Goh Nishitani

Multiple Plastids Collected by the Dinoflagellate Dinophysis mitra through Kleptoplastidy

ABSTRACT Kleptoplastidy is the retention of plastids obtained from ingested algal prey, which may remain temporarily functional and be used for photosynthesis by the predator. We showed that the marine dinoflagellate Dinophysis mitra has great kleptoplastid diversity. We obtained 308 plastid rbc L sequences by gene cloning from 14 D. mitra cells and 102 operational taxonomic units (OTUs). Most sequences were new in the genetic database and positioned within Haptophyceae (227 sequences [73.7%], 80 OTUs [78.4%]), particularly within the genus Chrysochromulina. Others were closely related to Prasinophyceae (16 sequences [5.2%], 5 OTUs [4.9%]), Dictyochophyceae (14 sequences [4.5%], 5 OTUs [4.9%]), Pelagophyceae (14 sequences [4.5%], 1 OTU [1.0%]), Bolidophyceae (3 sequences [1.0%], 1 OTU [1.0%]), and Bacillariophyceae (1 sequence [0.3%], 1 OTU [1.0%]); however, 33 sequences (10.8%) as 9 OTUs (8.8%) were not closely clustered with any particular group. Only six sequences were identical to those of Chrysochromulina simplex, Chrysochromulina hirta, Chrysochromulina sp. TKB8936, Micromonas pusilla NEPCC29, Micromonas pusilla CCMP491, and an unidentified diatom. Thus, we detected >100 different plastid sequences from 14 D. mitra cells, strongly suggesting kleptoplastidy and the need for mixotrophic prey such as Laboea, Tontonia, and Strombidium-like ciliates, which retain numerous symbiotic plastids from different origins, for propagation and plastid sequestration.

High-level congruence of Myrionecta rubra prey and Dinophysis species plastid identities as revealed by genetic analyses of isolates from Japanese coastal waters.

ABSTRACT We analyzed cryptophyte nucleomorph 18S rRNA gene sequences retained in natural Myrionecta rubra cells and plastid 16S rRNA gene and psbA sequences retained in natural cells of several Dinophysis species collected from Japanese coastal waters. A total of 715 nucleomorph sequences obtained from 134 M. rubra cells and 564 plastid 16S rRNA gene and 355 psbA sequences from 71 Dinophysis cells were determined. Almost all sequences in M. rubra and Dinophysis spp. were identical to those of Teleaulax amphioxeia, suggesting that M. rubra in Japanese coastal waters preferentially ingest T. amphioxeia. The remaining sequences were closely related to those of Geminigera cryophila and Teleaulax acuta. Interestingly, 37 plastid 16S rRNA gene sequences, which were different from T. amphioxeia and amplified from Dinophysis acuminata and Dinophysis norvegica cells, were identical to the sequence of a D. acuminata cell found in the Greenland Sea, suggesting that a widely distributed and unknown cryptophyte species is also preyed upon by M. rubra and subsequently sequestered by Dinophysis. To confirm the reliability of molecular identification of the cryptophyte Teleaulax species detected from M. rubra and Dinophysis cells, the nucleomorph and plastid genes of Teleaulax species isolated from seawaters were also analyzed. Of 19 isolates, 16 and 3 clonal strains were identified as T. amphioxeia and T. acuta, respectively, and no sequence variation was confirmed within species. T. amphioxeia is probably the primary source of prey for M. rubra in Japanese coastal waters. An unknown cryptophyte may serve as an additional source, depending on localities and seasons.

Encystment and Excystment under Laboratory Conditions of the Nontoxic Dinoflagellate Alexandrium fraterculus (Dinophyceae) Isolated from the Seto Inland Sea, Japan

S. Nagai, G. Nishitani, Y. Takano, M. Yoshida and H. Takayama. 2009. Encystment and excystment under laboratory conditions of the nontoxic dinoflagellate Alexandrium fraterculus (Dinophyceae) isolated from the Seto Inland Sea, Japan. Phycologia 48: 177–185. DOI: 10.2216/08-43.1. The sexuality (i.e. encystment and excystment) of the nontoxic dinoflagellate Alexandrium fraterculus, isolated from the Seto Inland Sea, Japan, was clarified for the first time under laboratory conditions. Sexual reproduction was by conjugation of isogametes, and plasmogamy was completed in 25–60 min after the conjugation started and produced a planozygote with one transverse and two longitudinal flagella, then a cyst was formed. Cysts were round or elliptical. The cyst size was 37.5–50.0 µm in diameter. The surface of cysts was smooth, and there was no paratabulation. Encystment through sexual reproduction was observed in two of 28 pairs, which included seven self-crossings, by use of seven nonaxenic clonal strains. No planozygote formation or encystment was found in any of the self-crossings, indicating that this species is heterothallic. Successful excystment was observed when the cysts, which had been preserved at 10°C in the dark for 3 mo, were incubated under light conditions at 25°C, thus showing that this species has a similar life cycle as reported in A. tamarense/catenella/tamiyavanichii.

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.

Swimming behavior of the spoon worm Urechis unicinctus (Annelida, Echiura).

Large numbers of swimming and stranding Urechis unicinctus were observed at night during low tide in Sasuhama, Miyagi Prefecture, northeastern Japan, during the periods from January to February in 2012 and 2013. Worms did not drift passively but swam actively, therefore hinting at a certain purpose for such behavior. As trochophore larvae of U. unicinctus were observed to occur simultaneously in the plankton, we infer the possibility that this is an event of reproductive swarming. Anatomical observations of both swimming and stranding U. unicinctus showed that none of the specimens had gametes, which may suggest that these were completely spent after spawning. Urechis unicinctus seemed to begin swimming after dusk and the observed swimming behavior occurred during the evening ebb tide throughout the night low tide during winter time. Stranding U. unicinctus have long been known in Japan and have been attributed to sea storms. The present study shows for the first time the possibility that U. unicinctus swims in order to reproduce at night and that this swimming behavior is closely linked to the stranding of U. unicinctus individuals.

And then there was one: Polydora uncinata and Polydora hoplura (Annelida: Spionidae), the problematic polydorid pest species represent a single species

It is necessary to monitor shell-associated polydorid worms owing to the risk that they pose to commercially important molluscs. This requires accurate identification, but is often hampered by morphological similarities among species, insufficient type specimens, and abridged species descriptions. Thus morphological characteristics and molecular sequences of the most harmful polydorid species, Polydora uncinata from Japan and Australia and Polydora hoplura from South Africa were compared to determine whether they represent two morphologically similar, but genetically distinct species, or a single species. A wide range of morphological variation (with respect to size, length of caruncle and pigmentation patterns) was observed in each species and population, and their variations largely overlapped and revealed them to be at the intraspecific level of a single species. This was confirmed by gene sequences of nuclear 18S and 28S rRNA that were completely identical for P. uncinata and P. hoplura . The mitochondrial 16S rRNA and cyt b gene analyses also showed no genetic differences between these two species. The tree topology of the mitochondrial cyt b gene did not reflect geographic differences but instead suggests artificial transportation of the species. We recommend the synonymization of P. uncinata with P. hoplura.

Development of compound microsatellite markers in the marine phytoplankton Alexandrium tamiyavanichii (Dinophyceae)

We isolated 24 polymorphic microsatellite markers from the toxic dinoflagellate A. tamiyavanichii. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 2 to 6, and the estimate of gene diversity was from 0.269 to 0.739 across the 24 microsatellites. We consider that these loci have a potentially to reveal the genetic structure and gene flow among A. tamiyavanichii populations.

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay, Japan.

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6×106 cells m-2 and 1.4×107 cells m-2, respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3×103 cells m-2day-1 and 1.4×104 cells m-2day-1, respectively. For A. pacificum, the highest cell density was 7.3×107 cells m-2 during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8×102 cells m-2day-1. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.

Morphological, developmental, and ecological characteristics of the suctorian ciliate Ephelota gigantea (Ciliophora, Phyllopharyngea, Ephelotidae) found on cultured wakame seaweed in Northeastern Japan

Wakame seaweed is an important aquatic resource in Iwate Prefecture. However, a suctorian Ephelota gigantea sometimes causes great damage to wakame culture. Since little is known about the biological characteristics of E. gigantea, its detailed morphology and temporal change of biological characteristics during the 2010 culture season were investigated. Scanning electron microscope observations showed that E. gigantea had different striation patterns on the stalk; there was a swell made of cement by which the stalk was attached to wakame firmly; and the buds had cilia arranged in concentric circles about a ring in the center of the ventral side. A suctorian parasite was found to infect E. gigantea, and the infection seemed to have decreased drastically the attached density of E. gigantea on wakame. Cell size of parasite-infected E. gigantea individuals was larger than that of uninfected individuals, probably because larger E. gigantea has larger surface area for attachment of the parasite. Cyst formation or conjugating individuals were not observed.

A New Primer Set to Amplify the Mitochondrial Cytochrome C Oxidase Subunit I (COI) Gene in the DHA-Rich Microalgae, the Genus Aurantiochytrium

This study was performed in order to develop a primer set for mitochondrial cytochrome c oxidase subunit I (COI) in the DHA-rich microalgae of the genus Aurantiochytrium. The performance of the primer set was tested using 12 Aurantiochytrium strains and other thraustochytrid species. There were no genetic polymorphisms in the mitochondrial sequences from the Aurantiochytrium strains, in contrast to the nuclear 18S rRNA gene sequence. This newly developed primer set amplified sequences from Aurantiochytrium and closely related genera, and may be useful for species identification and clarifying the genetic diversity of Aurantiochytrium in the field.