Hiroaki Tosuji

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.

Karyotyping of Female and Male Hediste japonica (Polychaeta, Annelida) in Comparison with Those of Two Closely Related Species, H. diadroma and H. atoka

Karyotypes of females and males of the brackish-water polychaete Hediste japonica sensu stricto, collected from the Ariake Sea, Japan, were examined by using regenerating tails. We used the Giemsa staining method and a computer-assisted image-analyzing system for the identification of each chromosome pair. The somatic chromosome number was 2n=28. The presence of an XX–XY (male heterogametic) sex chromosome system was determined from well-spread metaphase plates of somatic cells. The type of sex chromosomes related with phenotype exactly. The metacentric Y chromosome was much larger than the submetacentric X chromosome. All autosomes were metacentric. The karyotype of this species was compared with those of the other two closely related species (H. diadroma and H. atoka). The karyotypes of all the three species were similar to one another.

Reproductive Swarming of Sympatric Nereidid Polychaetes in an Estuary of the Omuta-gawa River in Kyushu, Japan, with Special Reference to Simultaneous Swarming of Two Hediste Species

Abstract Habitat differences and spatial and temporal separation in reproductive swarming among sympatric nereidid polychaetes were examined in an estuary of the Omuta-gawa River, Kyushu, Japan by annual periodical sampling from December 2003 to January 2005. Benthic adults of Tylorrhynchus osawai and Hediste diadroma occupied mainly the upper reaches of the estuary, whereas those of H. japonica usually inhabited the middle reaches, though their distributions overlapped. Reproductive swarming of mature adults occurred in the estuary just after high tide at night during spring tides in four nereidids: H. japonica (in the middle and lower reaches from late December to late February), H. diadroma (throughout the whole estuary from middle December to late April), T. osawai (in the middle reaches and another estuary from late October to late December), and Nectoneanthes oxypoda sensu Imajima, 1972 (in the lower reaches in late April and early May). This result shows that temporal separation of reproductive swarming may act as a reproductive isolation mechanism among these nereidids, except for H. japonica and H. diadroma. Simultaneous swarming and mass-spawning of the two Hediste species were commonly observed in the middle and lower reaches from late December to early February, suggesting the absence of a pre-spawning barrier to reproductive isolation between them. We found no difference in spawning behavior between H. japonica and H. diadroma. Males of both species seemed to participate in swarming earlier than females.

Calyculin A causes the activation of histone H1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor.

Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H(1) kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H(1) kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H(1) kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H(1) phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H(1) phosphorylation.

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.

Molecular Evidence for the Expansion of the Asian Cryptic Invader Hediste diadroma (Nereididae: Annelida) into the Northeast Pacific Habitats of the Native H. limnicola

We used previously established molecular methods to determine how far the Asian invader nereidid worm Hediste diadroma has spread into northeast Pacific estuaries that are inhabited by the native congener H. limnicola. Further, we analyzed the mitochondrial DNA of 702 Hediste specimens collected from 27 estuaries along 1,350 km of coastline in Washington, Oregon, and California, USA, to distinguish between the morphologically indistinguishable immature stages of these two species. In total, 377 specimens were identified as the invader H. diadroma and 325 were identified as the native H. limnicola. The invader H. diadroma was dominant at many sites in Puget Sound, Washington, and in the Columbia River estuary, Washington, and Oregon, suggesting that this species initially invaded estuaries in Washington or northern Oregon. In contrast, the native H. limnicola was dominant at intertidal sites in California and at subtidal sites in the Columbia River estuary. We also analyzed a partial nucleotide sequence from the mitochondrial cytochrome oxidase subunit I gene of H. diadroma in specimens collected from seven sites in the US and 11 sites in Japan, which showed no marked geographic differentiation between 18 US and 31 Japanese haplotypes. This finding suggests that H. diadroma have been introduced repeatedly into US estuaries from many regions in Japan.

Effect of calyculin A on the surface structure of unfertilized sea urchin eggs.

Calyculin A, a potent inhibitor of type 1 and type 2A protein phosphatases, induces contractile ring formation when applied to unfertilized sea urchin eggs [Tosuji et al., 1992: Proc. Natl. Acad. Sci. USA 89:10613-10617]. We report here the elongation of microvilli in the unfertilized eggs exposed to calyculin A. The elongated microvilli and associated sperm-egg binding sites (egg receptor for sperm) then became concentrated into a constriction site corresponding to the cleavage furrow. The egg receptor for sperm was also in close connection to the microfilaments. Okadaic acid is another known inhibitor of protein phosphatase type-1 and type-2A. Its effect, however, is about a hundredfold feebler for type-1 phosphatase than type-2A. Even after treatment with okadaic acid, no change was observed, suggesting that these morphological changes were induced by calyculin A solely though its inhibitory effect on the type-1 protein phosphatase.

Worldwide molecular phylogeny of common estuarine polychaetes of the genus Hediste (Annelida: Nereididae), with special reference to interspecific common haplotypes found in southern Japan

The nucleotide sequences of two mitochondrial genes (16S rDNA and COI) were compared among all species of Hediste, including five nominal and two cryptic species (H. atoka and H. diversicolor both consisting of two cryptic species, H. diadroma, H. japonica, and H. limnicola), as well as a newly found undescribed species (H. sp.), to estimate their phylogenetic relationships. The analysis using 16S rDNA sequence supported the monophyly of all five nominal species and H. sp., with no detection of the genetic differentiation between the two cryptic species in both H. atoka and H. diversicolor. However, analysis using COI sequence detected a marked differentiation between the cryptic species, and indicated that the two forms of H. atoka were separated into distinct clades; form A was included in a clade together with H. diversicolor, H. limnicola, and H. sp., whereas form B was included in another clade together with H. diadroma. Based on the topology of our phylogenetic analysis using the combined data set of 16S rDNA and COI, a hypothesis on the evolutionary history of the worldwide speciation in Hediste is proposed. This hypothesis seems to correspond well with the geographical distributions of current species and their morphological differentiation, supporting the previous hypothesis that the unique epitokous swarming and planktic larval development evolved independently in H. diadroma and H. japonica in eastern Asia. We also show that no or few interspecific substitutions have occurred in sequences of nuclear DNA (18S rDNA, 28S rDNA, and histone H3) in Hediste.

Cloning, sequencing of bone morphogenetic protein from sea urchin, Hemicentrotus pulcherrimus

A cDNA coding for bone morphogenetic protein (BMP) homolog of the sea urchin, Hemicentrotus pulcherrimus, was isolated from mid-gastrula using reverse transcription-polymerase chain reaction (RT-PCR) technique. The 2314 nucleotide sequence contains a 1383 open reading frame corresponding to a translation product of 461 amino acids. Comparison of the nucleotide and deduced amino acid sequence with BMP isolated from Strongylocentrotus purpuratus (SpBMP5-7; accession No. Z48313) shows a high degree of conservation. HpBMP seems to belong to the 60A subgroup as a result. A mRNA coding H. pulcherrimus BMP (HpBMP) was not detected in the unfertilized egg, but it was detected from blastula to prism stages.