DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: III. EFFECTS OF MITOMYCIN C ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

The "glucose-bleached" cells of Chlorella protothecoides, whoseplastids were profoundly degenerated containing no trace ofchlorophyll, were obtained by the method previously reported.Transferring the cells to the condition of re-generation ofchloroplasts (greening)—incubation in the light in a glucose-lessand nitrogen-rich medium—the effect of mitomycin C onthe recovery process was investigated. It was found that theantibiotic suppressed completely the cell division without affectingthe re-generation of chloroplasts. De novo formation of RNAand protein which has been observed to occur during the recoveryprocess was not affected by the antibiotic to any significantextent. It thus became clear that the re-generation of chloroplasts,accompanied by the formation of chlorophyll, RNA and protein,occurring under the said condition is not a phenomenon causedby the formation of new "normal" cells from previously degeneratedcells. As was expected, the antibiotic suppressed strongly theDNA synthesis, indicating that the new formation of DNA is nota necessary condition for the re-generation of chloroplastsin "glucose-bleached" algal cells. (Received March 1, 1965; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: IV. EFFECTS OF 5-FLUOROURACIL, DIHYDROSTREPTOMYCIN, CHLORAMPHENICOL AND ACRIDINE ORANGE ON THE PROCESSES OF GREENING AND DIVISION OF "GLUCOSE-BLEACHED" ALGAL CELLS

1. The "glucose-bleached" cells of Chlorella protothecoides, whichwere obtained by the method described previously, were transferredto a glucose-free medium containing basal mineral nutrientsalone in the dark, and after a certain period of time, the cellsuspension was supplied with urea and light to induce the greeningof cells. At different times before and after the provisionof urea and light, the inhibitors were applied to the cultureto test their effects upon the process of greening.
2. Markedgreening of the glucose-bleached cells occurred aftera lagperiod in the control culture. 5-Fluorouracil inhibitedthecell greening strongly when it was applied at differenttimesbefore the provision of urea and light. When applied aftertheprovision of urea and light, the suppressive effect of 5-fluorouracilgradually decreased with the delay of its application. No inhibitiveeffect was observed when the uracil analogue was added laterthan the 12th hr after the provision of urea and light, thetime around which the chlorophyll formation started in the controlculture. On the other hand, the cell division was much morestrongly affected by 5-fluorouracil. Even when it was appliedat the 18th hr after the provision of urea and light, the celldivision was completely halted, indicating that the greeningand division of the glucose-bleached cells are separate processes.Different mechanisms of action of the uracil analogue towardsthese two processes were suggested.
3. Dihydrostreptomycin showedits strongest suppressive effectwhen added at the beginningof the dark incubation of algalcells in the glucose-free medium,and with the delay of application,its effect was progressivelyreduced, even during the periodof the dark incubation. Thesuppression, however, was stillmarked when it was applied atthe 15th hr.
4. Chloramphenicol was found to inhibit stronglythe chlorophyllformation and protein synthesis, but, to a muchlesser extent,RNA synthesis. Acridine orange suppressed thecell greeningand division at such a low concentration as 1.5µg/ml.
5. Based on these observations it was concludedthat synthesesof nucleic acid and protein are essential processesfor thegreening of the glucose-bleached algal cells. Successiveeventsoccurring in the greening process were discussed.

(Received March 9, 1965; )     

Phylogenetic Relationships Among Microsporidia Based on rDNA Sequence Data, With Particular Reference to Fish-Infecting Microsporidium Balbiani 1884 Species.

Recently, large discrepancies have been identified between microsporidian systematics based on molecular and traditional characteristics. In the current study the 530f-580r region of the rRNA gene of eight microsporidian species was cloned and sequenced. Included were two unclassified species of Microsporidium Balbiani, 1884 and an unidentified microsporidian that infects the musculature of different sea bream species. Sequence identities in excess of 98% indicated that these three species almost certainly are members of the same genus. Phylogenetic analyses of all microsporidian sequence data available for this region of the gene (20 species) and for partial small subunit sequences (51 species of 21 genera) revealed these species to be distinct from the family Pleistophoridae Doflein, 1901 and closely related them to the genus Sproguea Weissenberg, 1976. This clade was found to comprise a sister taxon to that containing the vast majority of fish-infecting species. Broad cladistic divisions were found between terrestrial insect-infecting and fish-infecting species, which together are distant from the aquatic insect-infecting microsporidia. The rRNA gene of certain fish-infecting genera was found to be more highly conserved than previously reported. This has implications for its utility in diagnostic assays and phylogenetic studies at, or close to, the species level.     

RIBONUCLEIC ACIDS APPEARING DURING THE PROCESS OF CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Investigations were made on the modes of synthesis of differentspecies of RNA which appear during the greening (chloroplastregeneration) of the "glucose-bleached" cells of Chlorella protothecoidescontaining profoundly degenerated plastids.
2. RNAs were extractedfrom the algal cells which had been labelledwith ³²P for 1hr before harvesting at different stages of thegreening inthe light and in darkness, and subjected to columnchromatographywith methylated albumin-coated kieselguhr. Itwas found that,during the greening process, the elution profilesof RNAs, interms of the optical density at 260 mµ and³²P-radioactivity,changed profoundly.
3. Based on these and other results, it wasconcluded that duringan early phase of the chloroplast regenerationin the glucosebleachedalgal cells, there occurs an active formationof both ribosomalRNAs (rRNAs) and the RNAs corresponding tosoluble RNA (sRNA),the formation coming, however, later toa standstill when thesynthesis of chlorophyll has proceededto a certain level. Thequantity ratio of sRNA to rRNA was foundto be constant (30:70)at different stages of the greening (bothin the light and indarkness), with a few exceptions. The synthesisof the chloroplastribosomal RNA is markedly accelerated bylight, and its maximumrate is observed sometime later thanthat of the non-chloroplast("cytoplasmic") ribosomal RNA. Itwas suggested that there areat least two different sites ofsynthesis of ribosomal RNAs,one in the plastid and the otheroutside of it (most probablyin the nucleus).

¹A part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965. ² Present address: Institute for Plant Virus Research, Ministryof Agriculture and Forestry, Aoba-cho, Chiba.     

Secondary host generation of the gall aphid Cerataphis jamuritsu (Homoptera)

Colonies of a Cerataphis species with well‐developed horns were found on the rattan Calamus quinquesstinervis in southern Taiwan. The morphology of first instar nymphs from the colonies accorded well with the morphology of first instar nymphs laid by alates of Cerataphis jamuritsu from galls on Styrax suberifolia, indicating that the rattan aphids are the secondary host generation of C. jamuritsu. Although the aphid colonies were attended by ants, the sharp horns of the first instar nymphs suggest that they might attack predators.     

Determining aphid taxonomic affinities and life cycles with molecular data: a case study of the tribe Cerataphidini (Hormaphididae:Aphidoidea: Hemiptera)

Aphid taxonomy is often frustrated by the host alternation and extensive polyphenism displayed by many species. Here we examine the utility of using molecular data to assist in life cycle and taxonomic determination. We found that a relatively small amount of DNA sequence data can greatly assist in these tasks. Molecular data have identified the synonymy of five species: Tuberaphis plicator (Noordam) is a junior synonym of T.takenouchii (Takahashi), T.taiwana (Takahashi) is a junior synonym of T.coreana Takahashi, Hamiltonaphis styraci (Matsumura) is transferred to Tuberaphis Takahashi, Astegopteryx roepkei Hille Ris Lambers is transferred to Ceratoglyphina van der Goot, and A.vandermeermohri Hille Ris Lambers is transferred to Cerataphis Lichtenstein. We have elucidated the complete life cycles of five species: A.basalis (van der Goot) alternates between Styrax benzoin and bamboos, Ceratoglyphina bambusae van der Goot alternates between S.benzoin and bamboos, Pseudoregma sundanica (van der Goot) alternates between S.paralleloneura and Zingiberaceae, T.coreana alternates between S.formosana and Loranthaceae, and T.takenouchii alternates between S.japonica and Loranthaceae. In all cases the molecular data agreed with available morphological data. This analysis demonstrates the utility of DNA sequence comparisons for elucidating complex life cycles and the taxonomy of difficult insect groups.