Paragymnodinium inerme was similar to cells of P. shiwhaense in shape and planktonic lifestyle, but its nutritional mode was different. The presence of incomplete nematocysts was also a unique feature. A phylogenetic analysis inferred from concatenated SSU and LSU rDNA sequences recovered the two dinoflagellates in a robust clade with Paragymnodinium spp., within the clade of Gymnodinium sensu stricto. This evidence, together with their morphological similarities, made it reasonable to conclude that these two dinoflagellates are new species of Paragymnodinium.     

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.     

PREY SPECIFICITY AND FEEDING OF THE THECATE MIXOTROPHIC DINOFLAGELLATE FRAGILIDIUM DUPLOCAMPANAEFORME1

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.     

Feeding by the Pfiesteria-like heterotrophic dinoflagellate Luciella masanensis

To explore the feeding ecology of the Pfiesteria-like dinoflagellate (PLD) Luciella masanensis (GenBank Accession no. AM050344, previously Lucy), we investigated the feeding behavior and the kinds of prey species that L. masanensis fed on and determined its growth and ingestion rates of L. masanensis when it fed on the dinoflagellate Amphidinium carterae and an unidentified cryptophyte species (equivalent spherical diam., ESD=5.6 microm), which were the dominant phototrophic species when L. masanensis and similar small heterotrophic dinoflagellates were abundant in Masan Bay, Korea in 2005. Additionally, these parameters were also measured for L. masanensis fed on blood cells of the perch Lateolabrax japonicus and the raphidophyte Heterosigma akashiwo in the laboratory. Luciella masanensis fed on prey cells by using a peduncle after anchoring the prey with tow filament, and was able to feed on diverse prey such as cryptophytes, raphidophytes, diatoms, mixotrophic dinoflagellates, and the blood cells of fish and humans. Among the prey species tested in the present study, perch blood cells were observed to be the optimal prey for L. masanensis. Specific growth rates of L. masanensis feeding on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte, either increased continuously or became saturated with increasing the mean prey concentration. The maximum specific growth rate of L. masanensis feeding on perch blood cells (1.46/day) was much greater than that of A. carterae (0.59/day), the cryptophyte (0.24/day), or H. akashiwo (0.20/day). The maximum ingestion rate of L. masanensis on perch blood cells (2.6 ng C/grazer/day) was also much higher than that of A. carterae (0.32 ng C/grazer/day), the cryptophyte (0.44 ng C/grazer/day), or H. akashiwo (0.16 ng C/grazer/day). The kinds of prey species which L. masanensis is able to feed on were the same as those of Pfiesteria piscicida, but very different from those of another PLD Stoeckeria algicida. However, the maximum growth and ingestion rates of L. masanensis on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte were considerably lower than those of P. piscicida. Therefore, these three dinoflagellates may occupy different ecological niches in marine planktonic communities, even though they have a similar size and shape and the same feeding mechanisms.     

Comparative growth rates and yields of ciliates and heterotrophic dinoflagellates

Growth rates, ingestion rates and grazer yields (grazer volumeproduced/prey volume consumed) were measured for six protozoanspecies (ciliates: Favella sp., Strombidinopsis acuminatum,Uronema sp.; heterotrophic dinoflagellates: Amphidinium sp.,Gymnodinium sp., Noctiluca scintillans) in laboratory batchculture experiments. Comparative growth data indicate that theprymnesiophyte Isochrysis galbana, the prasinophyte Mantoniellasquamata, two cryptophyte species and several autotrophic dinoflagellatespecies were suitable foods for these grazers. When grown onoptimized diets at 13C, maximum ciliate growth rates (range0.77–1.01 day^–1 uniformly exceeded maximum heterotrophicdioflagellate growth rates (range 0.41–0.48 day^–1).A compilation of published data demonstrates that this growthrate difference persists across a range of ciliate and dinoflagellatetaxa and cell sizes. Comparison of volume-specific ingestionrates and yields for the six species studied here showed thatthere was no single explanation for this growth rate disparity.Heterotrophic dinoflagellates exhibited both low ingestion ratesand, in one case, low yields; ciliates were able to achievehigher growth rates via either higher ingestion rates or higheryields, depending on ciliate species. Volume yield increasedover time throughout the exponential growth phase in nearlyall experiments, suggesting variation in response to changingfood concentrations or long-term acclimation to culture conditions.Higher maximum ciliate growth rates mean that these grazershave the potential to exercise tighter control over incipientblooms of their prey than do heterotrophic dinoflagellates.     

颗石藻Pleurochrysis carterae抗捕食特征

颗石藻Pleurochrysis carterae是沿海水域中常见钙化微藻,易形成高密度水华,也是养殖环境致害种之一。抗捕食防御能力可能是其种群增殖优势的一个重要原因。以卤虫作为捕食者,分析了颗石藻P.carterae抗捕食现象,以及在捕食压力下的重要生理生化响应特征,以期为颗石藻P.carterea抗捕食机制研究及其高密度增殖机理提供参考。研究结果显示:(1)当颗石藻P.carterae比例增加时,卤虫对微藻的摄食率显著降低,且存活率显著下降,显示该藻具抗捕食能力。(2)以卤虫饵料微藻球等鞭金藻(Isochrysis galbana)为对照,比较研究发现,相同的捕食压力下,饵料金藻的叶绿素荧光参数(电子传递速率ETR和最大量子产率Fv/Fm)显著降低,但颗石藻P.carterae的ETR和Fv/Fm没有显著变化,显示颗石藻P.carterae对卤虫抗捕食作用。(3)相对于没有捕食压力的对照组,捕食压力下,饵料金藻I.galbana的脂类组成没有显著差异。但是,颗石藻P.carterae的脂类组成则发生了显著变化,主要表现在对细胞叶绿体有重要作用的单半乳糖甘油二酯(MGDG),双半乳糖甘油二酯(DGDG),磷脂酰甘油二酯(PG)含量上升,与促细胞分裂相关的二酰甘油(DAG)和磷脂酰肌醇(PI)也上升。这些脂类代谢物的变化可能在其种群水平上抵抗捕食并实现种群增殖中发挥作用。(4)培养介质中磷的状态对颗石藻P.carterae细胞二甲基巯基丙酸(Dimethyl sulfonio propionate,DMSP)含量有显著影响,且影响颗石藻P.carterae对卤虫的致害效应:缺磷条件下生长的颗石藻P.carterae首先使卤虫受害。当培养液中仅以ATP为磷源时,颗石藻P.carterae的卤虫致害效应则降低。研究证明,颗石藻P.carterae具有抗捕食能力,细胞的脂类代谢物质以及DMSP可能在抗捕食防御中发挥作用。     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

A New Harziane Diterpene,Harziaketal A,and a New Sterol,Trichosterol A,from the Marine-Alga-Epiphytic Trichoderma sp. Z43

One new diterpene, harziaketal A ( 1 ), and one new highly degraded sterol, trichosterol A ( 2 ), along with three known compounds, including one diterpene, harzianone ( 3 ), and two steroids, (22E,24R)-5α,6α-epoxy-ergosta-8(14),22-dien-3β,7α-diol ( 4 ) and isoergokonin B ( 5 ), were isolated from the culture of the marine-alga-epiphytic fungus Trichoderma sp. Z43 by silica gel column chromatography (CC), Sephadex LH-20 CC, and preparative thin-layer chromatography (TLC). Their structures and relative configurations were assigned by nuclear magnetic resonance (NMR) and high resolution electrospray ionisation mass spectrometry (HR-ESI-MS) data, and the absolute configuration of 1 was established by X-ray diffraction. Compound 1 features a hemiketal unit situated at the four-membered ring of harziane-type diterpenes for the first time, while 2 represents the rare occurrence of sterols with rings A and B being degraded. Compounds 1 and 2 displayed weak inhibition against the tested phytoplankton (Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense) with half maximal inhibitory concentration (IC₅₀) ranging from 14 to 53 μg/mL.     

Effect of Algal Food on Animal Prey Consumption Rates in the Omnivorous Copepod,Mesocyclops thermocyclopoides

We measured the food consumption rates in the omnivorous copepod Mesocyclops thermocyclopoides on different animal prey types in the presence of, and in the absence of one of the algal food types, the small, nonmotile Chlorella, or the large, motile Chlorogonium. Animal prey tested included different zooplankton species covering a size range of 88 to 1446 μm. The number of animal prey consumed was inversely proportional, but the total weight consumed was directly proportional, to the body size and dry weight of the prey item. There was a significant reduction in animal prey consumption in the presence of algae, being higher with cladoceran prey than with ciliates and rotifers, and in the presence of Chlorogonium than in the presence of Chlorella. Cannibalism in M. thermocyclopoides was low when algal food was available.